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Life history stage ratios of Iridaea cordate and factors controlling these ratios in intertidal populations Green, Lesley Gail 1989

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LIFE HISTORY STAGE RATIOS OF IRIDAEA CORDATA AND FACTORS CONTROLLING THESE RATIOS IN INTERTIDAL POPULATIONS B.Sc. University of British Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Botany We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA by LESLEY GAIL GREEN April, 1989 Lesley Gail Green 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 The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Two populations of Iridaea cordata (Turner) Bory in Vancouver Harbour, Vancouver, British Columbia were examined. Both of the populations alternate between d i p l o i d dominance in the winter and haploid dominance in the summer. There was a significant difference between the ratio of diploid to haploid plants at the two sites. The alternation in dominance observed in the population as a whole was also seen in three different size classes (<5cm, 5-15cm and >20 cm) examined. Clearance experiments done in the f i e l d indicate that i t can take up to 1 year for Iridaea to recolonize a disturbed area. When recolonization does occur the ratio of diploid to haploid plants mirrors the ratio at the time of clearing, not the ratio at the time of regrowth. This suggests that the spores s e t t l e when the rocks are cleared but remain dormant u n t i l conditions allow for growth and formation of upright plants. The influence of apomeiosis on the number of diploid plants is probably negligible, since only 2% of cultured tetraspores showed evidence of this phenomenon. Some physical characteristics of Iridaea, bearing on reproduction were also explored. Tetrasporophytes and gametophytes have significantly d i f f e r e n t surface areas, with the gametophytes being larger, and they have significantly different densities of reproductive structures, with the tetrasporophytes having more. As a result, both plant types have statistically the same net number of reproductive structures per plant. There was l i t t l e i i difference with respect to surface area or density of reproductive structures between the high and lov i n t e r t i d a l plants of either ploidy. i l l TASliE QF CONTENTS page Chapter 1. General Introduction 1 Chapter 2. Life History Stage Ratios of Introduction 10 Material and Methods 15 Results IS Discussion 31 Chapter 3. Settlement onto Cleared Substratum Introduction 39 Materials and Methods 48 Results 50 Discussion 56 Chapter 4. Occurrence of Apomeiosis in Iridaea cordata Introduction 64 Materials and Methods 69 Results 72 Discussion 75 Chapter 5. Quantitative Characteristics of Different Reproductive Stages of Iridaea cordata Introduction 79 i y Materials and Methods 84 Results 86 Discussion 97 Chapter 6. General Discussion 104 References 110 Appendix 1- Raw Data From Resorclnol Testing 119 Appendix 2- Results of Culturing Water Samples 126 Appendix 3- Observations of Cleared Rocks 129 Appendix 4- Raw Data From Kontron Image Analysis 137 V Table 3.1 Table 3.2 Table 4.1 Table 5.1 Table 5.2 Table 5.3 Table 5.4 Table 5.5 Table 5.6 Table 5.7 page Densities of Enteromoroha in field samples 54 Results of resorcinol testing of Iridaea  cordata sampled from regrowth on cleared substrata 55 Results of testing cultures of tetraspores for apomelosis 74 Density of reproductive structures and surface area of Iridaea cordata collected at Brockton Point. 90 Comparison of spores released by carposporangia and tetraspores 91 Results of statistical analysis for data on carposporophytes vs. tetrasporophytes. 92 Results of statistical analysis for data on tetrasporophytes. 93 Results of statistical analysis for data on tetrasporophytes, high vs. low intertidal 94 Results of statistical analysis for data on carposporophytes, high vs. low intertidal 95 Results of spore counts for carpospore and tetraspore release in September, 1987 96 v i LIST OF FIGURES page Figure 2.1 I r i d a e a cordata expressed as % gametophytic over time. 22 Figure 2.2 Reproductive populations of Iridaea cordata 24 Figure 2.3 Iridaea cordata >20 cm plants 26 Figure 2.4 Iridaea cordata 5-15 cm plants 28 Figure 2.5 Iridaea cordata <5 cm plants 30 v i i , ACKNOWLEDGEMENTS To my teacher, supervisor and frie n d , Dr. Robert E. DeWreede, I would like to express my most heartfelt thanks. Thanks also are extended to the other members of my committee who supported me, and gave excellent e d i t o r i a l comments- Dr. MiJce Hawkes and Dr. Paul G. Harrison. These three men formed a supportive, patient and meticulous group. I would also like to thank Patrick Harrison for so generously lending me his computer, and Dr. Kathleen Cole for the experience of being part of her team. The Botany Department is also deserving of my thanks for the patience and support over the years. For technical help I would like to thank Loyal Merhoff, who guided me through the mysteries of the Mainframe, and Michael Weiss, who t r i e d to teach me how to use the Kontron. Thomas Kong and Josee Trembley were of invaluable laboratory assistance. For surviving the rigors of field work with minimal complaint and enthusiastic e d i t i n g I would l i k e to thank my trusted and faithful friends Allen Davenport, Stephen Green and Neal Pollock. What remains of my sanity i s due to the help of my family and friends who always supplied me with help, encouragement, good meals and clean laundry. Special thanks to Hugh, Noreen, Michael, Stephen, Granny, Kate and Jennifer. And to Put Ang- thanks for the toast and hot chocolate. v i i i CHAPTER 1 INTRODUCTION Many algae exhibit an alternation of generations. This alternation can be either isomorphic (the two l i f e history stages being morphologically identical) or heteromorphic (morphologically dissimilar). An example of isomorphic alternation of generations can be found in the Rhodophyta in the alga Iridaea cordata (Turner) Bory, the subject of this study. Life histories in the Gigartinaceae typically consist of two isomorphic phases, with dioecious gametophytes (Bold and Wynne, 1985). The gametophyte and the tetrasporophyte can not visibly be distinguished from one another unless they bear reproductive structures. The r a t i o o f l i f e history stages of algae is significant in commercial production a n d u t i l i z a t i o n o f carrageenan-producing species. Craigie a n d P r i n g l e (1978) developed a technique for identifying the ploidy of nonreproductive thalli of the carrageenan-producing alga Chondrus crispus Stackhouse. This technique indicates the l i f e history stage by identifying the carrageenan it produces, haploid gametophytes producing K-carrageenan, and diploid tetrasporophytes producing L-carrageenan. The kappa carrageenan i s alpha 1,3-linked galactose-4 sulphate alternating with beta 1,4-linked anhydrogalactose, while the - 1 -lambda carrageenan is alpha 1,3-linked galactose or galactose-2-sulphate alternating with beta 1,4-linked galactose- 2,6-disulphate. The carrageenan of preference in the phycocolloid industry is K-carrageenan (McCandles, 1981). This resorcinol test identifies the anhydrose link and gives a positive colour response. Identifying the time when haploid t h a l l i are the most common plant type in a community indicates the best time to harvest for K-carrageenan. When an alternation between a d i p l o i d and haploid stage occurs, it has been argued by some that the diploid stage