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A preliminary electrophoretic stury on Bangia vermicularis Harvey (Rhodophyta) populations of British… Borgmann, Ira Elizabeth 1987

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P R E L I M I N A R Y E L E C T R O P H O R E T I C S T U D Y O N BANGIA VERMICULARIS H A R V E Y (RHODOPHYTA) P O P U L A T I O N S O F BRITISH C O L U M B I A by IRA E L I Z A B E T H B O R G M A N N A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF M A S T E R OF SCIENCE in Genetics Programme T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Botany We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH C O L U M B I A February. 1987 © Ira Elizabeth Borgmann, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Botany The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: February, 1987 ABSTRACT A preliminary electrophoretic study on Bangia vermicularis Harvey (Rhodophyta) along the coast of British Columbia was undertaken to determine whether enzyme banding patterns could be used to identify the relatedness of the populations which varied in chromosome number (3, 6 or 4) and life history (asexual or sexual). Material from nineteen sites was tested for seven enzymes: glutamate dehydrogenase (GDH); malate dehydrogenase (MDH); lactate dehydrogenase (LDH); superoxide dismutase (SOD), glucose 6-phosphate dehydrogenase (G6PDH); phosphoglucoisomerase (PGI); and phosphoglucomutase (PGM). Unlike higher plants, some of the banding patterns obtained in Bangia could only be explained if the enzymes SOD, G D H , L D H and PGI are monomers and the enzymes P G M , SOD, GDH, PGI, and G6PDH have only one cellular location. Generally, with the exception of two enzymes, PGI and G6PDH, which were highly polymorphic, only one to three bands were evident. Considerable amounts of variation in the mobility of the isoenzymes were detected between populations independent of chromosome number or sexuality. Contrary to expectations, often populations with three chromosomes or six ' chromosomes had the same number of loci and many of the three chromosome populations had some banding patterns indicative of diploids. Consequently, if there had been a polyploid origin of the six chromosome populations then there must also have been many gene duplications in the three chromosome populations. Alternatively, if the three chromosome populations arose by aneuploid reduction within a six chromosome population then there must also have been gene silencing and/or the formation of null alleles. Populations that were very close geographically differed to a large extent suggesting that there ii may be little gene flow between populations. The variability between populations could be explained by isolation over long periods of time during which changes could have occurred in the structure and regulation of the enzymes tested. iii T A B L E O F C O N T E N T S A B S T R A C T ii A C K N O W L E D G E M E N T S vii 1. I N T R O D U C T I O N 1 2. M A T E R I A L S A N D M E T H O D S 15 2.1. Collection and Culturing of Bangia 15 2.2. Chromosome Counts of Bangia 21 2.3. Electrophoresis of Bangia ... 22 3. A N A L Y S I S O F E L E C T R O P H O R E T I C D A T A 26 4. R E S U L T S 32 4.1. C H R O M O S O M E C O U N T S 32 4.2. C O N T R O L S 35 4.3. E L E C T R O P H O R E S I S O F BANGIA P O P U L A T I O N S 45 4.3.1. Phosphoglucomutase : 47 4.3.2. Superoxide Dismutase/Tetrazolium Oxidase 59 4.3.3. Malate Dehydrogenase 72 4.3.4. Glutamate Dehydrogenase 76 4.3.5. Lactate Dehydrogenase 91 4.3.6. Phosphoglucose Isomerase 97 4.3.7. Glucose 6-Phosphate Dehydrogenase 114 5. DISCUSSION 128 5.1. DISCUSSION O F T H E R E S U L T S O F V A R I O U S E N Z Y M E T E S T S O N BANGIA P O P U L A T I O N S 128 5.2. G E N E R A L DISCUSSION 147 6. L I T E R A T U R E C I T E D 155 iv List of Figures Fig. 1 Bangia vermicularis Harvey 3 Fig. 2 Sexual Life History of Bangia 2 Fig. 3 Asexual Life History of Bangia 4 Figs. 4-6 Collection Sites of Bangia 17 Fig. 7 The three chromosome numbers observed in the genus Bangia from the northeast Pacific 34 Fig. 8 Banding patterns obtained from Point No Point southwest beach material through one season 37 Fig. 9 Banding patterns obtained from Point No Point southwest beach material after various periods of time and various storage methods 39 Fig. 10 Banding patterns obtained from Point No Point southwest beach material after various periods of freezing time 41 Fig. 11 Banding patterns obtained from fresh Hope Bay material after three weeks in culture .-. 43 Fig. 12a-b Banding patterns of PGM - samples of > 100 filaments from 15 populations 50 Fig. 13a-g Banding patterns of PGM - clonal cultures from 6 populations 52 Figs. 14a-b Banding patterns of SOD - samples of >100 filaments from 15 populations 61 Figs. 15a-i Banding patterns of SOD - clonal cultures from 5 populations 63 Figs. 16a-b Banding patterns of MDH - samples of >100 filaments from 15 populations 74 Figs. 17a-b Banding patterns of GDH - samples of >100 filaments from 15 populations 78 Figs. 18a-k Banding patterns of GDH - clonal cultures from 6 populations 80 Figs. 19a-b Banding patterns of LDH - samples of >100 filaments 93 Figs. 20a-b Banding patterns of LDH - clonal cultures from 5 populations 95 Figs. 21a-b Banding patterns of PGI - samples of >100 filaments 100 Figs. 22a-l Banding patterns of PGI - clonal cultures from 6 populations 102 Figs. 23a-b Banding patterns of G6PDH - samples of >100 filaments from 15 populations 117 Figs. 24a-i Banding patterns of G6PDH - clonal cultures from 6 populations. 119 Fig. 25 Summary plate of banding patterns for PGM 131 Fig. 26 Summary plate of banding patterns for SOD 132 Fig. 27 Summary plate of banding patterns for MDH 133 Fig. 28 Summary plate of banding patterns for GDH 134 Fig. 29 Summary plate of banding patterns for LDH 135 Fig. 30 Summary plate of banding patterns for PGI 136 Fig. 31 Summary plate of banding patterns for G6PDH 137 Figs. 32a-e The number of enzymes out of the total examined that had the identical banding patterns 140 Figs. 33a-e The number of enzymes out of the total examined that had at least one isoenzyme in common but were not identical in their electrophoretic patterns 142 Figs. 34a-e The number of enzymes out of the total examined that had no common isoenzyme bands in their electrophoretic patterns 144 v List of Tables Table I. Characterization of Algal Enzymes 11 Table II. Algal Studies Using Isoenzymes as Taxomonic Aids 12 Table HI. Further Applications of Isoenzyme Electrophoresis in Algal Studies ... 13 Table IV. Collection Sites of Bangia 16 Table V . An Example of Some Possible Banding Patterns of Haploid and Diploid Populations 30 Table VI. Some Examples of Complexity in Banding Patterns in Diploid Populations due to the Different Mobilities of Homodimers 31 Table VII. Chromosome Number and Reproductive State of Bangia Populations in British Columbia 33 vi A C K N O W L E D G E M E N T S I am indebted to Dr. K Cole for the initiation of this project and her continual encouragement and support throughout my studies. I would like to express my gratitude to Dr. J . McPherson for teaching me electrophoresis and all my supervisory committee (Drs. K. Cole, A. Griffiths, and J . McPherson) for their constant availability to discuss any question that I brought to them. I wish to thank Dawn Renfrew for accompanying me on a lengthy collecting trip. Thanks also to Carol Tarn, Carol Park, Larry Golden, Sandy Lindstrom, Dawn Renfrew, Bev Hymes, Brian Oates and Ellen Rosenberg for joining me on collecting trips and/or for collecting material for me. I am grateful to Dr. M. Hawkes for many helpful discussions during this project. Special thanks to all my family and friends for their patience and encouragement, and for listening. vii 1. I N T R O D U C T I O N Bangia, (Rhodophyta, Bangiaceae) is a morphologically simple alga consisting of cells embedded in a firm gelatinous sheath forming an unbranched cylinder (Fig. lc,d). The cells contain a single large stellate chloroplast and are uninucleate. The filament is initially uniseriate becoming multiseriate with age and has a rhizoidal holdfast. Filaments may be asexual, male or female. Populations of marine Bangia may have either a sexual (Fig. 2) or an asexual (Fig. 3) life history. Bangia is found worldwide occurring on rocks or pilings high in the intertidal zone and is subject to wave action, wetting and drying (Cole, Hymes and Sheath 1983)(Fig. la,b). In North America it is found most often in marine locations but has also been reported in fresh water locations. In North America its distribution on the Pacific coast extends from Alaska to Mexico and on the Atlantic coast, from Labrador to the Gulf of Mexico (Sheath and Cole 1984). Fresh water Bangia has been found in the Laurentian Great Lakes (Sheath and Cole 1980). The identification of Bangia at the species level is difficult because there are few morphological characters that are taxonomically useful. Many characters, such as filament length and pigmentation may be altered under various environmental conditions (Sommerfeld and Nichols 1973). There are geographical trends in cell dimension: Bangia from the Pacific Coast (160 m diameter) is larger than that from the Atlantic Coast (92 m diameter) (Sheath and Cole 1984). Adding to the 1 CARPOSPORANGIA ^ 2 SEXUAL LIFE HISTORY OF BANG/A F i g . 1 Bangia v e r m i c u l a r i s Harvey A. g r o w i n g on rock B. growing on p i l i n g s C. female, male and a s e x u a l f i l a m e n t s D. s p o r e l i n g . 3 ASEXUAL LIFE HISTORY OF BANGIA I N T R O D U C T I O N / 5 difficulty of identification both marine and fresh water populations can change their salinity requirements over a few generations; originally fresh water populations can adapt to marine conditions and marine populations to fresh water conditions (denHartog 1972; Geesink 1973; Reed 1980; Sheath and Cole 1980). Hence, it has been proposed by Sheath and Cole (1984) that there are minimally two marine species in North America: B. atropurpurea (Roth) Ag. on the Atlantic coast and B. vermicularis Harvey on the Pacific coast. The fresh water form in the Great Lakes may be a variety of B. atropurpurea. A collaborative, comprehensive study was recently begun to investigate the biogeographic trends and systematica of the genus Bangia in North America (Sheath and Cole 1980, 1984). Bangia is very abundant along the British Columbia coast. Initially the life histories and chromosome numbers of various populations in British Columbia were examined and four categories of filaments identified: sexual four; sexual three; asexual three; and asexual six (Cole, Hymes and Sheath 1983). This has led to the question of how these populations may have arisen and are related and whether polyploidy, aneuploidy or aneuploid reduction may have occurred. A preliminary isoenzyme electrophoretic study was undertaken to try to answer this question. Isoenzyme electrophoresis is a technique which has been used successfully on higher plants to detect gene duplication, polyploidy, aneuploidy and aneuploid reduction. After staining for enzyme activity various bands may arise resulting from post-translational modifications, the presence of different alleles at a locus coding for products of different mobilities, or from multiple loci coding for I N T R O D U C T I O N / 6 products of different mobilities (Markert 1983, Weeden 1983a). The frequently used enzymes for electrophoretic studies are most often in essential pathways and do not undergo modifications. The exceptions are mitochondrial enzymes that are transported across the membrane into the mitochondria and then modified so that they become active. Prior to the modification they are inactive and are not detected by this staining method (Newton 1983). Therefore, the isoenzymes observed are usually present due to different alleles at a locus or different numbers of loci. The number of bands present will depend on the number of alleles at each locus, the number of loci, the number of subunits of the enzyme and whether or not the products of the loci interact to form heteromers (Markert 1983, Weeden 1983a). Some enzymes may be found in more than one cellular compartment (chloroplasts, mitochondria, and cytosol) and these are coded by different loci. Any or all of these loci may be duplicated by gene duplication, aneuploidy or polyploidy. Gene duplications may arise from unequal crossing over in meiosis (tandem duplications) or from reciprocal translocations and subsequent selfing (unlinked duplications) (Gottlieb 1981). This latter occurrence would probably indicate a monophyletic line since that particular translocation is not likely to arise more than once (Gottlieb 1981). Reciprocal translocations are often common in annual plants (Gottlieb 1977a) and may explain the high frequency of gene duplication within those that have been examined. Gene duplications have been detected in several higher plants including Clarkia (Gottlieb 1974a, Gottlieb 1974b), Layia (Warwick and Gottlieb 1985) and Zea (McMillin and Scandalios 1983) and have been used to examine and confirm lineages based on morphology (Gottlieb 1977b, I N T R O D U C T I O N / 7 Weeden and Gottlieb 1979, Pichersky and Gottlieb 1983). Both allopolyploidy and autopolyploidy have also been commonly detected in higher plants. Allopolyploidy is more readily detected because of fixed heterozygosity due to the pairing of homologous chromosomes, as in Tragopogon (Roose and Gottlieb 1976). Autopolyploidy does not necessarily result in fixed heterozygosity, as in Coreopsis where one hexaploid species shows no fixed heterozygosity (Crawford and Smith 1984). In the Liliaceae polyploidy has been the predominant mechanism for genome amplification with examples of both allopolyploidy and autopolyploidy (Oliver, Martinez-Zapater, Pascual, Enriquez, Ruiz-Rejon, and Ruiz-Rejon 1983). Duplication of gene loci may plaj' a very important role in the evolution of gene sequences. If there is only a single gene coding for an enzyme there are strong selection pressures to maintain the enzyme at its present state since many