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Intrapopulation compatibility in Gonium pectorale Müller (Volvocales:Chlorosphyceae) McCauley, Marion Joan 1974

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INTRAPOPULATION COMPATIBILITY IN GONIUM PECTORALE MULLER (VOLVOCALES, CHLOROPHYCEAE) by MARION JOAN MCCAULEY B.Sc., U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t he Department of BOTANY We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r , 1974 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by t h e Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my u i r i t t e n p e r m i s s i o n . Department of Botany The U n i v e r s i t y of B r i t i s h C o l u m b i a Vancouver, B r i t i s h C o l u m b i a Canada Date OjVh-hiA; /£> • /9 ?y i i ABSTRACT F o r t y c l o n e s o f Gonium p e c t o r a l e M'uller were c r o s s e d i n a l l com-b i n a t i o n s a t 20°C, and t h e r e s u l t a n t z y g o t e s examined t o determine t h e degree of s e x u a l c o m p a t i b i l i t y w i t h i n a s i n g l e p o p u l a t i o n . C l o n e s i s o l a t e d from the same s i t e but i n d i f f e r e n t y e a r s were i n c o m p a t i b l e , i n d i c a t i n g a l a c k o f gene f l o w between them. Two d i s t i n c t groups were found w i t h i n 31 c l o n e s i s o l a t e d from a s i n g l e mud sample, and a l l t h e c l o n e s i n one were i n c o m p a t i b l e w i t h a l l t h o s e i n t h e o t h e r . However, members o f both groups were c o m p a t i b l e w i t h t h o s e i n a t h i r d group. The e x i s t e n c e of a t l e a s t two complementary p a i r s o f mating t y p e s w i t h i n a s i n g l e G_. p e c t o r a l e p o p u l a t i o n , sensu S t e i n ( 1 9 5 8 b ) , i s c o n s i d e r e d i n t he c o n t e x t of o t h e r s e x u a l c o m p a t i b i l i t y r e s u l t s , and i s proposed as a r e a s o n f o r the o c c a s i o n a l i n a b i l i t y t o o b t a i n o p p o s i t e mating t y p e s from a mud sample from which o n l y a few c l o n e s have been i s o l a t e d . i i i TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES i u LIST OF FIGURES v v ACKNOWLEDGEMENTS • u i INTRODUCTION 1 MATERIALS AND METHODS 4 1. I s o l a t i o n 4 2. S t o c k s 4 3. C r o s s e s 6 4. G e r m i n a t i o n T e c h n i q u e s 9 RESULTS 11 DISCUSSION 23 SUMMARY 35 BIBLIOGRAPHY 36 i v LIST OF TABLES Page TABLE I . I s o l a t i o n dates and s i z e ranges of the M clones. 5 TABLE I I . S i z e ranges f o r the c o l o n i e s and c e l l s of the S clones provided by Dr. 3.R. S t e i n (personal communication). 12 V LIST OF FIGURES Page FIGURE 1. Intercrossing of the S clones. 14 FIGURE 2. Germination of zygotes produced by S clone crosses. 14 FIGURE 3. Intercrossing of the M clones at 20°C. 17 FIGURE 4. Varying i n t e n s i t y of mating response amongst the M clones. 19 FIGURE 5. Germination of zygotes from the M clone crosses. 22 FIGURE 6. Cross r e s u l t s from Stein (1966b) showing i n t e r -r e l a t i o n s h i p of clone S1.6 and Minnesota s t r a i n 23 with the remaining S clones. 25 u i ACKNOWLEDGEMENTS I would l i k e t o thank Dr. J a n e t R. S t e i n f o r her g u i d a n c e , p a t i e n c e and encouragement t h r o u g h o u t t h i s s t u d y . F i n a n c i a l s u p p o r t f o r t h i s work was p r o v i d e d by a g r a n t t o Dr. S t e i n (N-.R.C. A1035) and by t h e U n i v e r s i t y o f B r i t i s h C o lumbia Committee on R e s e a r c h . S i n c e r e t h a n k s a r e extended t o D r s . P.R. Gorham and M. Hickman o f the Department o f Botany, U n i v e r s i t y o f A l b e r t a f o r p r o v i d i n g space and equipment f o r the c o m p l e t i o n o f t h i s s t u d y . I am g r a t e f u l f o r t h e e n d l e s s s u p p o r t and encouragement g i v e n by my husband, V i c t o r . 1 INTRODUCTION During the past 20 years, there have been many s t u d i e s on the c o m p a t i b i l i t y of a l g a l populations, e s p e c i a l l y on the green f l a g e l l a t e s known as the Volvocales. I n v e s t i g a t i o n s have dealt both with popu-l a t i o n s from the same general area and with those widely separated g e o g r a p h i c a l l y . In these s t u d i e s (Brooks, 1966; Carefoot, 1966; Coleman, 1959; G o l d s t e i n , 1964; H a r r i s and S t a r r , 1969; S t e i n , 1958a, b, 1965a, 1966a, b) 10-15 clones were i s o l a t e d from a s i n g l e mud or water sample c o l l e c t e d from a p a r t i c u l a r l o c a t i o n at a given time. A l l such clones were considered as belonging to one popu l a t i o n . A f t e r c r o s s i n g a l l i s o l a t e s i n every combination, u s u a l l y two clones of opposite mating types were kept f o r f u r t h e r s t u d i e s on i n t e r p p p u l a t i o n c o m p a t i b i l i t y . For some populations opposite mating types were not obtainable w i t h i n the clones i s o l a t e d , t h e r e f o r e a s i n g l e clone was kept f o r f u r t h e r study. Subsequent crosses f r e q u e n t l y demonstrated that the l a t t e r clones were compatible with one or s e v e r a l clones from other populations, and t h e r e f o r e were sexual, p r o v i d i n g compatible mating types were present. However, i t was s t i l l unresolved whether populations with one apparent mating type a c t u a l l y c o n s i s t of (a) only one; (b) two opposite mating types, only one of which appears i n the 10-15 clones i s o l a t e d ; er (c) s e v e r a l incompatible mating types of g e n e t i c a l l y i s o l a t e d s t r a i n s . _ _ In the c o l o n i a l Uolvocales, a c u l t u r e s t a r t e d from a s i n g l e colony (Bold, 1942). 