will be favoured since heterozygosity may prevent manifestation of detrimental recessive genes. The only exception to this would be where certain recessive traits have a selective advantage (Bhattacharya, 1985). It has been hypothesised (Law, 1979) that there are limiting resources which dictate the traits observed in organisms and that l i f e history strategies evolve to make the most efficient use of available resources (Law, 1979). In Iridaea cordata the energetics of different l i f e history stages have not been studied. Whether it is more efficient to arise annually from perennial crusts or to recruit annually from spores is not known. Iridaea is in the family Gigartinaceae, order Gigartinales with a Pacific Rim d i s t r i b u t i o n from Japan to southern California and South America . The thallus of Iridaea is - 2 -composed of a perennial crustose holdfast, and a multiaxial upright blade (Hannach and Waaland, 1986). In the work completed for this study no perennial crusts were studied. Chromosome numbers have been reported for rridaea cordata (n=4) (Fralick and Cole, 1973). There are 7 species of Iridaea occurring in North America (Abbott, 1971). Iridaea cordata is the most widespread species in the northeastern Pacific (Abbott, 1972). There has been some controversy over the taxonomy of Iridaea cordata and Iridaea flaccida (Setchell & Gardner) Silva. It was found that Iridaea flaccida. which i s characteristically greenish in colour, takes on the purplish colour of Iridaea cordata when it is introduced to deeper waters (Foster,1982). Iridaea flaccida is mid to low i n t e r t i d a l with a tetrasporangial-free margin around the edge of reproductive tetrasporangial blades. Iridaea cordata is the most common species of Iridaea in the northeastern Pacific, and is purple-brown in colour. It tends to inhabit the low intertidal to subtidal (Abbott, 1971). The plants I studied all conform to the description of Iridaea cordata. Iridaea cordata also inhabits a wide v a r i e t y of wave environments as Iridaea cordata var. cordata in protected sites, and as Iridaea cordata var. splendens (S. & G.) Abbott in open coastal waters (Hannach and Waaland, 1986). In Washington waters competition with Laminaria saccharina sets the lower limit of Iridaea cordata (Hruby, 1976) and in California it sets the lower limit for Iridaea flaccida (Foster, 1982). In some Puget Sound populations Iridaea - 3 -cordata composed less than 5% of the seasonal cover in July and April, but sampling was sporadic (Thorn, 1980). Iridaea  flaccida forms 100% cover in some populations in California (Foster, 1982). A general review of the literature to date, including d i s t r i b u t i o n , ecology and p r o d u c t i v i t y of Iridaea was published by Hannach and Waaland (1986). Waaland (1977) has also Investigated the possibility of c u l t u r i n g Iridaea for commercial purposes finding that this red alga needs an environment with much wave action or aeration to grow well in culture. Sylvester and Waaland (1983) developed a method for cloning some red algae, including Iridaea, for commercial propagation. Mumford (1979) also attempted to culture Iridaea  cordata for commercial purposes. Potential for harvesting natural and a r t i f i c i a l populations of Iridaea cordata for commercial use was considered by Adams and Austin (1979). In Chile, Iridaea is the second most important commercial species of the Rhodophytes. In 1979, close to 2,700 dry tons of Iridaea spp. was exported to the U.S.A. (Santelices and Lopehadia, 1980). Some studies were made of Iridaea cordata populations before the use of the resorclnol test. Adams (1979) considered a Georgia Strait population and by examining reproductive plants concluded that there is an alternation in the dominant l i f e history stage at different times of the year, with the N - 4 -plants dominating in the summer months and 2N plants in the winter months. Populations of Iridaea cordata were examined in 1976 by Hansen and Doyle in central California. They found that the reproductive portion of the population was dominated by diploid tetrasporophytes. Hansen and Doyle (1976) proposed three mechanisms for the maintenance of a d i p l o i d dominance 1) the plants are a r i s i n g seasonally from d i p l o i d perennial crusts, not reproducing sexually; 2) tetrasporophytes are hardier and able to withstand harsh environmental conditions when carpospores and gametes cannot; 3) apomeiosis is occurring causing a cycling of 2N plants. In 1985, Dyck et al. investigated Iridaea populations in British Columbia, Oregon and California, including the sites that Hansen and Doyle (1976) studied. Dyck et al.'s (1985) study d i f f e r e d from the previous ones in u t i l i z i n g the resorcinol test to look at both reproductive and nonreproductive plants. They concluded that British Columbia populations were composed p r i m a r i l y of gametophytes in the summer months and that a gradient existed of increasing proportions of tetrasporophytes with decreasing latitude from Oregon to California. They also looked at high and low wave action sites in British Columbia and found an increase in the proportion of the thalli that were tetrasporophytic with increased wave action. - 5 -May (1986) published a paper on a Juan de Fuca population of Iridaea and concluded that the population there was primarily haploid and arose from perennial crusts. She, however, did not examine the structure of the population year-round. Thus, many investigators have studied the population structure of Iridaea cordata. yet most of the data are incomplete because only reproductive plants were examined, or population structure for only a specific time of the year was studied. The purpose of the f i r s t portion of this study i s to examine two populations of Iridaea cordata year-round, using the resorcinol test, to elucidate the ratio of N/2N plants. Sampling year-round will provide data on changes the population may undergo in this ratio. If ratio changes are found it may then be possible to Identify some factors important in determining population ploidy. Utilizing the resorcinol test will permit the examination of both reproductive and nonreproductive plants. The first hypothesis examined is: diploid and haploid plants are expected to occur in equal proportions in a given population. Once the populations as a whole have been studied i t i s possible to consider the mechanisms responsible for the observed N:2N ratios. These mechanisms include differential size class mortality, differential spore settlement, apomeiosis and differential morphological characteristics. - 6 -The f i r s t mechanism to he c o n s i d e r e d v i l l be d i f f e r e n t i a l m o r t a l i t y f o r d i f f e r e n t s i z e classes, which would r e s u l t i n a s p e c i f i c N:2N ratio. Therefore, the second hypothesis examined i s ; i f d i f f e r e n t i a l s i z e class mortality is occurring, different size classes are expected to have different proportions of d i p l o i d and haploid plants. The second mechanism to be considered is differential spore settlement. Iridaea cordata is considered a late successional species (Littler and Littler, 1980). The sequence of intertidal succession indicates that, in general, green algae are early successional and browns and reds are established later (Northcraft, 1948; Fahey, 1953; Murray and Littler, 1918; Littler and Littler, 1980). Seasonally occurring populations of Iridaea cordata may be established by spore settlement