of the alterations in controlling or structural regions would result in enzymes that would be less efficient. Only those alterations having no effect or improving the efficiency of the enzyme are likely to be maintained. However, in the case of gene duplication, aneuploidy or polyploidy, where there are at least two loci, one locus would be able to undergo changes without detrimentally affecting viabilitj'. It would be possible to get mutations which decrease the efficiency of the enzyme at one locus, change its specificity or change its regulatory control (Weeden 1983b). Gene silencing can be used to trace phytogenies (Ferris and Whitt 1977; Weeden I N T R O D U C T I O N / 8 1983b: Buth 1984) and has been studied in tetraploid fish. Phylogenies have been constructed based on the loss of duplicate gene expression; these are similar to phylogenies based on morphology (Ferris and Whitt 1977, 1978; Crabtree and Buth 1981; Buth 1983). The systematic use of duplicate gene silencing may be of limited use if genes are shown to be able to retetraploidize (Buth 1982). Chromosomal rearrangements can be the cause of rapid speciation and this appears to occur frequently. Species once interfertile maj' become intersterile while keeping the same alleles. This has occurred in Coreopsis (Crawford and Smith 1982) and in Stephanomera (Gottlieb 1973). In the Astereae, chromosomal rearrangements have occurred such that coding regions have been translocated to other chromosomes and the centromeres lost (aneuploid reduction). Species with nine chromosomes have undergone aneuploid reduction to give rise to species with four and five chromosomes. All species code for the same number of gene loci for each enzyme examined (Gottlieb 1981). In Crepis there has been a general trend in decreased amounts of DNA. The DNA/nucleus content, the number of chromosomes, and the number of genes specifying enzymes are not correlated (Roose and Gottlieb 1978). It appears that genomic rearrangements resulting in duplication of gene loci are a common occurrence, although, these often can not be detected using karyotypes alone. Consequently, electrophoretic studies to detect the number of gene loci may be very important. "The most important use of electrophoretic evidence in phylogenetic studies will probably not utilize genetic identity values based on presence/absence or frequencies of alleles. Instead the unique ability of I N T R O D U C T I O N / 9 electrophoresis to identify the number of gene loci that specify particular enzyme systems will be exploited" (Gottlieb 1981, p. 35). Detection of gene duplications, polyploidy and aneuploid reduction may aid in the reconstruction of lineages and may prove especially valuable when other characters (eg. morphological) are not available. Determining the number of gene loci from the electrophoretic bands is preferable to using measures of genetic identity, genetic distance, percent polymorphic loci and heterozygosity. Genetic identity and distance measures have many statistical problems involving data transformations (Buth 1984), choice of genetic distance measure (Smith 1977) and rooting of phylogenetic trees (Rogers 1984). There are also problems with the underlying assumptions of random mating, large population size (Hartl 1980) and constant evolutionary rate (Buth 1984). In addition, electrophoretic divergence across the present sample of loci is not random and is probably not representative of overall genetic divergence (Gottlieb 1981). Percent polymorphic loci and heterozygosity give limited information and are highly dependent on sample size and the number and kinds of enzymes used (Brown and Weir 1983). As well there are technical difficulties involving sampling errors and detection of different alleles that may have the same migration rate (Johnson 1977). Many of the above problems can be bypassed by counting gene loci. The number of loci detected will not be affected by population size, mating systems, founder effects or sample size. Increasing sample size may detect more alleles but more loci are not likely to be found (Gottlieb 1981). Also, it does not matter whether INTRODUCTION / 10 the same or different alleles are present in the population. To date, electrophoresis has not been applied extensively in algal studies. It has been used in all major groups of algae mainly for the isolation of enzymes to allow their characterization and to study their induction and inhibition (Table I). Electrophoretic banding patterns have also been examined for possible use as an additional taxonomic aid to distinguish between the major groups of algae (Table II). Electrophoresis has been used in very few cases to examine variation and gene flow between populations (Table Il ia), seasonal variation (Table 111b), and variation due to culturing and nutrition (Table IIIc). It has also been employed to examine variation between generation types of Ulva (Hoxmark 1976; Hushovd, Gulliksen and Nordby 1982), and the presence of sexual or asexual reproduction in Enteromorpha linza (Innes and Yarish 1984) and Heterocapsa pyguaea (Watson and Loeblich 1984). Using electrophoresis gene duplication and polyploidy have been discovered in only one genus, Chara (Grant and Proctor 1980). Al l twelve species of this green alga that were studied appeared to be polyploid. Isoenzyme electrophoresis was applied to different Bangia populations in an attempt to establish the relationship between three of the more readily available types in the northeast Pacific: sexual four; asexual three; and asexual six. Other techniques often used for determining relatedness between species with different chromosome number would be difficult in Bangia. The small size of the chromosomes does not readily allow visible banding patterns and D N A Table I: C h a r a c t e r i z a t i o n of A l g a l Enzymes 11 Group Chlorophyta Chiamydomonas Ent er omorpha I i nza Chi orel I a pyrenoidosa Chi orel l a s or oki ni ana Chi or el I a s p. O o c y s t i s alga Rhodophyta Cystoclonium pur pureum Chondr us cri spus Phaeophyta Lami nar i a di gi t at a Spatoglos s um pacifi cum AscophylI um nodosum Euglenophyta Euglena gracilis Eugl ena Enzyme adenyl sulphate 3'-phospho t r a n s f e r a s e peroxidase glutamate dehydrogenase c y s t e i n s t i m u l a t e d n i t r a t e reductase s p e c i f i c protease r i b u l o s e 1,5-diphosphate ca r b o x y l a s e glutamate dehydrogenase peroxidase hexose oxidase peroxidase r i b u l o s e 1,5-diphosphate c a r b o x y l a s e peroxidase ALA synthetase malate dehydrogenase p r o l i n i m i n o -p e p tidase aminopept idase g l y c o l a t e pathway enzymes phosp h o g l y c o l a t e , phosphoglycerate phosphatase Author Jender and Schwenn 1984 Murphy and OhEocha 1973a T a l l e y , White and Schmidt 1972 Ti s c h n e r and Schmidt 1984 Vedeneev, Kasatlana and Semenko 1983 Lee 1983 Murphy and OhEocha 1973b S u l l i v a n and Ikawa 1973 Murphy and OhEocha 1973c Yamada, Ikawa and Nisizawa 1979 V i l t e r 1983a, 1983b Beale and Fole y 1981 Davis and Me r r e t t 1973 S e n k p i e l , R i c h t e r and Barth 1974 S e n k p i e l , R i c h t e r and Barth 1978 Horrum and Schwartzbach 1980 James and Schwartzbach 1982 McCarthy, James and Schwartzbach 1981 Table I I : A l g a l S t u d i e s Using Isoenzymes as Taxonomic A i d s Group Chlorophyta Chaetomorpha 2 s p e c i e s U l o t r i c h a l e s and Kl ebsormidium spp. CI osierium ehrenbergii and C. moni I i fe r um Pandorina morum Tetraselmis Charophyceae, Chlorophyceae, Ulvapnyceae Chi orococcum\6 s p e c i e s and Tetracytis 3 s p e c i e s Protosi phone 32 i s o l a t e s Chiamydomonas 3 s p e c i e s C h l o r o s a r c i n a c e a n a l g a 7 s p e c i e s Rhodophyta eleven genera Porphyra 4 s p e c i e s Euchema 4 s p e c i e s Phaeophyta Laminariales 4 s p e c i e s and Fucales 5 s p e c i e s Fucus serralus and F. vesiculosus Pyrrophyta Cr ypt hecodi ni um cohni i Heterocapsa Peri dini um bal l i cum and Glenodinium foliaceum Author B l a i r , Mathieson and Cheney 1982 Fowzia, Al-Houty and S y r e t t 1984 Francke and C o e s e l 1985 F u l t o n 1977 Huber and Lewin 1986 S y r e t t and Al-Houty 1984 Thomas and Brown 1970a Thomas and Brown 1970b Thomas and D e l c a r p i o 1971 Thomas and Groover 1973 M a l l e r y and Richardson 1972 Miura, F u j i o and Suto 1978 Cheney and Babbel 1 978 Marsden, Callow and Evans 1984 Marsden, Evans, Callow and Keen 1984 Beam, Himes, Daggett and Nerad 1982 Watson and L o e b l i c h 1983 Whitten and Hayhome 1986 13 Table I I I : F u r t h e r A p p l i c a t i o n s of Isoenzyme E l e c t r o p h o r e s i s i n A l g a l Studies A. V a r i a t i o n and Gene Flow Between Populations Species Porphyra yezoensis(Rhodophyta) Thai assisi or a pseudonana and 7". fl uvi at i I i s (Chrysophyta) Asterionella formosa (Chrysophyta) Author F u j i o , Kodaka and Hara 1985 Murphy and G u i l l a r d 1976 Soudek and Robinson 1973 B. Seasonal V a r i a t i o n Species Author Euchema 4 s p e c i e s (Rhodophyta) Cheney and Babbel 1978 Skeletonema costatum and Thalassiora Gallagher 1980, 1982 ps eudonana(Chrysophyta) Peridinium ci net urn f . Wynne 1977 west ii (Pyrrophyta) C. C u l t u r i n g and N u t r i t i o n Species Enteromorpha Ii nza(Chlorophyta) Polytoma uve//a(Chlorophyta) Porphyra yezoensis(Rhodophyta) Skeletonema costatum and Thai as s i s i or a pseudonana(Chrysophyta) Euglena gracilis var. baci 11ari s(Euglenophyta) Author Innes and Y a r i s h 1984 Mangat 1979 Miura, F u j i o and Suto 1979 Murphy 1978 Chancellor-Maddison and N o l l 1963 INTRODUCTION / 14 quantification has not been perfected. As well, crosses between different sexual populations and examination of subsequent meiotic pairing behaviour has not yet been possible. It was hoped that isoenzyme electrophoresis and counting of gene loci would detect gene duplications, aneuploidy, polyploidy, or aneuploid reduction in the Bangia populations. The advantages of counting gene loci discussed earlier are especially important in the current study of Bangia. Asexual reproduction occurs in both sexual and asexual populations so that a population may actually have generated from only one or a few genetically different individuals. This would affect allele frequency but not the number of loci. Also, the only individuals that could be examined are those with an asexual life history from which clonal cultures can be obtained and to which the calculations of genetic identity or genetic distance could not be applied (because of the assumption of random mating). As well, because of the amount of time available for growth only small sample sizes of asexual clones of Bangia could be obtained in the study. Attempting to determine the number of isoenzyme loci present using electrophoresis provides the best means at present of detecting polyploidy or aneuploid reduction in Bangia. To date, electrophoresis has not been extensively employed for this purpose. Little is understood about the genetics of algae and their evolution and if successful this technique may offer an additional method to examine this. 2. MATERIALS AND METHODS 2.1. COLLECTION AND CULTURING OF BANGIA Bangia vermicularis Harvey was collected from a number of sites throughout the northeast Pacific (Table IV; Figs. 4-6). Collection sites were chosen by the availability of fairly large healthy populations and by accessibility. Most populations were limited to one or a few rocks or pilings within a small region. The majority of sites were on the Gulf Islands and Lower Vancouver Island. Two additional sites further north, Triple Island (British Columbia) and Sitka (Alaska), were also included in the study. Bangia was removed from boulders or pilings using tweezers and transported to the laboratory in plastic bags or glass vials on ice. The material was stored in a 4°C refrigerator until it could be set up in culture or could be prepared for electrophoresis, usually overnight. The size of filaments and their reproductive units were used to determine whether the populations were sexual or asexual (as outlined in Cole and Sheath 1980). Mature female filaments (n) release carpospores which germinate to form the conchocelis (2n) phase. This phase may reproduce itself by fragmentation and monospores (Fig. 2). Ultimately, under suitable conditions, conchospores are produced. On release these germinate into male and female filaments (n). It is 15 T A B L E I V : C O L L E C T I O N S I T E S O F BANGIA 16 L o c a t i o n S i t k a (Alaska) 53°03'N 135°18'W T r i p l e I s l a n d 54°17'N 130°53'W T h i r d Beach 49°17'N 123°07'W F r i d a y Harbor (Washington) 48°32'N 123°01'W Ogden Point 48°25'N 123°24'W Smuggler's Cove 48°25'N 123°24'W Sooke 48°22'N 123°44'W Po i n t No Point south beach 48°24*N 123°58'W Corresponding Map Number i n F i g s . 4-6 1 2 3 4 5 5 6 7 Reproductive Mode asexual sexual asexual asexual asexual asexual sexual P o i n t No Point southwest beach 48°24'N 123°58'W Fernwood 48°55'N 123°31'W S p o t l i g h t Cove 48°59'N 123°33'W Whaler's Bay 48°53'N 123°20'W S t u r d i e s Bay 48°54'N 123°19'W Miner's Bay 48°51*N 123°18'W Port Washington 48°49'N 123°19'W Hope Bay 48*48'N 123°16'W Roesland 48°48'N 123°18.5'W O t t e r Bay 48°48'N 123°18'W 8 9 10 1 1 12 13 14 15 15 sexual asexual asexual asexual asexual asexual asexual asexual asexual asexual L i t t l e Bay 16 asexual 48°45'N 1 2 3 ° 1 T W Date 5/5/85 20/3/85 18/5/85 23/11/84 16/5/84 26/4/84 7/5/85 26/4/84 12/4/85 10/4/84 26/4/84 22/5/84 12/4/85 25/4/85 12/4/85 25/4/85 25/5/85 23/5/85 23/5/85 23/5/85 22/5/85 24/5/85 22/5/84 24/5/85 27/4/84 8/7/84 27/4/84 23/5/84 8/7/84 24/5/85 24/5/85 17 F i g . 4 C o a s t a l r e g i o n of B r i t i s h Columbia and southern Alaska - the two most n o r t h e r l y c o l l e c t i o n s i t e s of Bangia. 