2 In an attempt to resolve t h i s question, compatibility t e s t s i n -volving many clones from a single population, were c a r r i e d out. Forty clones of Gonium pectorale Muller, i s o l a t e d from Lemon Cove, C a l i f o r n i a , were crossed at d i f f e r e n t temperatures. The production of viable . zygotes, as i n d i c a t e d by zygote germination, was considered the best c r i t e r i o n f o r the determination' of compatible mating types. Consequently, several techniques were tested to determine the most suitable for inducing zygote germination. The r e s u l t i n g method was used to determine those clones of G. pectorale which were compatible. The l i f e - c y c l e of G_. pectorale has been described i n d e t a i l by Stein (1958b). Karyological studies of t h i s species have been reported by Cave and Pocock ( l 9 5 l ) and u l t r a s t r u c t u r a l studies by Lang (1963) and Stein (1965b). The systematics of t h i s species i n North America i s treated by Pocock (1955). A colony of G_. pectorale i s a square or rhomboid curved plate composed of 16, sometimes 8, 4 or 2, b i f l a g e l l a t e d c e l l s interconnected by cytoplasmic strands. The four c e n t r a l c e l l s are surrounded by 12 marginal c e l l s , three on each side, a l l being enclosed within a gela-tinous matrix. Each ovoid to nearly spherical c e l l contains a single bowl-shaped chloroplast and one or two basal pyrenoids, and i s capable of d i v i d i n g to form a new colony. Each c e l l also may become a gamete which, upon fusion with a sexually compatible isogamete, forms a zygote. Germination of a smooth-walled zygote or zygospore produces four c e l l s or gones united i n a colony, each of which develops into a 16-celled colony. As meiosis occurs during germination, G_. pectorale i s haploid 3 irn the vegetative condition. Although homothallic clones have been reported for t h i s species, h e t e r o t h a l l i c clones are more commonly encountered (Stein, 1965a). The former are those clones which can form f e r t i l e zygotes by them-selves, and the l a t t e r are those which can do so only when mixed with another sexually compatible clone (see Coleman, 1962). Other terms used i n the present study are defined as follows: population - "Colonies i s o l a t e d from one c o l l e c t i o n s i t e at any one time are considered members of the same population s t r a i n and are referred to as either a s t r a i n or a population," (Stein, 1958b). sexual i s o l a t i o n - a condition i n which two populations are incapable of mating and producing viable o f f s p r i n g , interpopulation crosses - crosses between clones from two d i f f e r e n t populations intrapopulation crosses - crosses between clones from the same popu-l a t i o n . 4 MATERIALS AND METHODS 1. Is o l a t i o n s Thirty-one clona l cultures were i s o l a t e d from a dried mud sample according to the technique described by Stein (1958b) (hereafter re-ferred to as the M clones). I s o l a t i o n s were made by Miss Linda Minchin i n the summer and f a l l of 1970 from four d i f f e r e n t wetted portions of a dried mud sample (Stein Mud 274) c o l l e c t e d i n August, 1963 from Lemon Cove, C a l i f o r n i a , by Dr. 0. Proskauer and stored at room tem-perature. The 31 clones and th e i r i s o l a t i o n dates are l i s t e d i n Table I. An add i t i o n a l nine cultures, i s o l a t e d from dried mud samples c o l l e c t e d from the same area but i n d i f f e r e n t years, also were tested for compatibility with the newly i s o l a t e d clones. The sources and i s o l a t i o n dates of these nine clones (1.6, 13.2, 13.6, 14.1, 14.3, 15.1, 15.3, 57.1 and 57.17 - hereafter referred to as the S clones) are given i n Stein (1965a - see Table l ) . 2. Stocks Stock cultures were grown i n 8 oz j a r s with small, inverted p e t r i dish l i d s (65 mm diameter) at a temperature of 20 4= 2°C. They were grown i n a 16 hr l i g h t - 8 hr dark cycle and at an i n t e n s i t y of 300-350 f t - c . Stocks were transferred every 4-8 weeks. Throughout the study a modified version of Pringsheim's soil-water medium, with a pinch of CaCO^ added, (S t a r r , 1964) was used (hereafter r e f e r r e d to as mSWC). The medium was autoclaved at 15 psi for 15 min and allowed to cool for 5 TABLE I. I s o l a t i o n dates and size ranges of the M clones. I s o l a t i o n Date Clone No. Colony Size (jum) C e l l S i z B (pm) 26-vI-70 2 35-57 7-15 II 4 27-53 7-14 ll-X-70 10 35-64 8-15 23-X-7D 14 50-76 10-15 II 15 30»62 8-15 it 16 31-73 -85.17 II 17 64-84 15-17 II 18 50-70 8-17 II 19 35-72 8-17 II 20 43-80 10-17 it 21 27-68 7-17 it 22 50-70 10-17 ti 23 35-70 7-15 II 24 35-67 7-16 II 25 69-88 15-17 II 26 33-70 9-15 24-X-70 27 35-70 7-15 II 28 28-56 7-15 it 30 27-53 7-14 i i 33 45-58 10-14 II 36 30-65 8-15 n 41 50-70 10-17 II 43 28-64 8-15 n 45 28-56 7-14 II 46 35-79 7-15 II 47 38-75 7-15 II 49 43-82 9-17 II , 50 45-67 10-15 II 5.1 26-58 7-14 n 52 50-65 10-15 n 53 45-76 10-15 6 24 hr before being used, rather.than the usual steaming for one hour on three consecutive days (see Starr, 1964). This modification mas adopted to hasten media preparation as no differences were observed i n growth rate and mating a c t i v i t y during the preliminary crosses. 