each year, or by perennial c r u s t s v h i c h v e g e t a t i v e l y give r i s e to the populations each year. One would expect an intertidal cleared area to follow such a successional sequence. The interest would be i n how long it would take for the Iridaea to reappear after the clearing occurred, and then to compare the ratio of life history stages of the new population with the population at the time of clearing and with undisturbed populations at the time of reappearance. Amongst s e t t l i n g spores there could be differential settlement and survival by ploidy. Differential spore settlement and survival may result from one spore type being hardier or differentially dispersed than the other (Hansen and - 7 -Doyle, 1976). In order to examine the importance of spores in the life history strategies of Iridaea cordata areas of study sites were cleared and the ratio of the plants that reappeared were observed. Points examined included: 1. Length of time required for Iridaea to reappear after clearing, 2. Whether the N:2N ratio of plants which settle reflects the population ratio at the time of clearing, and, 3. Whether the ratio of plants which grow on cleared surfaces d i f f e r s from the ratio in the population at the time of sampling. Thus the third hypothesis i s : spore settlement onto cleared surfaces reflects the ratio of diploid to haploid plants in the population at the time of c l e a r i n g . The populations may be established by either annual spore settlement, or by asexual growth from perennial crusts. Within this scope there may be other factors which dictate the ratio of life history stages. Hansen and Doyle (1976) proposed that apomeiosis may be occurring which leads to cycling of diploid dominated populations. Apomeiosis has been reported for MtXthamnion sarnlensis, (Magne, 1966), Gjgartim stellate (Chen and Craigie, 1980), Rhodochorton concrescens Drew (West, 1970), and Callithamnion sp. (West and Norris, 1966). In order for apomeiosis to affect the N:2N ratio of an entire population it is necessary for it to be a common occurrence in the affected species. If this is the case, then many Iridaea tetrasporophytes should release spores which form blades which test negatively (i.e. are 2N) for the presence of - 8 -kappa carrageenan. Therefore, the fourth hypothesis is: apomeiosis occurs in populations of Iridaea cordata exhibiting diploid dominance. The final concept considered in this thesis is morphological or physiological differences between isomorphic forms of Iridaea cordata. This portion was an excercise in using new technology to address a specific question. Many algae exhibit differing morphologies for the different ploidies. - It has been hypothesized that the different forms exist in response to requirements to prevent herbivory or maximize growth (Littler and Littler, 1980). Little work has been done on measuring physical or physiological differences between isomorphic forms. It is possible that even though the two forms have an outwardly identical appearance, they have physiological or physical differences which optimize survivorship under a given set of environmental or ecological conditions. This concluding portion of my study will examine both diploid and haploid thalll to determine if there is a difference in the density of reproductive structures or a difference in surface areas of individual thalli. The plants will also be compared within a life history stage for differences between high and low intertidal plants. The final hypothesis considered will be: isomorphic phases of Iridaea cordata exhibit no difference in the number of reproductive structures per plant, surface area of individual thalli, or density of reproductive structures. - 9 -CHAPTER 2 LIFE HISTORY STAGE RATIOS OF IRIDAEA CORDATA INTRODUCTION This chapter addresses the hypothesis that the r a t i o of d i p l o i d to haploid plants i s expected to he egual in a given population. Since 1976 there have been a number of studies which examined populations of Iridaea cordata. Many of these studies examined which life history phase was dominant at a particular site and time. Most of the studies proposed mechanisms by which the population structure was established. Adams (1979) studied a population in Georgia Strait and concluded that from May to August gametophytes occurred in equal or greater number than tetrasporophytes. Tetrasporophyte dominated for the remainder of the year. Hansen and Doyle (1976) worked in California and found both gametangial and tetrasporangial stages present throughout the year. In reproductive plants the tetrasporophytes were most common. Their laboratory investigations led them to believe that dominance of the diploid stage occurred at the spore level with more carpospores surviving than tetraspores. Their field results indicated that the typical sexual alternation of - 10 -d i p l o i d and haploid stages had been replaced with perennial crusts and vegetative reproduction. Tetrasporophytic dominance has been noted for many red algae at the geographical l i m i t s of the populations; also, tetrasporic plants have been noted farther north than gametophytic plants for individual species (Dixon, 1965). Three explanations are proposed to account for tetrasporophytic dominance in populations of Callithamnion corYmbosum,. A n t i t h a f f l n i o n crucjatum, Hvpnear PolvsiDhonla. Gelidiumr and Gracilaria. These are: 1)tetrasporophytes live longer than gametophytes, 2)proportionally more carpospores survive than tetraspores, and 3)tetrasporophytes are hardier plants than gametophytes (Kain, 1982). The third explanation may apply where environmental conditions are harsh, since under such conditions tetrasporophytes are often found to dominate. Kain (1982) found tetrasporophytic dominance in an Irish Sea population of Plocamlum. She concluded that this dominance was due to perennation of the diploid thalli by vegetative means. Also, she felt that the tetraspores produced failed to survive on apparently available substrate, although she did not study spores. Tetrasporophytic dominance has also been reported for B r i t i s h populations of Ceramium shuttleworthium (Kutzing) Silva (Edwards, 1973). His studies indicated that some environmental factor was l i m i t i n g formation of gametangia. Dixon (1960) also considered populations of this plant in the - 11 -British Isles. He found that N and 2N plants occurred in egual proportions in some of the l o c a l i t i e s but in other localities the ratio varied from year to year. Barilotti (1971) discussed possible theoretical reasons for the observed diploid dominance in algae. He concluded that diploid plants would dominate populations where 1) a maximum number of genotypes are favoured, 2) phenotypes arising from genetic dominance (homozygous or heterozygous) are favoured, and 3) overdomlnance is favoured. These conclusions are theoretical and are without experimental substantiation. He felt that haploid individuals would be selected for only in environments where recessive homozygosity is advantageous. Hansen (1977) found that 90% of the population of Iridaea  cordata in California was sexually mature in summer-autumn aft e r which time the majority of the population senesced. She indicated that the pattern she observed might be due to plants arising from a perennial crust. Dyck et al. (1985) modified the resorcinol test developed by Cralgie and Pringle (1978) so it could more easily be used to study the ratio of gametophytes to tetrasporophytes for an entire