18 F i 9 « 5 Lower c o a s t a l r e g i o n of B r i t i s h Columbia -c o l l e c t i o n s i t e s of Bangia. I 2 4 ° W *t>' to' I 2 3 ° W MATERIALS AND METHODS / 20 not yet possible to induce the release of conchospores and their subsequent germination under culture conditions in this laboratory. As well, it is not possible to maintain field collected male and female filaments in culture for more than a few days. On the other hand, when mature asexual filaments are placed into culture these release spores that develop into filaments which then repeat the cycle. Hence, clonal cultures are readily obtained from asexual populations. Cultures of asexual material were initiated by first pulling single filaments through four percent agar to remove epiphytes, and then placing them into 60 x 20 mm plastic petri dishes containing Modified Provasoli Medium with ten mg/1 germanium oxide and antibiotics (McLachlan 1973) to eliminate diatoms and bacteria. The cultures were kept covered with four to six layers of cheesecloth in three available chambers that differed in temperature and in day/night length (10°C,12:12;8°C,16:8;15°C,8:16). After spore release the filaments were removed. Following the initial four to six weeks the cultures were maintained in Modified Provasoli Medium without germanium oxide or antibiotics and were refreshed every four weeks. If there was any contamination selected material was transferred repeatedly until free of contamination. The cultures grew equally well in all three chambers. Eighty to one hundred asexual filaments from each population sampled in 1984 were individually cultured up to fourteen months, however, relatively few of these cultures grew sufficiently to be used for electrophoresis. Reasons for the poor growth of Bangia in culture may be several. Bangia grows on rocks and pilings high in the intertidal splash zone and is subjected to drying and rewetting. M A T E R I A L S A N D M E T H O D S / 21 Critical amounts of dessication at certain temperatures, as well as the substrate upon which it is grown, may be important for rapid growth of Bangia. The amount and type of light, nutrients and aeration may also affect the growth rate. Those populations which did grow sufficiently to be used for electrophoresis included: Odgen Point; Smuggler's Cove; Roesland; Otter Bay; Friday Harbor; and Third Beach. 2.2. C H R O M O S O M E C O U N T S O F BANGIA Material for chromosome counts was collected and fixed approximately two hours after sundown (Cole, Hymes and Sheath 1983). If collection at this time was not possible, material was placed in medium in a culture chamber and was removed and fixed approximately two hours after the beginning of the dark cycle. Material was fixed in ethanohglacial acetic acid solution (3:1) for at least twenty-four hours and then stored in seventy percent ethanol. Chromosome squashes were made using the aceto-iron-haemotoxylin-chloral hydrate method (Wittmann 1965). Approximately three to six filaments were squashed on each slide and at least three and up to six slides from each population were examined under a Wild M20 microscope. Several chromosomal squashes from each population were photographed using a Nikon M-35S camera and high contrast Kodak Technical Pan film. M A T E R I A L S A N D M E T H O D S / 22 2.3. E L E C T R O P H O R E S I S O F BANGIA Fresh material was pulled through four percent agar to clean the filaments which were then placed in Modified Provasoli Medium (McLachlan 1973) with germanium oxide and antibiotics for two days prior to grinding. The material was ground with a porcelain mortar and pestle on ice using as small a quantity as possible of extraction buffer (0.05M tris pH 7.5, 15% dextrose). The slurry was transferred to a 400 microlitre microcentrifuge tube, spun in an Eppendorf Micro Centrifuge (model 5414) for six minutes and the supernatant was loaded onto the polyacrylamide gel. The time of six minutes was determined by comparing material spun for as little as two minutes and up to twenty minutes to ascertain which minimum time allowed for sufficient removal of cellular debris to avoid distortion in the banding. Cultured material was ground and centrifuged in a similar manner. Twenty samples on each of four 0.75 mm thick gels were run in a Bio-Rad Protean Dual Slab Cell and twenty samples on each of two 0.75 mm thick gels were run in a Bio-Rad Protean II Cell. The enzymes examined could be resolved on one of two gel systems used. One system employed a 5% pH 7.2 spacer gel and a 12% pH 8.8 resolving gel (no SDS). The other system employed a 5% pH 7.2 spacer gel and a 12% pH 7.5 resolving gel (no SDS). To determine these optimal gel percentages various percentages of polyacrylamide were attempted for the spacer gel and the resolving gel in all combinations. Spacer gels of 3,4,5, and 6% and resolving gels of 7,8,9,10,12 and 14% were tried. As well, gels of all one concentration (7,8,9,10,12, or 14%) were tested. The pH of M A T E R I A L S A N D M E T H O D S / 23 the gels at which most enzymes would not denature was determined after comparing gels using buffers with varying alkalinities (pH 7.5, 8.0, 8.5, and 8.8). The above systems proved to resolve the bands the most clearly while maintaining enzyme activity. The running buffer (tris-glycine pH 8.35) was chilled to 4°C. Bromophenolblue was used as the tracking dye. The gels were run in a 4 °C refrigerator for two hours at 35 ma through the spacer gel and then for three to four hours at 65 ma through the resolving gel, adjusted every fifteen minutes, using a Gelman Instrument Company Model 38206 power pack. To determine these optimal speeds at which to run the gels various running times were attempted for the spacer gel (0.5,1,1.5,2,2.5,3 and 4 hours) and for the resoving gel (2,3,4,5,6 and 7 hours) in all combinations that allowed the resolving gel to run for equal or longer times than the spacer gel. The above system resolved the gels the most clearly of the combinations attempted. The following enzymes commonly used in isoenzyme banding studies were examined: malate dehydrogenase (MDH); glutamate dehydrogenase (GDH); phosphoglucoisomerase (PGI); phosphoglucomutase (PGM); lactate dehydrogenase (LDH); glucose-6-phosphate dehydrogenase (G6PDH); and superoxide dismutase/tetrazolium oxidase (SOD/TO). The first four were run on the 12% pH 8.8 resolving gel and the remaining three were run on the 12% pH 7.5 resolving gel. The enzyme recipes are modifications of those found in Shaw and Prasad (1970). The changes include substituting the tetrazolium salt M T T for N B T (in M D H , GDH, PGM, L D H and G6PDH) and substituting the coenzyme NADP for N A D (in GDH and LDH). SOD/TO appears as reverse banding on the M D H , GDH and, most clearly, L D H gels. All chemicals were purchased from M A T E R I A L S A N D M E T H O D S / 24 Sigma. Other enzymes were also attempted but no banding could be obtained. These included: acid phosphatase; alkaline phosphatase; esterase; isocitrate dehydrogenase; peroxidase and sorbitol dehydrogenase. The spacer gel was removed and the resolving gel was stained for at least one hour at 37 °C in Pyrex loaf pans. The gels were then fixed in methanohglacial acetic acid:water in the proportions 4:1:5 for approximately two to four minutes. Measurements of migration distance were made in centimetres from the border of the spacer/resolving gel to the band. The distances migrated by the bromphenolblue and pigments (phycoerythrin, present in the extract) were also recorded. Photographs of the gels were taken with a Polaroid MP-3 Land Camera using Polaroid Positive/Negative Land Pack Fi lm Type 665. The gels were placed on a light table and covered with yellow cellophane to improve the contrast before photographing. Another set of photographs was taken with a Konica Autoreflex T camera using 35 mm Kodak Tungston 160 film (colour slides) under a Bencher Inc. Illuma System Quartz Light Control table. A l l controls which tested the effect of treatment of material on the electrophoretic bands were run on 5% pH 7.2 spacer gels with 7% pH 8.8 resolving gels, 1.5 mm in thickness, for four hours at 35 ma. To examine if changes in banding pattern occurred throughout the season, material from Point No Point, collected at three different times (April 10, 1984; Apri l 26, 1984; May 22, 1984) was M A T E R I A L S A N D M E T H O D S / 25 compared. To determine if changes in enzymes occurred following culturing, material from Hope Bay, fresh and after three weeks of culturing, was compared. To establish whether changes occurred as the result of different storage methods, material from Point No Point, fresh, refrigerated (4°C) for up to three weeks, and frozen for up to two months was compared. To determine if changes in the enzymes resulted from storage of the extract, extracts of material from Point No Point, fresh and frozen for up to six weeks, were compared. Male filaments and female filaments from Point No Point were compared to determine if there are any differences in the banding patterns of the two sexes. 3. A N A L Y S I S O F E L E C T R O P H O R E T I C D A T A The electrophoretic data obtained from Bangia is more restricted than that from most higher plants or animals. This is due to two reasons. Firstly, individual filaments of Bangia are too small to examine and clonal cultures can only be obtained from asexual populations after extensive culturing. Secondly, it has not yet been possible to make crosses within and between sexual populations due to difficulties in inducing Bangia to complete its life history and subsequently obtaining clonal cultures sufficiently large enough to test. Hence, only populations of Bangia filaments and individual clonal cultures obtained from asexual populations of Bangia could be examined in this study. In most higher plants, the examination of individuals and progeny from controlled crosses facilitates the identification of loci and their respective alleles. However, in some higher plants, although individuals can be examined, progeny crosses are not feasible due to a long generation time. It has been possible to obtain information regarding loci number in such plants by comparing the banding patterns of haploid pollen and diploid sporophytic tissue (Weeden and Gottlieb 1979). This comparison is possible because subunits of the enzymes do not dissociate and reassociate but rather remain stable in the extraction buffer. Hence, only homomers are present from haploid pollen whereas both homomers and heteromers are present from diploid sporophytic tissue. This analytical technique was applied to Bangia in order to estimate the number of loci present for different enzymes of the various populations. 26 A N A L Y S I S O F E L E C T R O P H O R E T I C D A T A / 27 If the populations with three chromosomes gave rise to those with four or six chromosomes via polyploidy or aneuploidy these may be compared similarly to the haploid and diploid tissue of higher plants. For example, if an individual with six chromosomes arose by polyploidy from an individual with three chromosomes that individual would be effectively diploid. Populations derived from this individual would not appear different in their electrophoretic banding patterns if this event was recent. Heteromers would not be detected unless there had been sufficient time for the accumulation of amino acid changes in proteins such that one allelic product would have a different mobility. If this event was not recent there would be a detectable doubling in all or the majority of loci such that the six chromosome populations would band as would be expected of diploid tissue of higher plants and the three chromosome populations as haploid tissue. If all the populations appear to have the same number of gene loci, recent autopolyploidy or aneuploid reduction may have occurred. Aneuploid reduction may have resulted in populations with six chromosomes giving rise to those with three or four chromosomes. If three chromosome populations gave rise to four chromosome populations via aneuploidy heteromers for some but not all enzymes may be detected (again if there had been sufficient time). If the six chromosome populations gave rise to the four chromosome populations via aneuploid reduction there would be no apparent difference in banding patterns. If gene duplication or aneuploidy had occurred some loci may appear duplicated or there may be a loss of a locus by gene silencing. When interpreting isoenzyme banding patterns several factors have to be considered, such as, how the enzymes are affected by external environmental A N A L Y S I S O F E L E C T R O P H O R E T I C D A T A / 28 conditions and how they are affected by internal conditions such as cellular localization, number of subunits and interaction of subunits to form heteromers. Thereafter attempts can be made to determine the number of gene loci coding for an enzyme. The enzymes used in this study have been found to be little influenced by environmental factors in higher plants and are usually always present since they are in major biochemical pathways (Gottlieb 1977a). Therefore, the different climatic conditions of various populations and the different conditions of culture should not influence the isoenzyme banding patterns. Controls comparing field collected and cultured material confirmed this (see Section 4.2). Enzymes may be localized in different compartments of the cell including the cytosol, mitochondria, chloroplasts, and microbodies. These compartments often have very different environments (for example, pH) and hence isoenzymes localized in different subcellular compartments may require different conditions of extraction or electrophoresis to be clearly resolved (Gottlieb 1981). There are certain common patterns to the number of subunits for a particular enzyme and the subsequent formation of heteromers. The number of subunits an enzyme has is consistent throughout both higher plants and animals with one exception (Weeden 1983b). In Drosophila pseudoobscura, most alleles of the esterase-5 locus form dimers but one allele was found only as a monomer and another allele will not form a homodimer (Cobbs 1976). Such exceptions can complicate the interpretation of banding patterns. Subunits from different subcellular compartments will not form hybrid multimers but within a compartment there are very few examples of multimeric isoenzymes that cannot A N A L Y S I S O F E L E C T R O P H O R E T I C D A T A / 29 