3. Crosses To determine the s u i t a b i l i t y of the autoclaved medium, observe and recognize the d i f f e r e n t phases i n the l i f e cycle of _G. pectorale, and e s t a b l i s h f a m i l i a r i t y with the techniques to be used, several preliminary crosses were tested. A l l S clones, and numbers 14 to 26 i n c l u s i v e l y of the M clones, were chosen for these crosses because of t h e i r healthy condition i n culture, The crosses were made i n s t e r i l e watch-glass plates (watch-glass suspended on a glass t r i a n g l e i n a deep p e t r i dish) and spot-plates ( t r i p l e depression s l i d e i n a deep p e t r i dish) (see Hoshaw and Rosowski, 1973; Figure 3-1) and each i n -cubated at 15, 20 or 25°C. Water was added to the p e t r i dishes to prevent excessive evaporation. Concurrently, the remaining clones were transferred every 10-15 days to fresh medium to improve the con-d i t i o n of the cultures. Upon completion of the t e s t s and development of vigorously-growing cultures, crosses for the main study were prepared by in o c u l a t i n g 1-2 ml from each of two 7-10 day old cultures into a test tube of fresh mSWC medium and incubating at each of the three temperatures s p e c i f i e d previously. S t e r i l e pipettes were used for each culture to avoid any possible cross-contamination. Tubes incubated at 15 and 25°C at the 7 University of B r i t i s h Columbia and a l l those at the University of Alberta, were kept i n test tube racks. Those incubated at 20°C at U.B.C. were suspended by wire hooks on horizontal rods (see Starr, 1973; Figure 11-4). Crosses were kept at the same l i g h t i n t e n s i t y and cycle as was used f or stock maintenance for the f i r s t 2-3 weeks. Subsequently, they were placed at room temperature i n complete darkness to permit maturation of any zygotes formed. A l l tubes were checked both macro- and microscopically f o r zy-gotes one to several weeks aft e r placing i n the dark. The presence of an orange ri n g on the i n s i d e of the tube at the air-medium i n t e r f a c e and/or of an orange scum on the surface of the medium was i n d i c a t i v e of extensive zygote production. At the University of B r i t i s h Columbia a l l 40 clones were crossed 2 i n every combination and incubated at each of the three t e s t tem-peratures. During the incubations the culture chambers at 15 and 25°C overheated. As i n s u f f i c i e n t time was available to repeat the 3200 crosses affected before moving to Edmonton, i t was decided to concentrate on the crosses at 20°C. A d d i t i o n a l l y , since preliminary crosses between the S and M clones showed them to be completely incom-p a t i b l e , the study was r e s t r i c t e d further to compatibility s o l e l y 2 As G. pectorale i s isogamous, every clone must be crossed with every other clone to determine mating type of each. A d d i t i o n a l l y , as there are no physical differences, the designation of the mating type, v i z . *+.' or *-', i s purely a r b i t r a r y . The mating type designation of each of the S clones follows that given by Stein (1965a), which also was a r b i t r a r y . B within the M clones. At the University of Alberta, the methods used were s i m i l a r except that the number of crosses possible at any one time was l i m i t e d , since stocks f o r crosses were grown only i n te s t tubes. Once the mating type of each clone was determined, clones of opposite mating types were crossed a second time. A d d i t i o n a l l y , two clones, M17 and M25, which had shown complete i n c o m p a t i b i l i t y the f i r s t time, 'were recrossed with a l l others i n every possible combination and, as a test f o r the ten-t a t i v e l y determined mating type of each clone, four of each type were again crossed with a l l of the other clones of the same mating type. The eight chosen generally were those showing vigorous zygote production 'in some i f not a l l of the f i r s t crosses, and included 'minus' mating types M21, M27, M46 and M53, and 'plus' mating types M4, M10, M18 and M49. A l l of the above crosses were incubated at 20°C only. The crosses made at the University of Alberta were allowed to dry partly and sometimes completely a f t e r being put into the dark and before checking f o r zygotes. In dry test tubes zygotes were e a s i l y recognized by using a dissecting microscope to scan the walls of the tube. The contents of a l l tubes also were checked by making a wet mount and examining with a compound microscope. As several of the tubes became contaminated with fungi, a drop of Cotton Blue (Dohansen, 1940) was added to each wet mount to d i f f e r e n t i a t e between fungal c e l l s and Gonium zygotes. A l l tubes which were found to contain zygotes were stored at room temperature i n the dark f or subsequent use. 9 4 i Germination Techniques To obtain s u f f i c i e n t zygotes for t e s t i n g germination techniques, crosses mere made i n s t e r i l e match-glasses suspended on glass t r i a n g l e s i n deep p e t r i dishes. A l l were incubated at 20°C and under the same l i g h t i n t e n s i t y and regime as used for the crosses described i n the preceeding section. Two weeks a f t e r the crosses were made, the plates, with the l i d s askew, were put i n the dark and l e f t to dry. The watch-glasses were stored at room temperature i n i n d i v i d u a l envelopes. Various methods have been found successful for inducing zygote germination i n the algae. To determine a suitable method for G_. pectorale, zygotes from six d i f f e r e n t crosses within the M clones were subjected to each, of f i v e d i f f e r e n t conditions and examined daily for a month for evidence of germination. In a l l instances germination was found to occur under only one of the f i v e conditions. This method involved placing the