population (including the nonreproductive blades). Using this modified procedure, Dyck et al. (1985) studied s i t e s in Georgia S t r a i t and from northern Oregon to California. Their results indicated gametophytic dominance for the same s i t e where Hansen and Doyle (1976) reported tetrasporophytic - 12 -dominance. In addition a gradient appeared to exist from tetrasporophytic dominance in Oregon to gametophytic dominance in California. However, Dyck et al. (1985) also found substantial variation in tetrasporophyte to gametophyte r a t i o s between closely adjacent sites. Gametophyte dominance was found in Georgia Strait, and studies done in Barkley Sound showed increasing tetrasporophyte dominance with increasing wave action (Dyck et al.,1985). This correlation with wave action supports the Hansen and Doyle (1976) idea that tetrasporophytes may be hardier in this respect than the gametophytes. There was no co r r e l a t i o n between tetrasporophyte to gametophyte ratio and tidal height (Dyck et al. 1985). A population of Jrjdflefl cordata in the San Juan Islands was found be 83% gametophytic (May, 1986), when sampled from June to August. May proposed that perennation and lower survival of gametophyte spores or sporelings maintain these populations. There are some data available for my study sites. DeWreede & Green (in prep.) has followed the population structure of Iridaea cordata and found that one site had 80-90% gametophytes in the summer and 10-20% gametophytes in the winter, while another s i t e was characterized by 60-70% gametophytes in the summer and 30-40% in the winter. Utilizing the resorclnol test two populations of Iridaea  cordata were studied. These two populations were examined to - 13 -e s t a b l i s h i f 1) previously i d e n t i f i e d seasonal changes in the N/2N ratios continue over a year and a half, 2) to examine the population in terms of size classes to look for evidence of size-specific mortality, and 3) to compare the structure of the populations based on sampling reproductive plants compared to the structure of the population based on nonreproductive plants. - 14 -MATERIALS AND METHODS Two populations of Iridaea cordata located at Brockton Point and The Figurehead, Stanley Park, Vancouver, British Columbia were examined in detail. The sites were about 1 km apart. At both sites X . cordata is found below the 0.8m tide height and the population sampled was from -0.2 to 0.8m (Anonymous, 1986; Anonymous, 1987; Anonymous, 1988). Populations were sampled whenever the tide was below 0.7m from June 1986 to December, 1988. Both sites appear to have perennial populations of Iridaea  cordata, a north-east exposure, and have been the subject of a preliminary study (Dyck et al., 1985; DeWreede & Green, in prep.). These sites have slightly different topographies. The Brockton Point site is very rocky with large boulders and is steep. The Figurehead site is essentially a mudflat with only a few scattered rocks u n t i l the 0.9m level where there is a band of small boulders before a slight drop off at the -0.3m level. For both Figurehead and Brockton Point, one 50 m transect was set up within each of four subsites. Each transect was parallel to the shore within the narrow band of exposed Iridaea  cordata. During each collection period one sample set was collected from each 50 m transect, each sample set consisting of 30-40 plants for each of four categories. The four - 15 -categories of Iridaea sampled were: <5, 5-15, >20 cm (measured from base to tip of blade) and reproductive. The samples were gathered using a leather hole punch which produced a 6 mm disk. The sampling covered the entire i n t e r t i d a l range of Iridaea cprdflta-In the laboratory disks were subjected to the acetyl resorcinol test (Craigie and Pringle, 1978) as adapted by Dyck et al. (1985). This involved air drying the collected disks, then placing them into individual test tubes. Acetyl resorcinol was added in 2 ml portions. Samples were then placed in a hot water (80° C) bath for 2-3 minutes, u n t i l a colour change could be observed in the positively reacting thalli. Gametophytes reacted strongly with the resorcinol resulting in a r i c h red-burgundy colour. Tetrasporophytes were colourless or a very pale pink. Reproductive tissue of known ploidy of Iridaea cordata was sampled and used in all the tests to ensure that the chemicals were giving the expected results. Variations of the standard resorcinol tests were performed in order to further refine the procedure. The volume was reduced to 1.5 ml, then 1.0 ml and tested on 10 replicates of both gametophytic and tetrasporophytic thalli. The other parameter examined was the concentration of HCl used. In the standard procedure full strength concentrated HCl is used. I examined 1/2 and 1/4 strength HCl mixed with the usual amounts of the other reagents. These solutions were then added to - 16 -gametophytic and tetrasporophytic tissue (25 replicates for each) in 2 ml aliquots. Statistical analysis consisted of paired t-tests. The data indicated homogeneity of variance and normal d i s t r i b u t i o n , except where indicated (Zar, 1984). Comparisons vere made within and between populations. - 17 -RESULTS Over the year and a half of sampling the populations showed an alternation from haploid dominance in summer to d i p l o i d dominance in winter. Results for the populations as a whole (all size classes together) can be seen in Figure 2.1. The peak of haploid dominance occurred at the Figurehead during August 1986 (80%) and the peak of diploid dominance in November 1987 (71%). Brockton Point had a peak of haploid dominance in May 1987 (78%), and diploid dominance in November 1987 (73%). Within a site there is no statistically significant difference between transects. Paired t-tests run on the Brockton Point transects indicate no significant difference (p=.05, d.f.=29, t=.534) The results show that the Figurehead site usually has a higher proportion of gametophytic plants than the Brockton Point site. A paired t-test (p=0.0005, df=41 t=-4.457) indicates that the two sites are statistically different in their ratios. The ratios of gametophytes to tetrasporophytes over the sampling period for the reproductive plants can be seen in Figure 2.2. Sampling of reproductive plants was not as frequent as that of the entire population because at times there were few or no reproductive plants available. - 18 -Gametophytes dominate the reproductive population for the summer months and tetrasporophytes dominate the winter months. There vas no difference between the pattern observed for nonreproductive plants and reproductive plants, except that the reproductive plants represent extremes of the measured r a t i o s . Different s i z e classes of the populations were also examined, and these followed the same N/2N pattern as the reproductive plants. Size classes did not show a progression of gametophytic dominance (e.g. gametophytic dominance of the <5 cm size class followed by a decrease In the proportion of gametophytes In this size class as these plants grow up into the 5-15 cm class). Size class data are shown in Figures 2.3-2.5. Individual size classes predominantly showed gametophyte dominance in summer and tetrasporophyte dominance in winter. The exception was the >20 cm size class at Brockton Point which in 1987 had a gametophyte peak in May and a tetrasporophyte dominance in August. This size class also showed an unexpected increase In number of gametophytes in December 1986. The 5-15cm size class shows both an increase and a decrease for this time period. Paired t-tests done on the Brockton Point data showed no significant difference between the <5 and >20cm size classes at the 0.05 p r o b a b i l i t y l e v e l . The Figurehead data, however, are significant at a probability level of 0.03 (t=-2.256, d.f.