exchange subunits (Weeden 1983b). This ability to form heteromers is retained nearly as long as catalytic activity remains (Weeden 1983b). There has been the suggestion that loss of the ability to form heteromers can be of systematic value because of its rarity (Buth 1984). Considering these factors, Table V illustrates some banding patterns that may be expected in populations of haploids and diploids when one or two loci (with two alleles each) are present and all the homomers are equidistant. When the enzymes are monomeric little information can be obtained regarding the number of loci present (see Table V). However, when the enzymes consist of several subunits it is possible to determine if a population is haploid or diploid. The electrophoretic patterns can become very complex even when only a few loci are involved. They could become even more complex when there are different and increasing numbers of alleles at each locus, with the possibility of null alleles. Table VI shows an example of how complex banding patterns may become depending on the mobilities of the homodimers in a population of diploids for a dimeric enzyme if two loci are present, with two alleles at each locus, which do not form heteromers between loci. Analysis can be further complicated if heteromers are formed between the products of different loci or if certain subunits act differently from other subunits, as in the case of the esterase-5 locus of Drosophila already mentioned. Electrophoretic data obtained from populations of Bangia were examined using these principles. Table V : An Examole of Some Possible Banding Patterns of Haploid and D i p l o i d Populati ons Number of one locus, two a l l e l e s two l o c i , two a l l e l e s two l o c i , two a l l e l e s each each Enzyme Subunits (subunits from the two l o c i (subunits from the two loc: do not i n t e r a c t ) do i n t e r a c t ) Haploid D i p l o i d Haploid D i p l o i d Haploid D i p l o i d - A l - A l ~ A l - A l monomer - A2 ~ A2 - A2 - A 2 ~ B 1 - B 2 ~ B l - B2 - A l A j — A A A A - V l — A A A A dimer - V i - V l A A - A 2 A 2 A A - A 2 A 2 - A 2 A 2 ' A 1 B 1 — A B - B 1 B 1 ' A 2 B 2 — A A - A 2 A 2 ' A 1 B 1 — A B — B B .A B — B^B22 — A XA 2 - V i — B 1 B 1 — B B 1 — B2 B2 - A 2A 2 - A 2 A 2 - B 2 B 2 - B 2 B 2 — V i V i A A A A — A A A A A 1 A 1 A 2 A 2 A 1 A 2 A 2 A 2 — A A A A A 2 A 2 A 2 A 2 — A 1 A 1 A 1 A 1 tetramer — A 2A 2A 2A 2 — A 2 A 2 A 2 A 2 — BJBJBJBJ — B 2B 2B 2B 2 — i 1 llllllll Table VI : Some Examples of Complexity i n Banding Patterns i n D i p l o i d Populations due to the D i f f e r e n t M o b i l i t i e s of Homodiners (2 l o c i , 2 a l l e l e s each, no in t e r a c t i o n between subunits of d i f f e r e n t l o c i ) P o s i t i o n of Homodimers Banding Pattern Position of Homodimers Banding Pattern V i -- B 1 B 1 A 2A 2 — - B 2 B 2 - A 1 A 1 - A 1 A 2 B 1 B 1 - A 2A 2 B l B 2 - B 2B 2 A 1 A 1 -A 2A 2 — — B l B l - B 2 B 2 — A A — A A* A„A_ B B - BJBJ - B2 B2 A A — - B 1 B 1 A A 2  - B 2B 2 - A 1 A 1 ~ B 1 B 1 - A 1 A 2 - B B - A 2 A 2 - B2 B2 A 1 A 1 -A 2 A 2 ~ " — B 1B 1 - B 2 B 2 - A 1 A 1 - A 1 A 2 - A 2 A 2 - B1 B2 - B 2 B 2 A 1 A 1 ~ — B 1 B 1 A 2A 2 -- B 2 B 2 - A 1 A 1 - A l A 2 - B B ~ A 2 A 2 B1 B2 - B2 B2 ~ B 1 B 1 A 1 A 1 -A 2A 2 -- B 2 B 2 - B L B L ~ A 1 A 1 - A 1 A 2 B 1 B 2 - A 2A 2 - B2 B2 4. RESULTS 4.1. CHROMOSOME COUNTS The populations of Bangia in British Columbia used in the current study were placed into one of three categories based on life history and chromosome number: asexual six; asexual three; or sexual four (Table VII, Fig. 7). Chromosome numbers from six of these sites (Triple Island, the two Point No Point beaches, Ogden Point, Smuggler's Cove, and Roesland) were previously determined by Cole, Hymes and Sheath (1983) and the results obtained in this study corresponded to these. Counts were not obtained from Third Beach or Frida3' Harbor material. According to past reports, Third Beach material belongs in the category asexual six (Cole, Hymes and Sheath 1983). The sexual three category populations described by Cole, Hymes and Sheath 1983 were not available during the period of this study. 32 Table V I I : Chromosome Populations P o p u l a t i o n Sitka (Alaska) T r i p l e Island Point No Point (SW) Point No Point (S) Sooke Ogden Point Smuggler's Cove Fernwood Hope Bay Port Washington L i t t l e Bay Otter Bay Roesland Miner's Bay Sturdies Bay Spotlight Cove Whaler's Bay 33 Number and Reproductive State of Bangia i n B r i t i s h Columbia and A l a s k a Reproductive State Chromosome Number asexual 3 sexual (male) 4 sexual (male) 4 sexual (male) 4 asexual 6 asexual 6 asexual 6 asexual 6 asexual 3 asexual 3 asexual 3 asexual 3 asexual 3 asexual 3 asexual 3 asexual 6 asexual 6 34 F i g . 7 The three chromosome numbers observed in the genus Bangia from the northeast P a c i f i c : four (A); three (B); or s i x ( C ) . R E S U L T S / 35 4.2. C O N T R O L S Due to the nature of Bangia and the shortness of its growing season it was necessary to store and culture material from the various populations. Various controls were run to determine the effect of time of collection, storage and culturing. Material was collected from the majority of locations only once because extensive sampling was not feasible. In order to determine whether banding patterns change during the season, Bangia v/as collected from Point No Point southwest beach at the beginning, middle and end of its season. The banding patterns did not change (Fig. 8). It was necessary to store material either by refrigeration or freezing. To test the effect of this material from Point No Point southwest beach was examined for changes in banding patterns upon refrigeration (for one to three weeks) and upon freezing (for one to two months). The banding patterns did not change. However, upon longer periods of storage (three weeks refrigeration, or two months frozen) the bands stained less intensely (Fig. 9). The effect of storage on the extract of material from Point No Point southwest beach was also tested by comparing extract, fresh and frozen for two, four and six weeks. Again the banding patterns did not change but the banding became less intense with increased periods of storage (Fig. 10). RESULTS / 36 To determine the effect of culturing on the banding patterns, Bangia from Hope Bay was compared fresh and after three weeks of culturing. There were no changes in the banding patterns (Fig. 11). This was not surprising because other studies involving Euchema found no change in the banding patterns after three months in culture (Cheney and Babbel 1978). Clonal cultures of Bangia used in this study were grown for a longer term, fourteen months, and it is possible this might affect the patterns. Comparisons of the banding patterns obtained from male and female filaments of Point No Point showed no differences betweenthe two sexes. In summary, the time of sampling during the season and the subsequent storage and culturing of Bangia does not affect the isoenzyme banding patterns observed. A t most, the banding patterns become less intense. 3 7 F i g . 8 Banding p a t t e r n s o b t a i n e d from P o i n t No P o i n t southwest beach m a t e r i a l t h r ough one season. Each s e t of t h r e e shows r e s u l t s from m a t e r i a l c o l l e c t e d on A p r i l 10, 1984 ( f i r s t l a n e ) ; A p r i l 26, 1984 (second l a n e ) ; May 22, 1984 ( t h i r d l a n e ) . PHOSPHOGLUCOMUTASE LACTATE Fig. 8 DEHYDROGENASE 39 F i g . 9 Banding p a t t e r n s obtained from Point No Point southwest beach m a t e r i a l a f t e r v a r i o u s p e r i o d s of time and v a r i o u s storage methods of f i l a m e n t s . Each set of f i v e shows r e s u l t s from m a t e r i a l : f r e s h ( f i r s t l a n e ) ; r e f r i g e r a t e d one week (second l a n e ) ; r e f r i g e r a t e d three weeks ( t h i r d l a n e ) ; f r o z e n one month ( f o u r t h l a n e ) ; f r o z e n two months ( f i f t h l a n e ) . PHOSPHOGLUCOMUTASE LACTATE Fig. 9 DEHYDROGENASE 41 F i g . 10 Banding p a t t e r n s o b t a i n e d from P o i n t No P o i n t southwest beach m a t e r i a l a f t e r v a r i o u s p e r i o d s of time of f r e e z i n g Bangia e x t r a c t s . Each s e t of f o u r shows r e s u l t s from e x t r a c t : f r e s h ( f i r s t l a n e ) ; and f r o z e n two weeks (second l a n e ) , f o u r weeks ( t h i r d l a n e ) , and s i x weeks ( f o u r t h l a n e ) . GLUTAMATE GLUCOSE-6 PHOSPHATE PHOSPHOGLUCOSE DEHYDROGENASE DEHYDROGENASE ISOMERASE 43 F i g . 11 Banding p a t t e r n s o b t a i n e d from Hope Bay m a t e r i a l from f r e s h f i l a m e n t s ( f i r s t l a n e of each s e t ) and a f t e r t h r e e weeks i n c u l t u r e (second l a n e of each s e t ) . 44 PHOSPHOGLUCOMUTASE L A C T A T E Fig . 11 DEHYDROGENASE R E S U L T S / 45 4.3. E L E C T R O P H O R E S I S O F BANGIA P O P U L A T I O N S In the following chapter each of the seven enzymes is treated individually. Each section includes information on the enzyme's function, where it is located and how many subunits it has, if known, according to data on higher plants and animals. The section contains two sets of plates: banding patterns from electrophoresis of samples containing more than 100 filaments per population (both sexual and asexual populations); and banding patterns from electrophoresis of clonal cultures (obtained from asexual populations). Interpretations of the banding patterns and any apparent trends are also included. The plates of the banding patterns from electrophoresis of populations of filaments contain two photographs and one diagram for each population. The first photograph shows the gel stained in solution containing substrate. This shows the isoenzymes present and any artifacts. The second photograph shows the gel stained in solution not containing substrate and shows the artifacts. The artifacts may be pigments (most often phycoerythrin) or may arise from interactions of SH groups with the tetrazolium salt. Artifacts for any one population will stain consistently at the same position. The diagram illustrates the final banding pattern of the enzymes for each population. The origin of all gels is located at the top of each photograph. Speckling or dashes on the diagrams indicate very faint banding. The superoxide dismutase/tetrazolium oxidase plates do not have the second photograph because controls could not be run. Superoxide dismutase/tetrazolium RESULTS / 46 oxidase appears as reverse banding on many of the gels. Specific substrates are not added in order to detect the bands. The plates of banding patterns from electrophoresis on the clonal cultures show a photograph of the gel stained in solution containing substrate and a diagram of the final banding pattern of the enzyme for each clonal culture. Due to insufficient material controls could not be run on the clonal cultures. Bands which may be artifacts were identified by comparing the results obtained from the clonal cultures to those obtained from the samples of filaments and to those obtained from different enzyme systems. Phosphoglucose isomerase and glucose 6-phosphate dehydrogenase never showed the presence of artifacts in sample of populations and therefore were assumed not to show artifacts in the clonal cultures. Glutamate dehydrogenase, malate dehydrogenase and lactate dehydrogenase show the same artifacts and therefore the results of the clonal cultures were compared to identify the artifacts. Identifying artifacts in phoshoglucomutase was difficult because they are often located near the isoenzyme bands. Al l clonal cultures were tested for the seven enzymes but the number of individuals that showed banding was highly variable between populations and enzymes. RESULTS / 47 4.3.1. Phosphoglucomutase Phosphoglucomutase catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate. Glucose 1-phosphate is formed from the phosphorolytic cleavage of glycogen and glucose 6-phosphate is converted to pyruvate or ribose 5-phosphate (Stryer 1981). Phosphoglucomutase usually has two isoenzymes, one in the cytosol and one in the chloroplast (Gottlieb 1981). Phosphoglucomutase is a monomeric enzyme and both the cytosol and the chloroplast forms have similar molecular weights (Weeden 1983a). The populations of Bangia examined showed many banding artifacts, making identification of isoenzymes difficult. One to three isoenzyme bands were detected in field material (Figs. 12a,b). The populations with three bands had four or six chromosomes. No populations with three chromosomes had three isoenzyme bands. The following populations showed the same banding patterns: the two Point No Point beaches; Ogden Point(6), Miner's Bay(3), and Spotlight Cove(6); Sturdies Bay(3) and Sitka(3); Hope Bay(3) and Port Washington(3). A l l other populations had unique banding patterns. Clonal cultures (Figs. 13a,b,f,g) of four populations had one or two bands and two populations commonly had more than two bands. One population, Roesland (Figs. 13e,f), had up to four bands. Another population, Third Beach (Figs 13c,d), had up to five bands. R E S U L T S / 48 Very often only one band was observed in populations and clonal cultures where at least two would be expected, if Bangia also has one cytosolic and one chloroplast isoenzyme. It may be that not a sufficient number of chloroplasts were disrupted during grinding of material from some populations or clonal cultures to allow detection of the chloroplast isoenzymes. However, phycobiliproteins found in the chloroplast were clearly visible on the gel of material from all the populations and clonal cultures. This suggests that the enzymes in many populations, if not all, has only one cellular location, either the cytosol or the chloroplast. The other possibility is that there are null alleles or that the conditions required to detect the isoenzymes in other cellular locations differed from those used in this study. If phosphoglucomutase is a monomer in Bangia as in higher plants, it would appear from results of the clonal cultures that more than one locus is present in some populations and that there could be more than one allele per locus. It could be suggested that because of the numerous bands that appeared on the gels from the Roesland (asexual three) clonal cultures this population has many loci for phosphoglucomutase (at least four if it is haploid). There must also be a minimum of two loci for the Third Beach (asexual 6) population. The two bands that are present in some of the clonal cultures of various populations may be the two isoenzymes from the cytosol and chloroplast, two alleles of one locus (if diploid), or the products of two loci (one allele/locus if haploid or diploid). The banding patterns in the field material from Ogden Point(6), Miner's Bay(3), and Spotlight Cove(6) were the same suggesting that these three populations may have the same number of loci, though they vary in chromosome number. RESULTS / 49 The results of the field collected and tested material from Otter Bay differed from those of the cultured material. The field collected material showed no banding for phosphoglucomutase whereas the cultured material showed one band. It may be that phosphoglucomutase was detected in the clonal cultures because the plants in culture produced more enzyme than those in the field. 