zygotes i n a s t e r i l e watch-glass plate, wetting them with mSWC and then placing them i n darkness at 37°C for 48 hr. Water was added to the p e t r i dish to prevent excessive evaporation. After two days at 37°C, the watch-glass plate was placed i n a 20°C culture chamber with a 16 hr l i g h t - 8 hr dark cycle and subsequent germination was observed within f i v e days. When zygotes were f i r s t subjected to a low temperature (-5°C) for 24 hr before being placed at 20°C, or were placed at.20°C immed-i a t e l y after wetting, no germination was observed. S i m i l a r l y , attempts to induce germination of zygotes on mSWC supernatant s o l i d i f i e d with 1% agar proved unsuccessful, either when exposed to 37°C for 48 hr, 10 and then 20°C, or when exposed to 20°C only. Although no attempt was made to quantify the per cent germination, i t probably was moderately high since microscopic examination revealed that the majority of the heavy-walled zygotes were empty after f i v e days at 20°C. To determine the true compatibility of the clones, the v i a b i l i t y . of the resultant zygotes was determined. During storage i n the dark a l l l i q u i d i n the tubes' dried up thereby eliminating any vegetative c e l l s . Consequently, rather than transfer the zygotes to s t e r i l e watch-glass plates, the successful germination method was applied d i r e c t l y to the zygotes i n the test tubes. The presence of colonies i n the tubes, as determined with a dissecting microscope, was considered i n d i c a t i v e of germination and hence com p a t i b i l i t y . 11 RESULTS Size ranges for the M clones are presented i n Table I. The values for each clone are from measurements made on at l e a s t 15 c e l l s and colonies from several cultures which varied i n age from 5-20 days. This resulted i n a t o t a l of 60-80 c e l l s and colonies being measured for every clone. The data on size ranges f o r the S clones (Table II) were provided by Dr. D.R. Stein from measurements made pr i o r to t h i s study, as during the present study these clones were destroyed as a r e s u l t of the equip-ment malfunctioning. A l l of the M clones appear to be G_. pectorale var. pectorale as opposed to var. praecox Pocock (Pocock, 1955). This i s indicated by: l ) the c e l l and colony sizes (Table I ) ; 2) the presence of a single pyrenoid; 3) the almost spherical c e l l shape; 4) the possession of the c h a r a c t e r i s t i c bright green colour; 5) the absence of a large c e n t r a l space i n the colony (coenobium); and, 6) the absence of precocious daughter colony development. The nine S clones are assumed to be var. pectorale also (Stein, personal communication). The r e s u l t s of the crosses between the S clones are given i n Figure 1. Despite the malfunctioning of the 15 and 25°C culture chambers during the main compatibility t e s t s , s u f f i c i e n t data were ava i l a b l e from the preliminary crosses that r e s u l t s are reported f o r 15 and 25°C as well as for 20°C. A p o s i t i v e r e s u l t i s reported only when zygotes were produced i n . a t l e a s t two r e p l i c a t e crosses. Clone 12 TABLE IT. Size ranges for the colonies and c e l l s of the S clones provided by Dr. 3.R. Stein, (personal communication). Clone No. Colony Size (Aim) C e l l Size (jum) •1.6 25-63 6-16 13.2 44-90 7-24 13.6 27-76 6-17 14.1 26-50 6-12 14.3 30-70 7-15 15.1 30-78 7-17 15.3 30-90 7-19 57.1 32-69 8-16 57.17 . 25-66 6-19 13 FIGURE 1. Intercrossing of the S clones. A - zygotes formed at 15 C; « - at 20°C; 18 - at 25°C. FIGURE 2« Germination of zygotes produced by S clone crosses. Clone S1.6 was omitted as i t f a i l e d to mate with any of the S clones. A-zygotes germinated from cross at 15°C; ® - at 20°C; S- at 25°C. 1 4 13.2 14.1 15.3 57.1 13.6 @ 14.3 ' • • • 15.1 • • • 57.17 @ A B 1.6 13.2 14.1 15.3 57.1 13.6 14.3 15.1 57.17 15 S1.6 ujas incompatible with any of the other eight. There was vigorous zygote production within each of the four populations, v i z . S13, S14, S15 and S57, at both 20 and 25°C. At 15°C mating was shown only by population S57. As indicated i n Figure 2, a l l of the crosses produced viable zygotes. The r e s u l t s of the crosses between the M clones at 20°C are shown i n Figure 3. There were no homothallic clones as none of the s e l f -crosses produced zygotes. Clones M17 and M25 were not compatible with any others i n t h i s population. Of the remaining 29 clones, 12 were of the 'minus' mating type and 1.7 of the 'plus' mating type. There are d i s t i n c t groupings within each mating type, based on a b i l i t y to mate. These have been delimited by the heavy l i n e s and each grouping given a l e t t e r designation, v i z . G, H, 3, K and L, to f a c i l i t a t e c l a r i t y and eliminate lengthy r e p e t i t i o n i n the discussion.. These l e t t e r designations are used i n discussing the crosses, i e . the G x 3 or H x L crosses, etc. Clones i n group G were compatible with those i n group 3 and, except for clone M2, compatible also with those i n group K. The group H clones were compatible with those i n both groups K and L, but no zygotes were ever produced from crosses between groups G and" L or from crosses between groups H and 3. As shown i n Figure 4, considerable v a r i a t i o n was observed i n the i n t e n s i t y of the mating response. The G x 3 crosses consistently produced large numbers of zygotes, so much so that the e n t i r e surface 16 FIGURE 3. Intercrossing of the M clones at 20°C. G, H, K and L designate groups of clones based on a b i l i t y to mate within each mating type. 9- zygotes. 