=38). It must be noted, however, that the Figurehead data for this comparison did not show homogeneity of variance. Transformation were - 19 -attempted, but proved unsuccessful. This makes this t-value of questionable significance. Variations on the standard resorcinol test indicated that the volume of the resorcinol reagent used was not c r i t i c a l . In all cases of decreased volume (to as low as 1.0 ml) the N and 2N thalli were clearly differentiated. Varying the concentration of HCl used did not give acceptable results. The 1/2 strength solution gave a very weak colour if left in the hot water bath for over 5 minutes, and the 1/4 strength solution showed no colour change at all. - 20 -FIGURE 2,1 Jrjjdaea. cordata expressed as % gametophytlc over time (mean of all size classes), June, 1986- November, 1981 (error bars represent standard error). - 21 -100 o a o E <a (D ' c o o <D CL 90 H 80 70 -I 60 50-40-30-20-10-Figurehead Brockton Point^'^P^f Sampling Date FIGURE 2.Z Reproductive populations of Iridaea cordata expressed as % gametophytic over time, from July, 1986- August, 1987 (error bars indicate standard e r r o r ) . - 23 -FIGURE 2.3 Iridaea cordata >20cm plants expressed as % g a m e t o p h y t i c over t i m e , J u n e , 1986- November, 1987 (error bars represent standard error). - 25 -9Z Percent Gametophytic _* 10 c a * oi co - » J co co o O O O O O O O O O O O I ' I I I L_ I I I I FIGURE 2.4 Iridaea cordata 5-15 cm plants, expressed as % gametophytic from June, 1986- December, 1987 (error bars represent standard error) - 27 -83 Percent Gametophytic _t 10 ca * cn o» co co o o o o o o o o o o o o I ' I I ' I I _1 I I I ^ W V W V V I FIGURE 2 .5 Populations of Iridaea cordata <5 cm plants, expressed as % gametophytic, from June, 1986- December, 1987 (error bars I n d i c a t e standard error). - 29 -100 80 70-60-60-40 30 H 20 10-.1 I "Jrfl| , , HPf f A. I Sampling Date Legend E3 BROCKTON POINT m FIQUREHEAO DISCUSSION Studies by other authors on Iridaea. and data reported here, show that either the N or 2N stage dominated the population. My study gives a more complete picture of two populations than previous studies because the data were obtained year-round and both reproductive and nonreproductive plants were sampled. As a result, new information was obtained, and some of the results presented by other authors can also be integrated with my data. Results of this study indicate that populations of Iridaea  cordata at Stanley Park are dominated by a different ploidy at different times of the year. In all but one case, gametophytes consistently dominate the summer months and tetrasporophytes dominate in winter. The single exception is the >20 cm size class at the Figurehead site in November 1986 where gametophytes dominate. This single point has an unaccountably high proportion of gametophytes and is interpreted as error, either in the f i e l d or laboratory. Since sampling was always done intertldally there were two periods each year when no sampling could be done because the tide was not low enough, February to April and October. It i s during these two periods that the populations appear to switch over from tetrasporophytic to gametophytic dominance in April-May and from gametophytic to tetrasporophytic dominance in October-November. - 31 -Iridaea may not only be changing in the ratio of diploids to haploids, it may also have a seasonal change in abundance in the I n t e r t i d a l community. Gelidium f for example, has a maximum cover in the spring and minimum cover in the winter (Jernakoff, 1985). It would be informative to establish some measure of r e l a t i v e abundance of the two p l o i d i e s to determine i f the populations follow seasonal abundance cycles. May (1986) found gametophytic dominance (83%) in a San Juan Island population of Iridaea cordata. Since her sampling was limited to summer months her results can only be applied to my summer data, where i t i s found to correspond well. If May had done winter sampling she might have found a winter tetrasporophytic dominance in her population. May's results and observations indicate that this consistent summer dominance of N plants was due to the presence of perennial holdfasts as well as differential spore and sporeling survival. May found 80% of the summer populations arising from perennial holdfasts. Hansen and Doyle (1976) found tetrasporophyte dominance of Iridaea cordata year round. Since they only examined reproductive plants, their result does not agree with the results of my study in which the N:2N ratio of my reproductive plants also changed seasonally. It is possible that Hansen and Doyle (1976) examined populations that were subject to high wave stress, which would likely result in a diploid dominated population, or the populations were recovering from some sort of a disturbance so the population structure would be a t y p i c a l . It i s also possible that populations in California do not - 32 -exhibit the same patterns in population structure that the British Columbia populations do due to different environmental, biotic or abiotic conditions. A summer collection from a Stanley Park site by Dyck et al. (1985) indicated that the population was 80% gametophytic. Elsewhere, they found large differences In the ratio of tetrasporophytes to gametophytes over a very small geographical distance and they proposed that the ratio is established at the spore level, possibly in conjunction with some physical factor or herbivory. These results agree with my findings, and the rational presented by Dyck et al. (1985) could apply to my study. My results also indicated a significant difference in the ratio of diploid to haploid plants in populations of Iridaea cordata between two sites situated close together (<lkm), although the overall seasonal pattern was maintained. Foster (1982) studied grazer effects on an Iridaea  flaccida population in northern California. The population he examined existed in a different habitat than the Stanley Park populations I considered because 1) the California populations a r i s e from substrata covered with barnacles, 2) Iridaea forms almost 100% cover in the California populations in the summer and 3) few algae other than Iridaea are in this area. By contrast the Stanley Park populations arise from bare rock with few associated barnacles, Iridaea grows in sparse clusters on the rock, and there are many other species in the area. Both the California and the Stanley Park populations have - 33 -reproductive blades year round, but the California population has a cycle of spring growth, late summer-fall reproduction and winter senescence. Over the winter small blade remnants were observed on perennial holdfasts. This idea of the populations arising each year from perennial holdfasts was also proposed by Hansen and Doyle (1976) for the central C a l i f o r n i a populations of Iridaea they considered. The applicability of this idea to the Stanley Park populations w i l l be considered l a t e r . In California, Foster (1982) found that invertebrates had l i t t l e e f f e c t on the reestablishment of Iridaea f l a c c i d a on cleared sites. In some Instances grazers have been known to either retard, by spore consumption, or enhance, by competitor removal, the reestablishment of algae (Lubchenco and Menge, 1978). Foster (1982) proposed that the presence of barnacles in the Iridaea zone was beneficial in some way because Iridaea commonly grows on barnacles and thus is not accessible to some grazers. Grazers do effect a decrease in the percent cover of Iridaea in the i n t e r t i d a l ; however, the magnitude of their effect on the unmanipulated population is unknown (Foster, 1982). The effect of herbivores on the s t r u c t u r i n g of Iridaea  cordata populations I examined was not considered in th i s study. Reproductive plants in the populations I studied, shown in Figure 2.2, follow a similar pattern of ploidy dominance as the non-reproductive plants. In an examination of nine species of red algae, studied on the Isle of Man, it was found that most - 34 -of the plants showed a peak reproductive period in the winter months (Sept.