50 FERNWOOD Fig. 12a S P O T L I G H T C O V E S T U R D I E S B A Y P H O S P H O G L U C O M U T A S E W H A L E R ' S B A Y fat-Ml s ft * 51 1 i HOPE BAY PORT WASHINGTON OTTER BAY LITTLE BAY MINER'S BAY TRIPLE ISLAND F i « - 1 2 b P H O S P H O G L U C O M U T A S E SITKA 52 Fig. 13a O G D E N P O I N T C L O N A L C U L T U R E S P H O S P H O G L U C O M U T A S E OGDEN POINT CLONAL CULTURES OGDEN POINT C L O N A L C U L T U R E S PHOSPHOGLUCOMUTASE Fig. 13c THIRD BEACH CLONAL CULTURES PHOSPHOGLUCOMUTASE 55 ROESLAND CLONAL C U L T U R E S ROESLAND CLONAL CULTURES PHOSPHOGLUCOMUTASE ROESLAND CLONAL CULTURES OTTER BAY CLONAL CULTURES PHOSPHOGLUCOMUTASE R E S U L T S / 59 4.3.2. Superoxide Dismutase/Tetrazolium Oxidase Superoxide dismutate and tetrazolium oxidase are the same enzyme; however the name superoxide dismutase is more commonly used. Superoxide dismutase protects against oxygen damage by removing the reactive superoxide radical (Newton 1983) and converting it to oxygen and hydrogen peroxide (Gottlieb 1981). According to Harris, Auffret, Northrop and Walker (1980) and Newton (1983) there are two subcellular locations for superoxide dismutase. The mitochondrial superoxide dismutase contains Mn and is tetrameric. The cytosolic superoxide dismutase contains CuZn and is a dimer. However, according to Gottlieb (1981) the Mn containing enzyme is a dimer located in the chloroplast envelope and the CuZn containing enzyme is also a dimer located in the chloroplast, the latter showing the most activity. Of the fifteen populations of Bangia examined, all showed only one clear band (Figs. 14a,b). This is probably the cj'tosolic superoxide dismutase because it would be the easiest to extract. The mitochondrial superoxide dismutase may not have been detected if insufficient mitochondria were disrupted. Each population had one band which migrated to one of three positions. There is no relationship between the chromosome number or life history and the position to which the bands migrated. The clonal cultures of five populations were examined (Figs. 15a-i); material from individuals of three populations each showed one band. Only six of forty-one RESULTS / 60 individuals from Third Beach (Figs. 15d-g) and three of five individuals from Friday Harbor (Fig. 15g) showed more than one band. However the observed patterns cannot be explained on the basis of dimeric or tetrameric enzymes. They can be explained if the various bands represent enzymes from different cellular locations. But the question then arises why this does not occur in other populations or in all individuals of a population. The other possiblitiy is that superoxide dismutase in Bangia is a monomer rather than a dimer or tetramer. If this is the case Third Beach material may have one locus with two different alleles, some individuals being homozygous for one of the alleles and others heterozygous. Friday Harbor material may have more than one locus in the one individual that had three isoenzymes for superoxide dismutase. 61 S P O T L I G H T C O V E STURD1ES BAY W H A L E R ' S BAY H O P E BAY P O R T W A S H I N G T O N Fig . 14a S U P E R O X I D E D I S M U T A S E / T E T R A Z O L I U M O X I D A S E 62 0 1 T E R BAY L I 1 II.! BAY M I N E R ' S BAY T R I P L E I S L A N D SITKA Fig. 14b SUPEROXIDE DISMUTASE/TETRAZOLIUM OXIDASE OGDEN POINT CLONAL CULTURES OCDEN POINT CLONAL CULTURES Fig. 15b SUPEROXIDE DISMUTASE/TETRAZOLIUM OXIDASE G9 THIRD BEACH CLONAL CULTURES FRIDAY HARBOR CLONAL CULTURES Fig. 15g SUPEROXIDE DISMUTASE/TETRAZOLIUM OXIDASE 4.3.3. Malate Dehydrogenase RESULTS / 72 Malate dehydrogenase catalyzes the oxidation of malate to oxaloacetate(Stryer 1981). L-malate + N A D * -•-•^ oxaloacetate + N A D H + H + Malate dehydrogenase is located in mitochondria, microbodies, cytosol and chloroplasts. The first three are N A D ^ dependent and the last one is N A D P dependent. In the mitochondria and chloroplast malate dehydrogenase is involved in the citric acid cycle, in the microbodies in photorespiration and the glyoxylate cycle and in the cytoplasm in non-autotrophic CO^ fixation to form malate. The mitochondrial and cytosolic malate dehydrogenases are relatively acidic (Newton 1983). Of the fifteen populations of Bangia examined eleven showed some banding, however these appeared as blurs and could not be resolved clearly (Figs 16a,b). The populations could be divided into two groups according to the migration of the region of staining. Only one region was detected in each population suggesting that either all four malate dehydrogenases migrate to the same region or that not all four malate dehydrogenases are present. It may be that, as in some lines of maize, only the mitochondrial form is present (McMillin and Scandalios 1983). There was no relationship between the chromosome number or the geographic location of the population and the region of malate dehydrogenase banding. No information could be obtained regarding the number of loci in these RESULTS / populations. No clonal cultures showed any banding for malate dehydrogenase activity. ZZ2 I zzzz POINT NO POINT POINT NO POINT SOUTH BEACH SOUTHWEST BEACH SOOKE FERNWOOD Fig. 16a SPOTLIGHT COVE STURDIES BAY MALATE DEHYDROGENASE 74 L i ODGEN POINT 2 2 WHALER'S BAY MINER'S BAY TRIPLE ISLAND SITKA MALATE DEHYDROGENASE 4.3.4. Glutamate Dehydrogenase RESULTS / 76 Glutamate dehydrogenase catalyzes the reversible oxidative deamination of glutamate to form o^-ketoglutarate. glutamate + N A D or N A D P ^ N H 4 + <*.-ketoglutarate + H^O + N A D H or N A D P H + H ^ When amino acids are in excess they are converted to major metabolic intermediates. The amino group is transferred to «K-ketoglutarate to form glutamate which is then deaminated by glutamate dehydrogenase to yield N H H . During amino acid biosynthesis the reverse occurs and N H ^ can enter into an amino acid (Stryer 1981). According to Gottlieb (1981) no isoenzymes (different loci) have been found. However, Weeden (1983b) and Newton (1983) state that there are several subcellular locations for glutamate dehydrogenase. Mitochondrial glutamate dehydrogenase is the major glutamate dehydrogenase isoenzyme and is N A D dependent(Newton 1983). The plastid and cytosolic glutamate dehydrogenase are either N A D dependent or activated by both N A D and N A D P (Newton 1983). Glutamate dehydrogenase is usually a tetramer or hexamer in higher plants (Newton 1983). Of all the populations that showed some banding (twelve of the fifteen) seven had one band and five had two bands (Figs. 17a,b). The second band always migrated slower and was stained less intensely than the faster RESULTS / 77 migrating band. The second band never appeared alone. One possible explanation is that the band that is always present is a cytosolic glutamate dehydrogenase and that the second band may be a glutamate dehydrogenase isoenzyme located in plastids (NADP was used as the coenzyme in this stain). The plastids would be more difficult to disrupt and the isoenzyme may appear in only some gels when sufficient plastids disrupt. However, in all the populations and clonal cultures phycobiliproteins were detected as a band on the gels suggesting that plastids were sufficiently disrupted to detect a plastid isoenzyme if it was present. The mitochondria may possibly have not been sufficiently disrupted and this second isoenzyme may be located in the mitochondria. If this is the case then the mitochondrial isoenzyme is N A D P dependent unlike that in higher plants and animals. The other possibility is that the second band arose by mutation and spread throughout the populations by asexual reproduction, explaining why it never appears alone. If it is a less efficient enzyme it may stain less intensely. Those populations not showing any banding, consistently showed the same results with different runs even though extracts run along side these stained for glutamate dehydrogenase. Examination of the individuals (Figs. 18a-k) revealed the same banding patterns as the populations. Only a few of the clonal cultures showed two bands. THIRD BEACH CLONAL CULTURES • I THIRD BEACH CLONAL CULTURES GLUTAMATE DEHYDROGENASE THIRD BEACH CLONAL CULTURES THIRD BEACH CLONAL CULTURES GLUTAMATE DEHYDROGENASE THIRD BEACH CLONAL CULTURES ti it- 18c THIRD B E A C H C L O N A L CULTURES GLUTAMATE D E H Y D R O G E N A S E T H I R D B E A C H C L O N A L C U L T U R E S T H I R D B E A C H C L O N A L C U L T U R E S G L U T A M A T E D E H Y D R O G E N A S E OTTER BAY CLONAL CULTURES OTTER BAY CLONAL CULTURES GLUTAMATE DEHYDROGENASE OTTER BAY CLONAL CULTURES OTTER BAY CLONAL CULTURES GLUTAMATE DEHYDROGENASE 86 OTTER BAY CLONAL CULTURES Fig. 18g OTTER BAY CLONAL CULTURES GLUTAMATE DEHYDROGENASE ROESLAND CLONAL CULTURES ROESLAND CLONAL CULTURES G L U T A M A T E D E H Y D R O G E N A S E ROESLAND CLONAL CULTURES ROESLAND CLONAL CULTURES GLUTAMATE DEHYDROGENASE SMUGGLER'S COVE CLONAL CULTURES SMUGGLER'S COVE CLONAL CULTURES GLUTAMATE DEHYDROGENASE FRIDAY HARBOR CLONAL CULTURES 18k OGDEN POINT CLONAL CULTURES G L U T A M A T E DEHYDROGENASE 4.3.5. Lactate Dehydrogenase RESULTS / 91 Lactate dehydrogenase catalyzes the reversible conversion between pyruvate and lactate. pyruvate + N A D H + x L-lactate + N A D This occurs in most higher organisms when the amount of oxygen is limiting. -t-This reaction allows the regeneration of N A D and the subsequent continuation of glycolj'sis under anaerobic conditions (Stryer 1981). Lactate dehydrogenase has been found as a tetrameric enzyme in cattle, fish and other animals (Markert 1983) and is located in the cytosol (Masters 1983). Of the fifteen Bangia populations only one, Sturdies Bay, showed no banding for lactate dehydrogenase(Figs. 19a,b). The majority of populations had only one band. Four populations showed two bands (Fernwood(6), Otter Bay(3), Little Bay(3) and Triple Island(4)) and one population (Sitka(3)) showed four bands. The few bands observed may be explained in several ways. The presence of one allele in the population at one locus would appear as one band in both haploid and diploid populations. The presence of two alleles in the population at one locus would appear as two bands for haploid and as two bands for diploids, if those diploids arose by autopolyploidy at least twice, once for each allele. Otherwise, if the diploids were heterozygous, they should show five bands since RESULTS / 92 lactate dehydrogenase is a tetramer. However, examination of the clonal cultures (Figs. 20a,b) shows two populations that contain individuals with two bands and this would not be expected according to the above explanation. A second possibility is that some populations may have another locus for lactate dehydrogenase and subunits from these loci do not interact. If this were the case, each individual of such a population would show two bands. Examination of the clonal cultures (Fig. 20) shows this not to be the case. The observed results can only be readily explained if lactate dehydrogenase in Bangia is a monomer rather than a tetramer as in animals. If this is the case no information can be obtained regarding the number of loci present in various populations unless individuals are detected with at least two bands. This is the case for Otter Bay and Roesland. There may be one locus, two alleles (if diploid) or two loci, one allele/locus (if haploid or diploid) present. Both of these populations have three chromosomes but show banding patterns that would be expected in diploids. Though there is not enough information to be certain, this may apply also to Fernwood, Little Bay, Triple Island, and Sitka populations. The other populations may also be diploids but are not detected because of their homozygosity. The four bands in the Sitka population material may be due to a gene duplication or more alleles at one locus than in other populations. 