17 J L 19 21 22 45 53 28 46 52 20 27 43 47 17 25 G 14 18 10 15 16 23 24 26 H 30 33 36 41 49 50 51 17 25 ® 1 8 FIGURE 4. varying i n t e n s i t y of mating response amongst the M clones. G-L: r e f e r to legend for Figure 3. O-' extensive mat of zygotes several alyers thick; © - scattered clumps of zygotes, mat rare; • - small clumps of 10-100 zygotes each, not v i s i b l e with the naked eye; B - very few (10-15) s o l i t a r y zygotes. 1 9 J - K • 4 - L — — * l +\ 19 21 22 45 53 28 46 52 2 0 27 43 4 7 2 O o o o o ( 4 o o o o o • a a 14 o o o o o m a , 18 o o o o o ® © a 10 15 m © o © @ O 16 m • o © O © 23 • • 24 • o © © © 2 6 • E l • o • © © © H 3 0 • 33 a • © © © © 3 6 m 9 o 9 © © : 41 4 9 5 0 m a ® © © © J L 5 1 m ® ® © © © 20 of the medium was covered with a mat several layers thick. Zygote production usually was l e s s vigorous amongst the H x L crosses. Though zygotes were always v i s i b l e on the surface of the medium, they usually occurred i n clumps and r a r e l y formed a mat. In most instances zygotes could not be seen without microscopic examination of test tubes of the crosses involving the K clones though some showed a f a i n t r i n g at the air-medium i n t e r f a c e . Examination with a dissecting microscope revealed small clumps of zygotes (10-100 each.) on the walls of the tubes. In other tubes an extensive search produced only 10-15 s o l i t a r y zygotes. Although the number produced was low, zygote production was consistent. Despite the variable mating i n t e n s i t y , germination occurred i n a l l zygote-producing crosses (Figure 5), thereby i n d i c a t i n g true sexual com p a t i b i l i t y . Possible exceptions may be clones M20 and M50; however, crosses involving these two i n which no germination was observed, always were contaminated with fungi. As the hyphae completely enveloped, the zygotes, they may have had an adverse physical e f f e c t on germination. No zygotes were produced by the crosses between the S clones and the M clones. 21 FIGURE 5. Germination of zygotes from the M clone crosses. G-L: r e f e r to legend for Figure 3. g - zygotes from at l e a s t two crosses germinated; • - zygotes from only one cross germinated. 22 \ H 3 —  J - K >H L - N 21 22 4 5 53 28 46 52 2 0 2 7 43 4 7 IT 2 ' • m m G si a v. 10 ® ® E3 a ES m 15 ® • m 16 ® • • • m 23 m 24 • • • m El m 26 • m • m m H 30 m m m • 33 n a ® • m m a 36 3 a n • m m «> 41 m m • m m 4 9 m a m m a m » 5 0 • • • ® • 51 w 12 n ® 23 DISCUSSION The degree of sexual compatibility within the S clones reported here, i n both i n t e r - and intra-population crosses, i s d i s t i n c t l y l e s s than that obtained by Stein (1966b) for the same clones. For comparison, her r e s u l t s are reproduced i n Figure 6. She reported a l l intrapopula-tion crosses to produce zygotes at TO, 15, 20 and 25°C. In the present study three of the populations, v i z . S13, S14 and S15, did not produce zygotes when crossed at 15°C. No crosses were tested at 10°C. Stein reported f i v e interpopulation crosses, u i z . S13.6 x S14.1, S13.6 x S15.3, S14.3 x S15.3, S14.3 x S57.1, and S15.1 x S57.1, that resulted i n the production of zygotes, with two of these occurring at two temperatures. In the present study no interpopulation crosses produced zygotes; thus suggesting that the populations are now sexually i s o l a t e d . This decrease i n sexual compatibility may be a r e s u l t of main-tainin g the clones i n culture for many years. The S clones had been i n culture at le a s t six years when used by Stein, and were at le a s t 12 years old when used i n the present study. The period i n culture appears to be the only s i g n i f i c a n t variable between the two studies. The culture methods employed i n the present study are e s s e n t i a l l y i d e n t i c a l to those used by Stein. The only difference i n technique involved the methods of s t e r i l i z a t i o n of the culture medium; however, tests showed there to be no difference i n the growth and mating of the clones. 24 FIGURE 6. Cross r e s u l t s from Stein (.1966b) showing i n t e r r e l a t i o n s h i p of clone S1.6 and Minnesota s t r a i n 23 (clones 2 3 u l and 23.5) with the remaining S clones. • - zygotes formed at 10°C; H - at 15°C; O - at 20°C; and O- at 25°C 0 25 y 1 3 2 14.1 15.3 57.1 23.1 13.6 n a s o o o ^ @ o 14.3 • a ® o © si a • a ® 15.1 • m ® o B m a ® o 57.17 • SB © O 1.6 • o 23.5 • S3 ®0 • m ® o 26 There are two other reports for the Volvocales i n which compati-b i l i t y test r e s u l t s d i f f e r between two studies. Brooks (1966) reported several s t r a i n s of Astrephomene qubernaculifera Pocock (Astrephomena-ceae) and Carefoot (1966) reported two s t r a i n s of V/olvulina s t e i n i i P l a y f a i r (Volvocaceae) i n which mating was observed i n i t i a l l y but sub-sequently could not be induced. With the exception of two of the Astrephomene s t r a i n s , which apparently l o s t t h e i r capacity for sexual reproduction within several months of i s o l a t i o n , a l l had been kept i n culture for approximately eight years (Stein, 1958a). The i n c o m p a t i b i l i t y of clone S1.6 with the other eight S clones was noted also by Stein (1965a, 1966b). Although she found that i t lacked the a b i l i t y to mate with clones i s o l a t e d from nearby areas i n C a l i f o r n i a , she found i t to be compatible with 'minus' clone 23.1 from Minnesota, which i n turn was compatible with 'plus' clones S13.6, S14.3 and S15.1. Her data for crosses of Minnesota s t r a i n 23 with the C a l i f o r n i a s t r a i n s (S clones) are included