- Feb.) for the tetrasporophytes (Kain, 1982). This study by Kain also found that there were reproductive plants year round (5%), but during the winter season there was a dramatic increase(95%). This was also observed in Iridaea  cordata populations I studied except these populations have an added phase of gametophyte dominance. When considering changes of dominance in ploidy there is no evidence in my data of one si z e class having, for example, predominantly haploid plants, followed by the next larger size class having this characteristic in the next sampling period. One would expect that at the end of the summer the larger gametophytes would be senesclng and the smallest size class would be dominated by tetrasporophytes. There are indications of a si z e class being dominant seasonally, however. My data show that the smallest size class has a higher proportion of gametophytes, compared to the other size classes, in winter; at this time the population as a whole has tetrasporophyte dominance. One possibility is that these small plants grow to be the largest size class in the summer populations of gametophytes. These small winter plants could arise from either perennial crusts in the winter or from spores d i s t r i b u t e d by the winter reproductive tetrasporophytes; these germinated tetraspores may then take u n t i l summer to reach maturity as gametophytes. Further studies, such as measurements of absolute density, must be done to obtain a more complete picture of what is happening as a function of size - 35 -class. The data collected give comparative measures for specific size classes, indicating if individual classes are different relative to each other. Interpretation of the data collected on the different size classes is dependent on a series of assumptions. These assumptions are 1) the samples collected of one size class are representative of the entire population of that size class, 2) as the plants in one size class grow into the next higher class, those already in that size class will have grown into their next higher class, and 3) all size classes suffer mortality at the same rate. There was a statistical difference between the two sites in terms of overall composition of the populations. No reason for this difference has been discerned from this study. It i s possible that topographical variation accounts for the consistent difference I observed from Febuary 1986 to July, 1988. Topographical characteristics of the sites may result in a difference in wave action at the two sites. Wave action effects were not quantified in this study; however, Dyck et al. (1985) found an increase i n tetrasporophytic dominance with increased wave action which corresponds with the results of t h i s study. It i s also possible that once the ratios of recruitment are set up by some disturbance or stress the proportions continue to cycle themselves in very specific areas. The final - 36 -p o s s i b i l i t y that I considered was that the populations a r i s e from perennial crusts and i t i s these crusts which dictate these ongoing populations. Observations of f i e l d populations and observations of collected plants indicated that there i s rapid blade senescence following spore release. The gametophytic portions of populations in the 5-15 and the >20 cm size classes decreased in proportion to the rest of the population aft e r they dominated in the summer. The <5 cm size class drops off more slowly. It seems l i k e l y that i t i s these smaller plants that grow up into the winter populations of the gametophytes. I have established that populations of Iridaea switch over from d i p l o i d winter dominance to haploid summer dominance in Vancouver Harbour populations, but the question of what establishes these populations must be considered. Maggs and Guiry (1985) found some correlation between temperature and daylength and the type of spores which were formed and released by Schmitzia hiscockiana (Maggs and Guiry). It is possible that there is some environmental factor, such as temperature or daylength, which favours gametophytes in the summer and tetrasporophytes in the winter. Another possible mechanism which may determine N:2N ratios is apomeiosis, or reproduction without meiosis. In the case of Iridaea this would manifest itself in tetraspores released from the tetrasporophytes growing back into 2N, tetrasporophytic plants, rather than having undergone meiosis in the sporangia. This, was proposed as a mechanism for structuring Iridaea population by Hansen and Doyle (1976). Apomeiosis will be considered in more detail in Chapter 4. Thus, there are many possible explanations for the observed N:2N ratios of Iridaea populations including perennial holdfasts, spore settlement, apomeiosis, differential size class mortality, and grazer effects. Some of these factors will be considered in more detail In the following chapters. In this chapter I have established some general characteristlcs of two populations of Iridaea cordata. In subsequent chapters I will examine more specific characteristis of these populations. Specifically, I will examine the ratio of plants which s e t t l e out of the vater column onto cleared surfaces, and how closely these settling ratios reflect the population structure at the time of clearing. Also, I will consider the influence of apomeiosis on the populations, and the reproductive effort of the N and 2n life history stages. - 38 -CHAPTER 3 SETTLEMENT ON CLEARED SUBSTRATE  INTRODUCTION The objective of this part of my research is to investigate rates of settlement of Iridaea cordata onto cleared substrata and whether sites cleared at different times of the year have plants of different life history stages settling onto them. Two theories of how Iridaea populations are seasonally established are 1) populations arise seasonally from perennial crusts or 2) populations result from seasonal spore settlement. This chapter addresses the second theory. Spore settlement can greatly influence the structure of benthic algal populations. This is because spores can be the primary source of recruitment for such populations. Spores are subject to a number of biotic and abiotic factors, such as wave action, boundary layer phenomena, availability of substrata, sinking rates, and availability of gametes in the water column. The l a t t e r i s important since the absence of gametes would result in a lack of fertilization and ulimately nonproduction of spores. A factor which may act to dictate life history strategy by influencing rate of fertilization and spore production Is - 39 -c r i t i c a l gamete