94 MINERS BAY TRIPLE ISLAND SITKA Fig. 1 » » L A C T A T E D E H Y D R O G E N A S E OTTER BAY CLONAL CULTURES u M ROESLAND CLONAL CULTURES L A C T A T E DEHYDROGENASE 96 • THIRD BEACH CLONAL CULTURES OGDEN POINT CLONAL CULTURE SMUGGLER'S COVE CLONAL CULTURES Fig. 20b L A C T A T E DEHYDROGENASE RESULTS / 97 4.3.6. Phosphoglucose Isomerase Phosphoglucose isomerase is the enzyme in glycolysis that catalyzes the conversion of glucose 6-phosphate to fructose 6-phosphate (Stryer 1981). There are usually two cellular locations, the cytosol and the chloroplast, and only one band for the plastid phosphoglucose isomerase. Phosphoglucose isomerase is a dimeric enzyme (Gottlieb 1981). The banding patterns for the Bangia populations are very complex; three to ten bands were detected (Figs. 21a,b). The banding patterns of material from the two Point No Point beaches were the same, as were those of the Sooke and Spotlight Cove populations. Al l other banding patterns were unique to the populations. The banding patterns of the clonal cultures were highly variable in most of the populations (Figs. 22a-e). For example, Smuggler's Cove material had as few as two and up to eight bands, Third Beach material had one to six bands, Ogden Point material had one to seven bands and Roesland material had one to five bands. The patterns observed within the populations could not be easily explained as variable alleles at one or two loci. The only population that is readily explained is Otter Bay, in which there appears to be only one allele at one locus. It may be that all individuals in a population do not contain the same number of loci, RESULTS / 98 that there are many null alleles and/or that the chloroplast isoenzymes show up only occasionally, complicating the patterns. However, since phycobiliproteins were present on all the gels it is likely that the chloroplasts had been disrupted and the chloroplast isoenzymes would be detected equally in all individuals of a population. Some individuals of the clonal cultures of all populations except Otter Bay must have more than one locus for phosphoglucose isomerase. Material from three individuals of each of two populations examined, Ogden Point(6) and Roesland(3) showed a second faster migrating region in which bands were detected. These regions may indicate another locus or loci coding for isoenzymes with different properties or may indicate isoenzymes located in an organelle. These individuals are not frequent and it may be that these second regions are recently formed or that the individuals with them are disappearing from the populations. In the other populations in which clonal cultures were examined the bands are spread out more and there are no distinct regions. Examination of the simpler banding patterns in some of the clonal cultures suggests that phosphoglucose isomerase in Bangia is a monomer and not a dimer as in higher plants. If this is the case many gene duplications for the phosphoglucose isomerase enzyme must have occurred in order to account for the complex banding patterns observed. Clonal cultures often show only one isoenzyme. This suggests that there may not be two cellular locations for phosphoglucose isomerase, but rather only one. On the other hand, one or both of the cellular locations may have null alleles so that it may appear as if one isoenzyme is not present. Also, the conditions RESULTS / 99 required to detect the other isoenzymes may be different from those used in this study. The complex banding patterns for phosphoglucose isomerase found in all the populations would suggest that the asexual three and asexual six populations have the same, or similar numbers of loci and alleles and have more than one locus with two alleles. Though this cannot be positively determined without performing crosses, the results of the clonal cultures reinforce this conclusion. 100 FERNWOOD Fig. 21a SPOTLIGHT COVE STURDIES BAY PHOSPHOGLUCOSE 1S0MERASE WHALER'S BAY 101 MINER'S BAY TRIPLE ISLAND SITKA Fig . 21b P H O S P H O G L U C O S E I S O M E R A S E - f 102 1 OGDEN POINT CLONAL CULTURES 3 I Fig. 22a OGDEN POINT CLONAL CULTURES P H O S P H O G L U C O S E I S O M E R A S E 103 OGDEN POINT CLONAL CULTURES I 1. 1 4 Fig. 22b OGDEN POINT CLONAL CULTURES PHOSPHOGLUCOSE ISOMERASE 104 OGDEN POINT CLONAL CULTURES i OGDEN POINT CLONAL CULTURES PHOSPHOGLUCOSE ISOMERASE n 106 4 THIRD BEACH CLONAL CULTURES • Fig. 22e THIRD BEACH CLONAL CULTURES PHOSPHOGLUCOSE ISOMERASE THIRD BEACH CLONAL CULTURES THIRD BEACH CLONAL CULTURES PHOSPHOGLUCOSE ISOMERASE 108 THIRD BEACH CLONAL CULTURES i ft. 1 • 1 ft 1 THIRD BEACH CLONAL CULTURES F i g . 22g P H O S P H O G L U C O S E I S O M E R A S E 109 THIRD BEACH CLONAL CULTURES THIRD BEACH CLONAL CULTURES Fig. 22h PHOSPHOGLUCOSE ISOMERASE ROESLAND CLONAL CULTURES 110 F i g . 22i ROESLAND CLONAL CULTURES PHOSPHOGLUCOSE ISOMERASE ROESLAND CLONAL CULTURES ROESLAND CLONAL CULTURES PHOSPHOGLUCOSE ISOMERASE 1 12 Fig. 22k S M U G G L E R ' S COVE CLONAL CULTURES PHOSPHOGLUCOSE ISOMERASE 113 OTTER BAY CLONAL CULTURES FRIDAY HARBOR CLONAL CULTURES Fig. 221 PHOSPHOGLUCOSE ISOMERASE 4 . 3 . 7 . Glucose 6-Phosphate Dehydrogenase RESULTS / 114 Glucose 6-phosphate dehydrogenase is an enzyme in the pentose phosphate pathway which catalyzes the dehydrogenation of glucose 6-phosphate to form 6-phosphoglucono-g-lactone (Stryer 1981). glucose 6-phosphate + NADP ^ x 6-phosphoglucono-S-lactone + H + + NADPH There are two cellular locations, one in the cytosol and one in the chloroplast. Glucose 6-phosphate dehydrogenase is a dimer (Gottlieb 1981). There was a great deal of variation in the glucose 6-phosphate dehydrogenase banding detected amongst Bangia populations (Figs. 23a,b). The following populations had identical banding patterns: the two Point No Point beaches and Fernwood; Sooke and Triple Island; Hope Bay and Sturdies Bay; Port Washington and Otter Bay. All other populations had unique banding patterns. Examination of the clonal cultures suggests that there may be a great deal of variation between the number of loci in different populations (Figs. 24a-i). Some populations (Smuggler's Cove, Third Beach, Friday Harbor) have only one locus whereas others have several loci (Ogden Point, Otter Bay). In some populations up to eight or nine bands may be present in an individual. These can be explained by as few as two loci. Up to six bands would be present if the subunits coded by the separate loci did not interact RESULTS / 115 or up to ten bands if the subunits coded by the separate loci did interact. There must be a minimum of two loci (two alleles each) but more loci or alleles may be present. Material from the two Point No Point beaches, Fernwood, Spotlight Cove and Miner's Bay have similar banding patterns to those found in the clonal cultures of Smuggler's Cove, Third Beach, and Friday Harbor. These may also have only one locus for glucose 6-phosphate dehydrogenase. The other populations probably have more than one locus The clonal cultures of Roesland are unusual because they have one to three bands. It may be that the extra third band is a chloroplast isoenzyme and that the individuals are actually similar to those of Smuggler's Cove, Third Beach, and Friday Harbor. However, phycobiliproteins from all clonal cultures and populations formed a band in the gel suggesting that chloroplasts were disrupted and isoenzymes localized in the chloroplast should be detected. Another possibility is that some individuals of this population have one locus for glucose 6-phosphate dehydrogenase whereas other individuals have two loci. It is also possible that all individuals have two loci, and those with one apparent locus have null alleles. The same situation occurs in Otter Bay material with one to five bands. Some of the clonal cultures of Third Beach, Smuggler's Cove, Roesland, Otter Bay and Friday Harbor showed only one isoenzyme for glucose 6-phosphate dehydrogenase. This would not be expected if there were two RESULTS / 116 cellular locations for glucose 6-phosphate dehydrogenase in Bangia as in higher plants. This suggests that there may be only one location for glucose 6-phosphate dehydrogenase or that one of the two locations have null alleles present or that the conditions of electrophoresis used would not detect all isoenzymes. The results obtained from the clonal cultures and the Field collected and tested material from Ogden Point are not the same. The clonal cultures showed up to nine bands whereas the field collected material showed only five. This may be explained by differences in culture conditions, by differences in the material obtained from two different years, or by undetectable quantities of the enzyme in field collected material. 117 • POINT NO POINT POINT NO POINT SOUTH BEACH SOUTHWEST BEACH SOOKE ODGEN POINT PERNWOOD SPOTLIGHT COVE STURDIES BAY WHALER'S BAY Fig. 23a GLUCOSE-6 PHOSPHATE DEHYDROGENASE 118 HOPE BAY PORT WASHINGTON OTTER BAY LITTLE BAY m = MINER'S BAY TRIPLE ISLAND SITKA Fig. 23b GLUCOSE-6 PHOSPHATE DEHYDROGENASE — : M • • OGDEN POINT CLONAL CULTURES = In • • i OGDEN POINT CLONAL CULTURES G L U C O S E - G P H O S P H A T E D E H Y D R O G E N A S E OGDEN POINT CLONAL CULTURES i OGDEN POINT CLONAL CULTURES Fig. 24b GLUCOSE-6 PHOSPHATE DEHYDROGENASE • • OGDEN POINT CLONAL CULTURES 4 OGDEN POINT CLONAL CULTURES GLUCOSE-6 PHOSPHATE DEHYDROGENASE i • OGDEN POINT CLONAL CULTURES 4 OGDEN POINT CLONAL CULTURES GLUCOSE-6 PHOSPHATE DEHYDROGENASE 1 2 3 rsr-1 • • t ! 1 1 M 1 ' • OGDEN POINT CLONAL CULTURES i Fig. 24e THIRD BEACH CLONAL CULTURES GLUCOSE-6 PHOSPHATE DEHYDROGENASE THIRD BEACH CLONAL CULTURES >m SMUGGLER'S COVE CLONAL CULTURES GLUCOSE-6 PHOSPHATE DEHYDROGENASE ROESLAND CLONAL CULTURES 125 ! t Fig. 24g ROESLAND CLONAL CULTURES GLUCOSE-6 PHOSPHATE DEHYDROGENASE 126 ROESLAND CLONAL CULTURES 4 ROESLAND CLONAL CULTURES Fig. 24h GLUCOSE -6 PHOSPHATE DEHYDROGENASE OTTER BAY CLONAL CULTURES FRIDAY HARBOR CLONAL CULTURES GLUCOSE -6 PHOSPHATE DEHYDROGENASE 5. D ISCUSSION 5.1. D ISCUSSION O F T H E R E S U L T S O F V A R I O U S E N Z Y M E T E S T S O N BANGIA P O P U L A T I O N S The previous chapter dealt with results produced with each enzyme individually. This next chapter will address recurring points that appeared throughout the results from various enzyme tests (eg. the number of cellular locations and subunits) and the amount of variability between populations and groups of populations. Several enzymes (PGM, SOD, G D H , PGI, G6PDH) appear to have only one cellular location; often only one band would be detected in a population or in clonal cultures. Higher plants have several cellular locations for these enzymes and hence several bands. This was observed in clonal cultures from Ogden Point (PGM, SOD, G6PDH), Otter Bay (PGM, SOD, G6PDH), Friday Harbor (PGM, G6PDH), Roesland (SOD, PGI, G6PDH), Smuggler's Cove (SOD, G6PDH), and Third Beach (PGI, G6PDH). Another explanation may be that there are many null alleles present or that the conditions under which some of the isoenzymes would be detected were different from those used in this investigation. Or the organelles were not disrupted, releasing the other isoenzyme. This has already been discussed in the previous chapter and is improbable because of the presence of phycobiliprotein bands on the gels indicating that chloroplasts were disrupted. Banding patterns for SOD, G D H , L D H and PGI could be more readily explained 128 DISCUSSION / 129 if these enzymes were monomers rather than multimers as in higher plants. For example, lactate dehydrogenase is a tetramer in animals (Markert 1983) but observed results in Bangia can only be explained if lactate dehydrogenase is a monomer. Phosphoglucose isomerase is a dimer in higher plants but many of the banding patterns of the clonal cultures of Bangia can not be readily explained if phosphoglucose isomerase is a dimer. In higher plants glutamate dehydrogenase is a tetramer or hexamer. However, if the second observed band is not a chloroplast isoenzyme then this enzyme in Bangia is probably not a tetramer or a hexamer. Superoxide dismutase is another enzyme that may be a monomer in Bangia rather than a dimer or tetramer as in higher plants. It is also of interest that these enzymes should all appear as monomers. There are several other possible explanations for the banding patterns. One is that these enzymes in Bangia undergo modifications resulting in a different number of bands than expected; however this does not occur in higher plants. Another possibilitj' is that there may be null alleles or certain combinations of heteromers that are inactive. Or there are consistently certain banding artifacts that result in a different number of bands than expected. In some populations of one chromosome type entirely it appeared that certain individuals had different numbers of loci present for a particular isoenzyme (PGI, G6PDH) than the majority of individuals. This occurred in both asexual three and asexual six chromosome populations. Clonal cultures within a population may have one to many bands suggesting that there may be one to more than one locus for that enzyme present. This occurred in material from Odgen Point (PGI, G6PDH); Third Beach (PGI); Roesland (PGI, G6PDH); and Otter Bay (G6PDH). DISCUSSION / 130 An explanation for this phenomenon could be that: the populations were established by more than one spore with variable numbers of loci; or that the populations have been established for a long time and null alleles have arisen. The latter is more likely since no populations were detected with a mix of chromosome numbers as would be expected in at least some populations if two or more different individual had established the population. In addition to the foregoing three major recurring observations in the results, the degree of variability amongst the enzymes and populations is also of great interest. It should be noted firstly that there is no seasonal variation (see section 4.2) and that the two closely located Point No Point populations always showed identical banding patterns for all the enzymes. The four chromosome population of Point No Point south beach was first detected four years ago though extensive sampling was done before this time, suggesting that this population was recently established. This would indicate that the techniques used gave reproducible and reliable results. Many of the enzymes examined were conservative in the number of bands detected (SOD, M D H , G D H , LDH) (Figs. 26-29) with usually only one or two bands. One enzyme, P G M , was variable amongst the populations but still had only a few bands (Fig. 25). In contrast PGI and G6PDH showed many bands in most populations (Figs. 30,31). These patterns were difficult to interpret. The variability may be due in part to post translational modifications. However, there were some populations and clonal cultures with one or few bands suggesting, that it is unlikely that modifications are the explanation for the large amount of Fig. 25 P H O S P H O G L U C O M U T A S E P O I N T N O P O I N T S O O K E O G D E N F E R N W O O D H O P E P O R T OTTER S O U T H S O U T H W E S T (6) P O I N T (6) BAY W A S H I N G T O N BAY B E A C H B E A C H (6) (3) (3) (3) (4) (4) L I