i n Figure 6. This i n t e r -r e l a t i o n s h i p of Minnesota clone 23.1 with the S clones suggests that clone S1.6 may not be as g e n e t i c a l l y d i f f e r e n t from the other S clones as i s suggested by the r e s u l t s of the present study. Had an opposite mating type to clone S1.6 been obtained i t might have shown t h i s through crosses with some of the S clones. Stein (1966b) points out that "not a l l interpopulation crosses are r e c i p r o c a l between the two mating types of two populations" which i s evident from the S clone crosses, wherein 'plus' S14.3 x 'minus' S15.3 produced zygotes but 'minus' S14.1 x 'plus' S15.1 did not (Figure 6). Thus non-reciprocal mating may be 27 a factor i n the i n c o m p a t i b i l i t y of clone S1.6 with the other S clones. Non-reciprocal mating between two s t r a i n s of Pandorina morum Bory (In-Bl II and Io-72), led Coleman (1959) to suggest that only one mating type was common to the two pairs of complementary mating types. Since more recent studies (Stein, 1965a, 1966b) in d i c a t e that sexual i s o l a t i o n i n G_. pectorale i s not as prevalent as reported previously,. i t seems more probable that both of Coleman's s t r a i n s represented a single pair of mating types. The s i t u a t i o n concerning clone S1..6 discussed i n the precedinig paragraph, adds support to the contention that Coleman's non-reciprocal mating more l i k e l y was between two s t r a i n s with the same complementary pair of mating types, than between two s t r a i n s each representing a d i f f e r e n t pair of mating types. • f the 31 M clones only two, M17 and M25, -could not be induced to mate with any other clone or with each other. This may be because these two represent a mating type for which the opposite and comple-mentary mating type was not i s o l a t e d and/or did not survive i n culture, as only 31 o f the o r i g i n a l 55 clones i s o l a t e d by Minchin survived to produce viable cultures. The i n c o m p a t i b i l i t y also could be a r e s u l t of the temperature at which crosses were incubated, as both these clones grew at a much slower rate than did the others. A l l crosses were i n -cubated at 20°C, which may d i f f e r considerably from the optimum tem-perature for these two clones. Two a d d i t i o n a l p o s s i b i l i t i e s e x i s t . Whilst clones M17 and M25 were i s o l a t e d from rewetted mud, suggesting t h e i r sexual origin"^, the p o s s i b i l i t y cannot be excluded that they became s t e r i l e p r i or to use i n t h i s study, as did two Astrephomene 28 s t r a i n s mentioned previously (Brooks, 1966). A l t e r n a t i v e l y , morpho-l o g i c a l differences may have been responsible, as clones M17 and M25 were larger than most of the others (Table i ) . Similar c e l l size d i f -ferences as well as several other morphological v a r i a t i o n s have been reported by Stein (1958b - see Table 6) for 13 G_. pectorale s t r a i n s . However, both the r e s u l t s of that and l a t e r studies (Stein, 1965a, 1966b) f a i l e d to demonstrate any c o r r e l a t i o n between morphological , c h a r a c t e r i s t i c s and s t r a i n i n t e r c o m p a t i b i l i t y . Subsequent examination of her clones revealed the morphological v a r i a t i o n reported to be i n s i g n i f i c a n t (Stein, personal communication). The occurrence of two d i s t i n c t mating groups within the remaining 29 M clones suggests that the population consists of two pairs of complementary mating types, members of each pair being incompatible with those of the other. These have been designated + a and - a and +k and -k; groups G and • comprising the former pair and groups H and L the l a t t e r . The v a r i a t i o n i n mating response (Figure 4) gives an i n d i c a t i o n of the degree of compatibility of the clones within each pa i r . The G and 3 clones were highly compatible suggesting a marked ^ I t i s generally assumed that Gonium can survive for extended periods i n dried mud samples only as thick-walled zygotes or zygospores. However, the p o s s i b i l i t y cannot be excluded that t h i s alga i s capable of surviving also i n the vegetative state, as Trainor and McLean (1964) and McLean (1967) have reported that vegetative c e l l s of Sponqiochloris  t y p i c a Trainor and McLean (Chlorococcales) were viable a f t e r one year i n a dry state. 29 s i m i l a r i t y i n t h e i r genetic make-up. The s l i g h t l y lower numbers of zygotes produced by the majority of L':x H crosses may be i n d i c a t i v e of a l e s s e r degree of compatibility between the 17 clones involved, and/or of the culture conditions not being as favorable for mating as they were for the G and 3 clones. Considerable v a r i a t i o n in i n t e n s i t y of mating reaction was ob-served also i n sexual compatibility studies of Platydorina caudata Kofoid (Harris and Starr, 1969). Again, t h i s probably i s a r e f l e c t i o n of the genetic s i m i l a r i t i e s of the s t r a i n s , as higher proportions of zygotes always resulted from matings between h e t e r o t h a l l i c pairs from the same e o l l e e t i o n s i t e than from matings between pairs from d i f f e r e n t l o c a t i o n s . Except for the difference i n numbers of zygotes generally produced by each of the two pairs of mating types (+ a and - a , +^  and ~ ' D ) , i n t h i s study, no other c h a r a c t e r i s t i c was observed that might f a c i l i t a t e t h e i r separation. Goldstein (1964) found that of the f i v e i n t e r c r o s s i n g 4 groups of Eudorina s t r a i n s which he studied, three exhibited a 'clumped' zygote arrangement and two a 'scattered' zygote arrangement. There was almost complete sexual i s o l a t i o n between the clumped' and 'scat-tered' s t r a i n s , as evidenced by the f a c t that only two of the 104 intercrosses recorded involved s t r a i n s with d i f f e r i n g zygote arrangements. S i m i l a r l y , Coleman (1959) observed that i n t e r c r o s s i n g occurred only 4 Four species and one variety were included; three groups were composed of one species, one. of three species, and one of one species and one v a r i e t y . 