density (Gerrltson, 1980). The c r i t i c a l gamete density i s the minimum density necessary for sexual reproduction to occur. If the number of gametes available for sexual reproduction drops below this critical density then selection should favour asexual reproduction. In other words, when there are so few gametes of opposite mating types available that they may not meet and successfully reproduce, asexual reproduction is favoured. Thus the timing and occurrence of sexual reproduction may be determined by the optimal conditions for gamete survival. Since density may be low at the periphery of a population, and thus also the gamete density, peripheral populations may be more likely to show asexual reproduction while populations at the center of an organism's range are more likely to reproduce sexually. It is believed that asexual reproduction increases the chance of successful recruitment and the elimination of the problems of mating encounters (Gerrltson, 1980). Sexual reproduction results In greater genetic variability in the progeny thus allowing for greater potential for adaptation and evolution (Gerrltson, 1980). Since much of the subject of this chapter Is spores, It i s important that some background on spores and spore dispersal be discussed. In s i t u research on spores has been limited due to the difficulty of collecting and identifying spores in the water column. The settlement process, and germination, of most spores Is also not well known. Some progress In the study of - 40 -spore attachment and germination was made in 1972 when Neushul et a l . observed these phenomena using an underwater microscope. Settling behavior of spores of some algae was examined by Coon et al. (1972). They pointed out that interactions between spores and the water are c r i t i c a l in determining what settles and grows to form the benthic community. Microvideo was also u t i l i z e d by Okuda and Neushul (1981) as a technique for observing spore behavior. Using this technology a re l a t i o n s h i p between spore size and sinking rate was observed, in that large spores sink faster than smaller ones. In the case of Iridaea  cordata both tetraspores and carpospores are approximately the same size (20 micrometers) so would be expected to sink at equal rates. Algal spores are theoretically adapted for sinking, and some cells aggregate to facilitate sinking (Coon et al., 1972). Since the spores are adapted for sinking i t might be expected that the benthic community would be similar over time in species composition since the species present shed spores which sink to recolonize the immediate area. Hruby and Norton (1979) investigated the ability of algae to colonize rocky shores in Scotland. Using glass slides they found that those propagules which were most abundant in the water column were also most common on the slides . Patchiness was seen i n the settlement on the glass slides. Hruby and Norton (1979) attempted to relate diversity of colonizing species to abundance of algal propagules in the water, and to elucidate the e f f e c t s of location, level on the shore and the presence of overlying seaweed canopy on colonization. They - 41 -found that the primary factor which determined whether a species appears on the shore is the availability of propagules from the parent plants. Propagules of most littoral species are patchily distributed in seawater as a r e s u l t of the characteristics of the physical environment (Hruby and Norton, 1979). Propagules will be released into the water only when the parent plants are submerged. Water turbulence may often be sufficient to mix propagules into a fairly homogeneous suspension In the water overlying the shore, but this suspension may then be dispersed as a batch by water currents (Hruby and Norton,1979). The supply of propagules was not, however, the only factor which influences colonization for, regardless of the inoculum available, only those species normally found in the littoral fringe or at the top of the eulittoral zone colonized the slides placed at these levels. Differences in temperature, rainfall, or Insolation that occur from day to day may determine whether a propagule survives after settling. Both diversity and abundance of colonists were found to be higher under a canopy than out in the open (Hruby and Norton, 1979). The hypothesis that some seaweeds have spores which may spend relatively long periods of time d r i f t i n g i n the middle or upper layers of the water column, and therefore have correspondingly larger dispersal shadows, and that other seaweeds produce spores which remain close to the bottom and have small dispersal shadows has also been Investigated (Amsler - 42 -and Seaxles, 1980). The location of spozes in the water column reflects the dispersal potential of the taxa. They found spores of the Rhodophyta concentrated near the bottom, which they interpereted as in d i c a t i n g a small dispersal shadow. Spores collected in the upper and middle parts of the water column originate from taxa which can be considered ephemeral, fugitive species or facultative epiphytes. These opportunistic species are characterized by large dispersal shadows. Unequal distribution of spores in the water column is presumably a function of differences in spore density, shape and s i z e (Amsler and Searles, 1980). In a study to establish dispersal distances of viable spores Anderson and North (1965) transplanted Macrocystls into open areas so that juveniles growing up around the parent plants would indicate the dispersal distance. Average l i b e r a t i o n rates for spores was a minimum 500,000 spores per plant per hour, and the maximum distance juveniles were found from isolated single transplants was 5 meters. This indicates that though high numbers of spores are released, they have a limited dispersal distance. With a goal of developing a precise method for comparing algal spore size with sinking rates, Coon et al. (1972) examined settling behavior in red algal spores. They found that spores from a single plant, or from a single species, showed only slight variations in diameter. However, spores from different species showed a great deal of variation. Where - 43 -both carpospores and tetraspores of a single species were examined the carpospores were consistently larger. The density of the spores was always greater than the density of water and the size range of the spores was 17.3-96.5 micrometers. The size of planktonic organisms is thought to be important in influencing nutrient absorption, susceptibility to predation and time of suspension (Coon et al., 1972). Some of these factors may also apply to spores. Even taking into account wave action and boundary layer phenomena, both of which can Increase dispersal by moving nonmotile spores, the relative dispersal distance of red algal spores from the parent plants is small (Coon et al., 1972). The number of spores which potentially could be released and the number which have been actually observed to be released are guite d i f f e r e n t . In some red algae only 22% of the potential spores were in fact released (Boney, 1960). These observations were done in the laboratory, so in the natural environment higher or lower spore release may occur. Recolonization of denuded rocky intertidal areas has been considered by many authors (Bokenham and Stephenson, 1938; Northcraft, 1948; Fahey, 1953; Connell, 1972; Connell and Slatyer, 1977; Murray and Littler, 1978; Littler and Littler, 1980; Southgate et al., 1984). According to these authors