T T L E M I N E R ' S W H A L E R ' S T R I P L E BAY BAY S T U R D I E S BAY S P O T L I G H T I S L A N D (3) (3) BAY ( 6 ) C O V E (4) (3) (6) SITKA (3) Fig. 26 S U P E R O X I D E D I S M U T A S E / T E T R A Z O L I U M O X I D A S E P O I N T N O P O I N T S O O K E O G D E N F E R N W O O D H O P E P O R T OTTER L I T T L E M I N E R ' S W H A L E R ' S T R I P L E SITKA S O U T H S O U T H W E S T (6) P O I N T (6) BAY W A S H I N G T O N BAY BAY BAY S T L K D I E S BAY S P O T L I G H T I S L A N D (3) B E A C H B E A C H (6) ( 3 ) ( 3) (3) (3) (3) B A Y (6) C O V E (4) (4) (4) (3) (6) Fig. 27 MALATE DEHYDROGENASE 7ZZL 7ZZL TZH ZLZL 7Z21 ZZZ2 7ZZZ. W2Z P O I N T N O P O I N T S O O K E S O U T H S O U T H W E S T (6) B E A C H B E A C H (4) (4) O G D E N F E R N W O O D H O P E P O R T OTTER P O I N T (6) BAY W A S H I N G T O N BAY (6) (3) O) (3) L I T T L E M I N E R ' S W H A L E R ' S T R I P L E SITKA BAY BAY S T U R D I E S BAY S P O T L I G H T I S L A N D (3) (3) BAY (6) C O V E ( 4 ) (3) (6) (3) Fig. 28 G L U T A M A T E D E H Y D R O G E N A S E P O I N T N O P O I N T S O O K E O D G E N F E R N W O O D H O P E 1 P O R T OTTER S O U T H S O U T H W E S T (6) P O I N T (6) BAY W A S H I N G T O N BAY B E A C H B E A C H (6) ( 3 ) (3) (3) (4) <<4) L I T T L E M I N E R ' S W H A L E R ' S T R I P L E SITKA BAY BAY S T U R D I E S BAY S P O T L I G H T I S L A N D <3) (3) (3) BAY ( 6 ) C O V E ( 4 ) (3) (6) Fig. 29 L A C T A T E D E H Y D R O G E N A S E I I I J I I I I I I I i I ) i I ! I I I I L \ I I ) I J J . P O I N T N O P O I N T S O O K E O G D E N F E R N W O O D H O P E PORT OTTER L I T T L E M I N E R ' S W H A L E R ' S T R I P L E SITKA S O U T H S O U T H W E S T (6) P O I N T (6) BAY W A S H I N G T O N BAY BAY BAY S T U R D I E S BAY S P O T L I G H T I S L A N D (3) B E A C H B E A C H (6) (3) (3) (3) (3) (3) BAY ( 6 ) C O V E (4) (4) (4) (3) (6) Fig. 30 PHOSPHOGLUCOSE ISOMERASE P O I N T N O P O I N T SOOKE O G D E N F E R N W O O D H O P E P O R T OTTER L I T T L E S O U T H S O U T H W E S T (6) P O I N T (6) BAY W A S H I N G T O N BAY BAY B E A C H B E A C H (6) (3) (3) (3) (3) (4) (4) M I N E R ' S W H A L E R ' S T R I P L E SITKA BAY S T U R D I E S BAY S P O T L I G H T I S L A N D (3) (3) BAY (6) C O V E (4) (3) (6) Fig. 31 GLUCOSE-6 PHOSPHATE DEHYDROGENASE P O I N T N O P O I N T S O O K E O G D E N F E R N W O O D H O P E PORT OTTER S O U T H S O U T H W E S T (6) P O I N T (6) BAY W A S H I N G T O N BAY B E A C H B E A C H (6) ( 3) ( 3) ( 3) L I T T L E M I N E R ' S W H A L E R ' S T R I P L E SITKA BAY BAY S T U R D I E S BAY S P O T L I G H T I S L A N D (3) (3) BAY (3) (6) C O V E (6) (4) (3) DISCUSSION / 138 variability observed. The summary plates, figures 26 to 31 indicated above, are arranged to compare the results of the electrophoretic banding patterns of the population runs. The distance bands migrated varied due to slight differences in the distance gels ran and shrinkage in staining and fixing solutions. Therefore, on all the gels material from Point No Point was run as a standard. On the summary plates the bands are lined up relative to one another but are not drawn to scale. Below each drawing is recorded the population and its chromosome number in brackets. There was no relationship detected between the number of enzyme loci present and the chromosome numbers of the various populations. Material from Ogden Point and Spotlight Cove with six chromosomes and Miner's Bay with three chromosomes, appears to have the same number of loci coding for phosphoglucomutase. Two populations, Otter Bay and Roesland, have banding patterns for lactate dehydrogenase indicative of diploids, though both have only three chomosomes. Fernwood with six chromosomes, Little Bay and Sitka with three chromosomes, and Triple Island with four chromosomes also have banding patterns indicative of diploids for this enzyme. Al l populations, except Otter Bay, appear to have more than one locus for phosphoglucose isomerase. Smuggler's Cove, Spotlight Cove, Fernwood and Third Beach with six chromosomes, Miner's Bay and Roesland with three chromosomes, the two Point No Point beaches with four chromosomes and Friday Harbor have only one locus for glucose 6-phosphate dehydrogenase present whereas other populations appear to have more than one locus. It may be that many of the populations, if not all, are banding for glutamate dehydrogenase as would be expected of diploids with one locus and two DISCUSSION / 139 alleles. These results suggest that aneuploid reduction and not polyploidization had occurred at least to some extent in Bangia, but the results are too complex to be explained by aneuploid reduction alone. It suggests that gene silencing and/or gene duplications may have also frequently occurred. In order to obtain an overview of the degree of variability amongst various populations tables were prepared comparing populations pairwise with respect to the number of enzymes that had the identical banding patterns (Fig. 32a-e), at least one band in the same position (Fig. 33a-e), or no bands in common (Fig. 34a-e). There are five tables within each figure: one comparing all the populations; one comparing the three chromosome populations; one comparing the six chromosome populations; one comparing the three and six chromosome populations; and finally a summary table. The number of enzymes in each table is expressed as a fraction because, due to occasional lack of banding in some populations, not all seven enzymes were always available for comparison. It should be noted that the number of bands staining varied with the enzyme and this affected the tables. For example, SOD shows only one band per population and therefore populations will either have the same pattern or will not whereas PGI with up to eight bands is likely to show at least one band in common but few identical patterns. Generally, there is no greater similarity between the populations of the same chromosome number (three or six) than between populations of different chromosome numbers with respect to the isoenzymes tested (Figs. 32e, 33e, 34e) As well, there is no greater difference between populations of different 140 POINT NO POINT ////< 1/5 SOOKE 2 / 7 1/5 OGDEN POINT 2 / 6 1/5 0 / 6 FERNWOOD 2 / 7 2 / 5 1 / 7 2 / 6 HOPE BAY 1/7 2 / 5 1/7 2 / 6 A / 6 PORT WASHINGTON 1/6 0 / 6 1/4 0 / 6 3 / 5 3 / 6 3 / 5 OTTER BAY 1/5 1/6 1/6 1/6 2 / 6 2 , 3 LITTLE BAY 2 / 7 2 / 5 4 / 7 1/6 2 / 7 2 / 6 1/6 2 / 6 MINER'S BAY 1/4 0/4 0/4 0/4 1/4 0/4 0 / 3 0 / 4 0 / 4 Fig. 32a The number of enzymes out of the total examined that had the identical banding patterns; a pairwise comparison of a l l populations. STURDIES BAY 2 / 7 2 / 7 2 / 5 1/7 2 / 6 4 / 7 2 / 7 3 / 6 3 / 6 1/6 2 / 7 0/4 WHALER'S BAY 2 / 5 4 / 7 1/6 2 / 6 1/6 0 / 7 2 / 5 2 / 7 0 / 6 1/7 1/6 2 / 6 5/7 0/4 2 / 7 SPOTLIGHT COVE 1/6 1/6 1/7 0/4 1/7 1/7 TRIPLE I8LAND 2 / 7 0 / 5 0 / 7 1/6 2 / 7 1/6 2 / 6 0 / 6 0 / 7 2 / 4 2 / 7 0 / 7 1/7 A. HOPE BAY 4 / 6 3 / 6 1/6 2 / 7 1/4 2/7 PORT WASHINGTON 3 / 5 2 / 6 2 / 6 0 / 4 1/6 Fig. 32b OTTER BAY 2 / 5 1/6 0 / 3 2 / 6 The number of enzymes out of the total examined that had the identical banding patterns; a pairwise comparison of populations with three chromosomes. LITTLE BAY 2 / 6 0/4 0/6 MINER'S BAY 0/4 0 / 7 STURDIES BAY 2 / 4 141 Fig. 32c The number of enzymes out of the total examined that had the identical banding patterns; a pairwise comparison of populations with six chromosomes. •OOKE 1/5 1 / 5 2 / 5 2 / 5 OGDEN POINT 0 / 6 1/7 4 / 7 FERNWOOD 2 / 6 1/6 WHALER'S BAY 2 / 7 Fig. 32d The number of enzymes out of the total examined that had the identical banding patterns; a pairwise comparison of populations with three chromosomes (top) versus populations with six chromosomes (side). SOOKE OGDEN POINT FERNWOOD WHALER'S BAY SPOTLIGHT COVE 2 / 5 2 / 5 1/4 1/5 2 / 5 0/4 0 / 5 1/7 1/7 0 / 6 1/6 4 / 7 0/4 2 / 6 2 / 6 3 / 5 1/6 1/6 0/4 4 / 7 3 / 6 3/6 1/6 2 / 7 0/4 2 / 7 2 / 6 1/6 2 / 6 5/7 0/4 0 / 7 1/6 2 / 7 0 / 7 Fig. 32e A SUMMARY-The number of enzymes with identical electrophoretic banding patterns and their frequency amongst the pairwise comparisons of populations; DUMBER OF RHYMES PAIRWISE COMPARISONS or: ALL POPULATIONS POPULATIONS WITH THREE CHROMOSOMES POPULATIONS WITH SIX CHROMOSOMES POPULATIONS WITH THREE CUROHOSOKES VERSUS POPULATIONS WITH SIX CHROMOSOMES 0 2 0 /SI 6/21 1 / 1 0 5/35 i 3 0 / 9 1 4 / 2 1 4 / 1 0 1 0 / 3 5 i 3 1 / 9 1 8 / 2 1 4 / 1 0 1 0 / 3 5 i 5 / 9 1 2 / 2 1 0 / 1 0 3 / 3 5 A 4 / 9 1 1/21 1 / 1 0 2 / 3 5 5 1 / 9 1 0 / 2 1 0 / 1 0 1 / 3 5 P O I N T N O P O I N T 2/5 S O O K E 2/7 2/5 O G D E N P O I N T 3/6 3/5 4/6 F E R N W O O D 2/7 2/5 3/7 4/6 H O P E B A Y 2/7 2/5 3/7 4/6 2/6 P O R T W A S H I N G T O N 3/6 3/4 4/6 2/5 3/6 2/5 O T T E R B A Y 4/6 2/5 3/6 4/6 3/6 1/6 3/5 3/7 2/5 2/7 4/6 3/7 2/6 4/6 3/6 3/6 2/4 1/7 2/5 3/4 3/4 1/4 2/4 2/3 2/4 3/4 L I T T L E B A Y M I N E R ' S B A Y Fig. 33a S T U R D I E S B A Y The number of enzymes out of the t o t a l examined that had at least one isoenzyme band i n common, but were not i d e n t i c a l i n th e i r electrophoretic patterns; a pairwise comparison of a l l populations. 3/7 2/6 2/7 2/6 3/6 3/6 3/7 2/4 W H A L E R ' S B A Y 3/7 2/5 2/7 4/6 4/7 2/5 3/7 4/6 3/7 2/6 4/6 3/6 2/7 3/4 3/7 4/7 3/6 4/6 2/6 3/7 2/4 4/7 S P O T L I G H T C O V E 3/7 T R I P L E I S L A N D 4/7 3/5 5/7 4/6 3/7 3/6 3/6 3/6 5/7 2/4 3/7 5/7 4/7 H O P E B A Y 2/6 3/6 2/5 3/6 1/6 2/6 2/4 3/5 4/6 2/3 L I T T L E B A Y 3/6 2/4 P O R T W A S H I N G T O N Fig. 33b The number of enzymes out of the t o t a l O T T E R B A Y examined that had at least one isoenzyme band i n common, but were not i d e n t i c a l i n the i r e l e c trophoretic patterns; a pairwise M I N E R ' S B A Y comparison of populations with three S T U R D I E S B A Y chromosomes. 3/7 1/4 3/4 3/7 3/6 3/6 3/6 5/7 2/4 143 Fig. 33c The number of enzymes out of the t o t a l examined that had at lea s t one isoenzyme band i n common but were not i d e n t i c a l i n t h e i r e l e c trophoretic patterns; a pairwise comparison of populations with s i x chromosomes. SOOKE j 2/5 3/5 2/5 2/5 OGDEN POINT 4/6 3/7 2/7 FERNWOOD 2/6 4/6 WHALER'S BAY 3/7 Fig. 33d The number of enzymes out of the t o t a l examined that had at le a s t one isoenzyme band i n common but were not i d e n t i c a l i n t h e i r electrophoretic patterns; a pairwise comparison of populations with three chromosomes (top) versus populations with s i x chromosomes ( s i d e ) . 800KE 2/5 2/5 3/4 2/5 2/5 2/4 3/5 OGDEN POINT 3/7 3/7 4/6 3/6 2/7 3/4 5/7 FERNWOOD 4/6 4/6 2/5 4/6 4/6 3/4 4/6 WHALER'S BAY 2/7 2/6 3/6 3/6 3/7 2/4 3/7 SPOTLIGHT COVE 3/7 2/6 4/6 3/6 2/7 3/4 5/7 Fig. 33e A SUMMARY-The number of enzymes with at le a s t one isoenzyme band i n common and t h e i r frequency amongst the pairwise comparisons of populations; HUMBER OF EHYKES PAIRWISE COMPARISONS OF: ALL POPULATIONS POPULATIONS WITH THREE CHROMOSOMES POPULATIONS WITH SIX CHROMOSOMES POPULATIONS WITH THREE CHROMOSOMES VERSUS POPULATIONS WITH SIX CHROMOSOMES 0 0/91 0/21 o/ i n 0/35 I 3/91 2/21 0/10 0/35 2 31/91 7/21 5/10 12/35 3 36/91 10/21 3/10 14/35 4 18/91 1/21 2/10 7/35 s 3/91 1/21 0/10 2/35 P O I N T N O P O I N T 2/5 S O O K E 3/7 i/5 O G D E N P O I N T i/< 1/5 2/6 F E R N W O O D 3/7 1/5 3/7 0/6 H O P E B A Y 4/7 1/5 3/7 0/6 0/6 P O R T W A S H I N G T O N 2/6 0/4 2/6 0/5 0/6 0/5 O T T E R B A Y 2/6 2/5 2/6 1/6 2/6 3/6 0/5 L I T T L E B A Y 2/7 1/5 1/7 1/6 2/7 2/6 1/6 1/6 M I N E R ' S B A Y Fig. 34a 0/4 2/4 1/4 1/4 2/4 2/4 1/3 2/4 1/4 B T U R D I E S B A Y The number of enzymes out of the t o t a l examined that had no common isoenzyme bands i n th e i r e l e c t r o p h o r e t i c patterns; a pairwise comparison of a l l populations. 4/7 1/5 3/7 2/6 1/7 1/6 0/6 2/6 2/7 2/4 W H A L E R ' S B A Y 2/7 1/5 1/7 1/6 2/7 2/6 1/6 1/6 0/7 1/4 2/7 S P O T L I G H T C O V E 3/7 1/7 1/5 2/5 2/7 2/7 2/6 2/7 2/7 2/6 1/6 3/6 3/7 2/4 2/ 3/ T R I P L E I S L A N D 1/6 2/6 1/6 3/6 2/7 0/4 2/7 2/7 2/7 H O P E B A Y 0/6 P O R T W A S H I N G T O N Fig. 34b The number of enzymes out of the t o t a l examined that had no common isoenzyme bands i n th e i r e l e c t r o p h o r e t i c patterns; a pairwise comparison of populations with three chromosomes. 