30 betu/een £. morum s t r a i n s possessing the same type of zygote aggregation, however many st r a i n s possessing the same aggregation pattern were -sexually incompatible. In A_. qubernaculifera i n t e r c r o s s i n g s t r a i n s possessed the same chromosome number, there being no i n t e r c r o s s i n g betu/een s t r a i n s with d i f f e r e n t chromosome, numbers (Brooks, 1966). Although the possible existence of multiple pairs of mating types has been shown for some a l g a l species (Coleman, 1959; Starr, 1959; Stein, 1965a), and may be i n f e r r e d from other published data (Goldman, 1964; Brooks, 1966; Carefoot, 1966), t h e i r occurrence within a single population has been studied only once (Brandham and Godward, 1965). Within the 47 clones of Cosmarium b o t r y t i s Meneghini tested, they found two d i s t i n c t pairs of mating types, which they designated + and 1 1 - and + and - , and each pair was incompatible with the other. Their clones were i s o l a t e d from nine d i f f e r e n t s i t e s , thus c o n s t i t u t i n g nine d i f f e r e n t populations according to the d e f i n i t i o n of 'population' used i n the present study. However, within the ten clones i s o l a t e d from Chobham, Surrey and c o n s t i t u t i n g a single population, two were of the 1 + mating type, two of the - mating type and six of the - mating type. 1 The + mating type may not have been present at a l l i n the Chobham population or may have been missed during the i s o l a t i o n procedure. As only ten clones were i s o l a t e d the l a t t e r appears more probable. They pointed out that i f the clones from the Chobham l o c a l i t y were 1 considered alone the six of the - mating type would be assumed s t e r i l e . This s i t u a t i o n p a r a l l e l s that found i n the present study regarding the reproductive status of clones M17 and M25. f 31 Similar also to t h e i r f i n d i n g s , the pairs of mating types i n the present study were completely incompatible. However, i n contrast, ab each pair was compatible with an intermediate mating type (- ). This intermediary consists of the three clones of group K (Figure 3) a b which cross with three of the four + and a l l of the + mating types. It i s possible that the exception, clone M2, possesses a fundamental genetic difference which prevents zygote formation-. The lack of i n t e r -mediate + clones may be because they were not i s o l a t e d and/or did not survive i n culture, or because they were i n v i a b l e (e;g., due to the presence of a sex-linked l e t h a l gene). The production of variable numbers of zygotes, and very few zygotes i n half the crosses in v o l v i n g the K clones, suggests that i n general the K clones have a very low compatibility with the G and H clones. Thus the K clones may have been produced by intercrosses between the two p a i r s of complementary mating types. If so, they would be very susceptible to s l i g h t v a r i a t i o n s i n culture conditions, hence the varied r e s u l t s . A l t e r n a t i v e l y , they may act as the intermediary stage towards the development of a separate interbreeding group of clones a r i s i n g from either complementary p a i r , e.g., H and L from G and 3 or vice versa. However, t h i s would be very u n l i k e l y unless the K clones consistently produced large numbers of zygotes i n crosses with either the G or H clones, thereby ensuring the production of a vigorous population i n which subsequent mutations could lead to the e s t a b l i s h -ment of a new and i s o l a t e d interbreeding population. Instead, the r e s u l t s support the contention that the K clones originated as a r e s u l t 32 of previous interbreeding between the two presently g e n e t i c a l l y i s o l a t e d pairs of mating types. The genetic s i m i l a r i t y of the two pairs of mating types (+ a and - a and +'3 and - ^ ) , indicated by t h e i r common compatibility with the K clones, suggests that they arose from the same stock, either one from the other or both from a common parent. I t i s possible that both occurred i n d i f f e r e n t (but possibly overlapping) niches i n the pond thus enabling both to co-exist. Support for t h i s contention i s the d i f f e r i n g degrees of compatibility observed, i n that the niche for the + a and - a clones may be more cl o s e l y approximated by the laboratory conditions than i t may be for the +^  and clones. This then could explain the d i f f e r i n g numbers of zygotes consistently produced by the crosses. The f i v e a d d i t i o n a l populations comprising the S clones were included i n the compatibility tests with the M clones because they originated from the same or nearby areas. Three of the f i v e , S13, S14 and S57, were c o l l e c t e d from the same pond but i n d i f f e r e n t years, v i z . 1951, 1955 and 1956 re s p e c t i v e l y . The M clones also were c o l l e c t e d from t h i s pond, but i n .1963. Consequently, these four populations may be considered as a ' l o c a l population', or community of p o t e n t i a l l y interbreeding i n d i v i d u a l s at a given l o c a l i t y (Mayr, 1970). However, according to Stein (1958b), the difference i n c o l l e c t i o n dates would negate the four being considered as belonging to the same population, and therefore they would be outside the scope of a s t r i c t 'intrapopu-l a t i o n ' study. 33 Although Stein (1966b) found that the three s t r a i n s , S13, S14 and S57, were compatible to a certain degree, (Figure 6, modified from Ste i n , 1966), Coleman (1959) reported i n P_. morum two sexually i s o l a t e d groups e x i s t i n g amongst the seven clones or s t r a i n s i s o l a t e d on four d i f f e r e n t occasions from a pond designated 'Bloomfield I 1 . This pond was sampled repeatedly to determine i f mating types were of constant occurrence and Coleman found that two of the c o l l e c t i o n s contained a d i f f e r e n t pair of mating types. Two c o l l e c t i o n s were made from each of f i v e other ponds and i n every instance the two pairs of mating types were incompatible, suggesting to her that, the coexistence of two sexually i s o l a t e d Pandorina populations i n a single pond i s not an uncommon occurrence." The r e s u l t s of crosses of G_. pectorale have already demonstrated the i n a b i l i t y of the S clones to interbreed and t h i s i n c o m p a t i b i l i t y i s extended to crosses with the M clones. The S clones, except for clone S1.6, o r i g i n a l l y were i n t e r f e r t i l e suggesting that they a l l belong to one pair of mating types. This pair then, may be incompatible with the pairs of mating types present i n the M clones. A l t e r n a t i v e l y , the population i n the pond represented by the M clones may be sexually i s o l a t e d from the populations which existed there seven dr more years previously, simply as a r e s u l t of evolutionary change. The f a c t that multiple pairs of mating types m'aysex'isth.ujithin single populations suggests a reason for the occasional i n a b i l i t y to i s o l a t e opposite mating types from some populations. I t i s conceivable that 34 a more extensive study might show the existence of an unlimited number of reproductively i s o l a t e d units within a single population. 35 SUMMARY The present study has reaffirmed the existence of multiple, reproductively i s o l a t e d breeding units i n Gonium pectorale Muller, previously demonstrated by Stein (1965a), within populations from diverse geographical l o c a t i o n s . In contrast to her r e s u l t s , two incompatible pairs of complementary mating types are reported within a single population, represented by 31 clones i s o l a t e d from a dried mud sample. 36 BIBLIOGRAPHY Bold, H.C. 1942. The c u l t i v a t i o n of algae. Bot. Rev-. 8: 69-138. Brandham, P.E. and Godiuard, M.B.E. 1965. The inheritance of mating type i n Desmids. New Phytoloqist 64: 428-435. Brooks, A.E. 1966. The sexual cycle and i n t e r c r o s s i n g i n the genus Astrephomene. 3_. Protozool. 13: 367-375. Carefoot, 3.R. 1966. Sexual reproduction and i n t e r c r o s s i n g i n Volvulina s t e i n i i . 3_. 'Phycol. 2: 150-156. Cave, M.S. and Pocock, M.A. 1951. Karyological studies i n the Volvocaceae. flm. 3. Bot. 38: 800-811. Coleman, A.W. 1959. Sexual i s o l a t i o n i n Pandorina morum. 3. Protozool. 6: 249-264. 1962. Sexuality. Ln: Lewin, R.A. (Ed.), Physiology and Biochemistry of Algae. Academic Press, New York, pp. 711-729. Goldstein, s M. 1964. Speciation and mating behavior i n Eudorina. 3. Protozool. 11: 317-344. Harris, D.O. and Starr, R.C. 1969. L i f e history and physiology of reproduction of Platydorina caudata Kofoid. Arch. Protistenk. I l l : 138-155. Hoshaw, R.W. and Rosowski, 3.R. 1973. Methods for microscopic algae. In: Stein, 3.R. (Ed.), Handbook of Phycoloqical Methods. Cambridge University Press, Cambridge, pp. 53-67. 37 Dohansen, D.A. 1940. Plant Microtechnique. McGraw-Hill Book Co., Neuj York. 523 pp. Lang, N.3. 1963. Electron microscopy of the Volvocaeeae and Astrephomenaceae. Am. 3_. Bot. 50: 280-300. Mayr, E. 1970-. Populations, Species, and Evolution. Belknap Press, Cambridge, Mass. 453 pp. McLean, R.J. 1967. Dessication and heat resistance of the green alga Spongiochloris t y p i c a . Can. 3_. Bot. 45: 1933-1938. Pocock, M.A. 1955. Studies i n North American Uolvocales. I. The genus Gonium. MadrorTo 13: 49-64. Starr, R.C. 1959. Sexual reproduction i n ce r t a i n species of Cosmarium. Arch. Protistenk. 104: 155-164. 1964-. The culture c o l l e c t i o n of algae at Indiana University. Am. 3. Bot. 51: 1013-1044. 1974. Apparatus and maintenance. Ijn: Ste i n , 3.R. (Ed.), Handbook of Phycoloqical Methods. Cambridge University Press^ Cambridge, pp. 171-179. Stein, 3.R. 1958a. A morphological study of Astrephomene qubernaculifera and Uolvulina s t e i n i i . Am. 3_. Bot. 45: 388-397. 1958b. A morphologic and genetic study of Gonium pectorale. Am. 3_. Bot. 45: 664-672. 1965a. Sexual populations of Gonium pectorale (Uolvocales). 'Am. 3. Bot. 52: 379-388. 38 Stein, 3.R. 1965b. On cytoplasmic strands i n Gonium pectorale (Volvocales). 3_. Phycol. 1: 1-15. 1966a. Growth and mating of Gonium pectorale (Volvocales) i n defined media. 3_. Phycol. 2: 23-28. 1966b. E f f e c t of temperature on sexual populations of Gonium pectorale (Volvocales). Am. 3. Bot. 53: 941-944. Trainor, F.R. and McLean, R.3. 1964. A study of a new species of Spongiochloris introduced into s t e r i l e s o i l . Ajn. 3_. Bot. 51: 57-60. 

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