'A f/fA 0/6 0/5 O T T E R B A Y 2/6 3/6 0/5 L I T T L E B A Y 2/7 2/6 1/6 1/6 M I N E R ' S B A Y 2/4 2/4 1/3 2/4 1/4 B T U R D I E S B A Y 2/7 2/6 1/6 3/6 2/7 0/4 145 Fig. 34c The number of enzymes out of the BOOKE | 2 / 5 1/5 1/5 1/5 t o t a l examined that had no common OGDEN POINT 2/6 3/7 1/7 isoenzyme bands i n t h e i r e l e c t r o p h o r e t i c patterns; a pairwise comparison of FERNWOOD 2/6 1/6 populations with s i x chromosomes. WHALER'S BAY 2/7 BOOKE 1/5 1/5 0/4 2/5 1/5 2/4 2/5 OGDEN POINT 3/7 3/7 2/6 2/6 1/7 1/4 2/7 FERNWOOD 0/6 0/6 0/5 1/6 1/6 1/4 1/6 WHALER'S BAY 1/7 1/6 0/6 2/6 2/7 2/4 2/7 SPOTLIGHT COVE 2/7 2/6 1/6 1/6 0/7 1/4 2/7 Fig. 34d The number of enzymes out of the t o t a l examined -that had no common isoenzyme bands i n th e i r electrophoretic patterns; a pairwise comparison of populations with three chromosomes (top) versus populations with s i x chromosomes ( s i d e ) . Fig. 34e A SUMMARY-The number of enzymes with no common isoenzyme bands and t h e i r frequency amongst the pairwise comparisons of populations; NUMBER OF CHYMES PAIRWISE COMPARISONS OF: ALL POPULATIONS POPULATIONS WITH THREE CHROMOSOMES POPULATIONS WITH SIX CHROMOSOMES POPULATIONS WITH THREE CHROMOSOMES VERSUS POPULATIONS WITH SIX CHROMOSOMES 0 0/91 0/21 0/10 0/35 1 3/91 2/21 o / i o 0/35 a 31/91 7/21 5/10 12/35 3 36/91 10/21 3/10 14/35 4 18/91 1/21 2/10 7/35 3 3/91 1/21 0/10 2/35 DISCUSSION / 146 chromosome numbers than between populations of the same chromosome number. No pattern or trend was observed between the chromosome numbers and banding patterns of the various populations. The populations that appear the most similar in their electrophoretic patterns are Spotlight Cove (six chromosomes), Miner's Bay (three chromosomes), and Ogden Point (six chromosomes); Spotlight Cove and Miner's Bay are the most similar. The next set of populations that show the greatest similarity are Otter Bay (three chromosomes), Hope Bay (three chromosomes), and Whaler's Bay (six chromosomes); Otter Bay and Whaler's Bay are the least similar. The population that appears least similar to all others is Sturdies Bay. However, banding patterns of only four enzymes were available for comparisons; the other enzymes failed to stain. Only three populations (Triple Island and the two beaches of Point No Point) with four chromosomes were available in this study; these two populations are geographically widely separated. They shared no identical banding patterns and had no bands in common for three of seven enzymes tested. Unfortunately, comparing populations with different chromosome numbers (3, 4 or 6) as discussed in section 3 was to a large extent not possible because there were often few if any identical bands shared between populations. DISCUSSION / 147 5.2. G E N E R A L DISCUSSION This electrophoretic study on the genus Bangia in the northeast Pacific produced some information regarding the number of subunits and number of possible cellular locations of seven enzymes, the number of loci involved and the geographic distribution trends of the isoenzyme banding patterns. As well, it presents data which can be used in formulating hypotheses on the chromosomal evolution within the genus. This preliminary investigation also has provided direction for further studies that are needed. The major point of interest in this study was whether the number of enzyme loci present in the various Bangia populations could be used to help explain the origination of the different chromosome numbers. However, no general relationship between chromosome number and the number of loci could be established from the data (see section 5.1). According to karyotype analysis the six chromosome populations are diploid while the three chromosome populations are haploid (Cole, Hymes and Sheath 1983). However, many asexual three and asexual six chromosome populations have the same number of isoenzyme loci (LDH, PGI, G6PDH, GDH) and both banded in some cases as would be indicative of diploids. There are two possible explanations for this: the three chromosome populations arose from the asexual six chromosome populations by aneuploid reduction; or half the chromosomes are non-functional in the asexual six chromosome population and there have been many gene duplications. D N A quantification would help in determining whether DISCUSSION / 148 aneuploid reduction had occurred or if the six chromosome populations contain twice the D N A of the three chromosome populations have. The two following hypotheses may explain the incongruity between the electrophoretic results and karyotype analysis. In Porphyra, a closely related genus, it has been observed that diploid conchocelis will at times give rise to diploid blades rather than haploid blades (Ma and Miura 1984). If this occurred in sexual three populations of Bangia the filaments would have six rather than three chromosomes and these may be asexual due to the presence of both "mating types" (assuming Bangia has alleles for determining sex). Subsequently, aneuploid reduction may have given rise to the asexual three populations, and possibly the sexual four, if one of the mating alleles was damaged, altered or lost. Alternatively, half the chromosomes in the six chromosome filament may be inactivated. If this is the case then gene duplications must have occurred for many of the enzymes to explain why the asexual three and asexual six chromosome populations frequently band as would be expected of diploids. Either that or the inactivation is not complete. This would suggest determination of form, filaments versus chonchocelis, may be a dosage effect. The three chromosome populations may have arisen from loss of most,if not all, the inactivated DNA. Both of these possibilities could lead to very interesting future research. How is sexuality controlled in Bangial By a single locus or several? Are these loci present or absent in asexual populations? If present, are populations asexual because 'male' and 'female' determining loci are present in both populations? How DISCUSSION / 149 do sexual populations with different chromosome numbers compare with respect to these alleles? What controls differentiation of Bangia into conchocelis or filaments? Is this a dosage effect? If yes, how does dosage affect morphology? Do diploid filaments arise when half the chromosomes are inactivated in conchocelis? What would cause this inactivation? The answer to these questions would do much to help elucidate how these populations with different chromosome numbers and life histories may have arisen. Another aspect to consider in this study of Bangia is the distribution of the electrophoretic banding patterns in populations along the B.C. coast and whether there are any geographic trends. The present coast has not changed much since the glacier retreat about 10,000 years ago (Thomson 1981). Al l the sites examined are connected by either the flood or ebb tidal streams or a combination of these (Thomson 1981). Sturdies Bay and Miner's Bay populations lie on opposite sides of Active Pass but they are never identical in any enzyme banding patterns. Similarly Spotlight. Cove and Fernwood populations, lying on opposite sides of Trincomali Channel, are only identical in one of the enzyme banding patterns examined. These results may be explained if the strength of the tidal streams does not permit much, if any, cross channel water movement, even during slack. Populations in the two coves, Whaler's Bay and Sooke, are fairly unique in their banding patterns differing from most of the other populations. These coves are located to the side of strong tidal streams, prime locations for backeddies that may prevent mixing of water and introduction of spores and individuals from other populations. There is more similarity among populations within regions of slower tidal streams (for example, Pender Island) than among DISCUSSION / 150 populations within regions of strong tidal streams (for example, Point No Point and Ogden Point). Populations, such as Little Bay, located within regions of minimal tidal streams tend also to be fairly unique in their isoenzyme banding patterns. Two sets of populations were particularly interesting in their electrophoretic patterns. In five of the seven enzymes tested the bands observed from Triple Island material were the same as those from Sitka material. However, the Sitka population had some additional bands to those found in Triple Island material. This is the most northerly region where sexual four chromosome populations are detected and it has been suggested that they may have originated in this region (Cole, Hymes and Sheath 1983). It is interesting that Triple Island material shows very little similarity to the other four chromosome populations, however, they are geographically far apart. Triple Island material shows a great similarity to the three chromosome population, Sitka. Spotlight Cove (6) and Miner's Bay (3) populations, also with different chromosome numbers, had the same banding patterns in five of the seven enzymes examined. Considering all the above points it would be useful to examine closely a single channel, its tidal streams and their strengths, amount of cross current water movement, and backeddies and how these factors affect gene flow between populations. One study on Euchema suggested that there is limited gene flow even within a population (Cheney and Babbel 1978). Therefore it could be expected that little gene flow would be observed between populations of Bangia and this may explain why the populations of Bangia show little similarity to each other. If the populations of Bangia have been established for a long period of time and if there is little gene flow between the populations this can explain DISCUSSION / 151 many of the observed results. Asexual three and asexual six chromosome populations show the same number of loci for many but not all enzymes, and the number of loci within a population is not always the same. If the populations had been established for a long period of time gene silencing and null alleles may have formed. Over time different enzyme mobilities can arise from the accumulation of amino acid changes and this may explain the large amount of variation observed within and between populations. It is necessary to know the number of subunits in an enzyme in order to interpret the electrophoretic data. This has been determined in higher plants and animals by extensive studies and the number of subunits remains consistent. However, this information is not available for the red algae. Present information suggests that the red algae diverged early from the branches that gave rise to higher plants and animals (Mohn 1984) and therefore the red algae may be very different in biochemical features such as the number of subunits in an enzyme. Results obtained in this study suggest that, for at least some isoenzymes, the number of subunits in an enzyme may not be the same in Bangia as in higher plants and animals. It has also been suggested by the results of these studies in Bangia that the cellular locations for various isoenzymes may not be as numerous in red algae as they are in higher plants. However, the question of whether or not Bangia, or any of the red algae, have the same number of enzyme subunits or the same cellular locations as higher plants requires further study. DISCUSSION / 152 This electrophoretic study on Bangia is the most extensive one done within the algae regarding the number of populations (19 sites) and number of individuals examined. To obtain a sufficient slurry upwards of one hundred filaments were simultaneously examined in fifteen field populations. Examinations of single individuals was limited to clonal cultures that were obtained after extensive culturing. There have been only four published electrophoretic studies on red algae examining different populations. The most extensive one included eight populations of four Euchema species from six different sites examined for five different enzymes (Cheney and Babbel 1978). Euchema, unlike Bangia, is sufficiently large that individual plants from the field could be examined and a large number of individuals in each population were tested. As with Bangia, the inablility to perform progeny studies limited the amount of information that could be obtained and conservative assumptions had to be made. They suggested removal of two of the species from the genus Euchema, confirming morphological data. Again as with Bangia, there were no seasonal differences in banding patterns and no difference in banding patterns after three months of culturing. A study on Porphyra yezoensis examined material from eleven field localities and nine cultured strains for five enzymes (Miura; Fujio and Suto 1979). It was determined that cultured populations were not as polymorphic as wild populations. However it was not stated how the number of individuals from each strain were generated. It may be that the individuals were obtained via asexual reproduction and then a decrease in polymorphism would be expected relative to wild DISCUSSION / 153 populations. Two other studies suffered greatly from limited sample size. Mallery and Richardson (1972) examined eleven genera of red algae, one individual per genus, and decided that enzymes (esterase, aminopeptidase, acid phosphatase), soluble proteins and biliproteins could be used to separate genera. In examining three isolates of Rhodochorton purpureum, each from a different site (Alaska, Washington, and Chile), and four cultures, each of a different species of Acrochaetium, for esterase, total protein and phycobiliproteins, Richardson and Mallery (1973) concluded that the different forms of protein may have adaptive value, that there may have been incipient speciation, that the two genera are a complex and not two separate genera and that there is high variabil i ty within the complex. The conclusions of both studies appear to be premature given the data that were presented. The purpose of the current study was to establish isoenzyme banding patterns in Bangia populations along the northeast Pacific, to examine the degree of variation and the number of enzyme loci present and then to see if these data could be used to explain the origination of the various chromosome numbers amongst these populations. The electrophoretic data showed a great deal of variability both within and between populations possibly due to isolation. It is suggested that the various chromosome numbers probably arose by aneuploid reduction or by loss of inactivated D N A . However, the complexity of the results suggest gene silencing, null alleles, and gene duplications may have occurred frequently. This study also indicated that the number of subunits and cellular locations for an enzyme may DISCUSSION / 154 not be the same in Bangia as in higher plants and animals. The use of isoenzyme electrophoresis in the red algae is still in its infancy. There is a need for further indepth and comprehensive studies before any patterns will become apparent. Some basic questions that require answers include: what amount of variability can be expected within and between populations; what numbers of subunits does an enzyme have; what are the cellular locations of the various enzymes; and are there any geographic trends with respect to variability. 6 . L I T E R A T U R E C ITED Beale, S.I. & Foley, T. 1981. Regulation of A L A synthetase activity in Euglena gracilia. Plant Physiology 67(4 suppl):33. Beam, C.A., Himes, M., Daggett, P-M. & Nerad, T.A. 1982. Electrophoretic analysis of soluble enzymes of the Crypthecodinium cohnii. J . Protozoology 29(3):494. Blair, S.M., Mathieson, A .C. & Cheney, D.P. 1982. Morphological and electrophoretic investigation of selected species of Chaetomorpha (Chlorophyta, Cladophorales). Phycologia 21(2): 164-172. Brown, A.H.D. & Weir, B.S. 1983. Measuring genetic variability in plant populations. In Tanksley, S.D. & Orton, T .J . (Ed.) Isozymes in Plant Genetics and Breeding: Part A. 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