the primary colonizers are usually green algae, though the primary colonizers are not the same for s i t e s cleared at d i f f e r e n t times of the year (Fahey, 1953). The f i r s t major colonizer was - 44 -found to be d i f f e r e n t in 3 successive years by Kain (1975) which i s guite d i f f e r e n t from Fahey's (1953) opinion that the f i r s t major colonizer should remain constant regardless of when the s i t e was cleared and Northcraft's (1948) results that the season of the year had l i t t l e effect on the regular sequence of algal settlement. Kain (1975) did find that the first colonizers were consistently green algae, but different species. Since red algae have non-motile spores their ability to disperse into new s i t e s may be l i m i t e d . Connell (1972) examined the issue of recolonization of denuded surfaces from the perspective of space, longevity, and growth rates of plants. He concluded that opportunistic, initial settlers were successful because of their ability to grow quickly and reproduce quickly. They occupy space and then die off. Such opportunistic species have many spores in the water column, have large dispersal shadows, and are thus able to. take advantage of available space. If later successional species with smaller dispersal shadows are able to colonize space they will do so and remain in that space longer than the shorter-lived, opportunistic ones. Succession on rocky i n t e r t i d a l systems has been c l a s s i f i e d as having three phases by Fernandez et al. (1980). These phases are: 1) Diatom film with macrophyte germlings - 45 -2) Dominated by algae of the Ulvacean group- during which time there is settling of macroalgae which drives the community to its typical composition 3) Normal structure Models of succession that have been proposed are usually patterned after t e r r e s t r i a l communities, not rocky i n t e r t i d a l ones. Since the algae are settling onto rock surfaces there is l i t t l e interaction below the surface of the substratum. The algae do not directly compete for nutrients, since they are in the water column, but do compete for space and sunlight. Of the three mechanisms, or models, proposed for succession by Connell and Slatyer (1977), two are more applicable than the third. The facilitation model indicates that early colonizers must somehow alter the habitat to make it more suitable for later successlonal species. The facilitation model is unlikely to apply to the intertidal zone since the rock surface cannot be altered. If shading is considered alteration of the habitat then possibly it is applicable to the intertidal situation. The tolerance model assumes differential abilities of species to u t i l i z e . r e s o u r c e s . I f e a r l y and late successlonal species have evolved different means of u t i l i z i n g resources i t i s possible that early successlonal species can take advantage of primary space with fast growth and frequent reproduction; however, once they start to die back the later successlonal species maintain space by virtue of longer life spans. The inhibition model, which deals with the issue of space, i s the - 46 -most likely model to apply to the Intertidal situation. This model argues that any colonizer will resist invasion of competitors, but when the initial colonizers die back and space becomes available the later successional species can u t i l i z e the space to reach maturity. This chapter considers settlement on cleared surfaces, with the purpose of determining factors which may be affecting the settlement of Iridaea cordata in the Intertidal zone, and establishing whether Iridaea populations arise annually from spores. - 47 -MATERIALS AND METHODS To examine what settles in the intertidal zone on cleared substrata rocks were selected at both study sites in Stanley Park (Vancouver, B.C.). Rocks were selected based on the following criteria: 1) some Iridaea cordata had to be present to ensure that Iridaea was capable of growing on them and 2) they had to be fairly large (at least large enough to not be easily displaced by wave, tide or human intervention). Once rocks were selected they were cleared by scraping with a paint scraper, burning with a blow torch, and then by scrubbing with a steel brush. The entire exposed surface of smaller rocks was cleared, and the remaining larger rocks were only partly cleared. Patches that were cleared were usually 0.5 m x 0.5 m. Various methods were t r i e d to mark the rocks for easy recognition, but most were unsuccessful. Among the methods t r i e d were: spray paint, flagging tape, quick dry epoxy with sand mixed in, bright plastic markers, wet/dry epoxy (Pro-line technical coating) with marker tags and, finally, with epoxy capable of setting underwater (Poxy Quick Sea Coin'). Five rocks were cleared at both Brockton Point and Figurehead in June 1986 (rocks I-V), November 1986 (VI-X) and January 1987(XI-XV). - 48 -The rocks were examined periodically (once per month when tides allowed) and the species growing on the rocks were noted and photographed. When Iridaea cordata appeared blades were sampled and the ploidy of the nonreproductive plants was determined by the resorcinol method. To establish what spores were present in the water, water column samples were collected whenever the rocks were cleared. Sampling was done by two methods. First the water was sampled by a 20 micrometer plankton net. Material collected by net was allowed to settle onto glass disks which were cultured in the laboratory. Though most of these cultures suffered premature death, some were quantified for the numbers of plants which grew in them. The second method involved collecting a jar full of water (2 L) and settling the contents onto glass slides or disks, then culturing whatever settled out. In both cases spores were grown in P.E.S. (McLachlan, 1973) using seawater c o l l e c t e d from the s i t e and f i l t e r e d through Whatman GF/C glass fibre and either Gelman or Sartorius .45 micrometer filters. Chambers were set at 14° C and 12/12 hour light/dark cycle. Cultures were observed weekly and whenever possible the small algae were i d e n t i f i e d . Hannach and Waaland (1986) found maximum photosynthetic rates in Iridaea cordata to occur at 150 micro Einsteins per meter sguared. My l i g h t was as close to this as I could duplicate using Sylvania/Lifeline (F48T12/CW) bulbs. - 49 -RESULTS Some problems vere encountered vhile carrying out this experiment. Marking the rocks proved to be difficult and hence some rocks vere not easy to relocate. However others, mostly at Brockton Point, vere easily found; the majority of rocks at the Figurehead s i t e were much more d i f f i c u l t to relocate. The Brockton Point rocks thus became the focus of t h i s study. Another, more general, problem vas that very little Iridaea cordata regrev on the study rocks during the two years of the experiment. However, some information was collected on the pattern of regrowth on cleared substrates. The Iridaea that did regrow were sampled and the data presented in this chapter. These data are very limited, and can only be interpreted as representative of the response of a larger sample size. Data that vere collected on regrovth of other species are given in Appendix 3. Observations of rocks over the course of the experiment indicated that there vas a pattern of settlement on cleared rocks. Rocks are covered with a film of diatoms after one month. These appear as a brovn slimy mat. Next the diatoms thinned out and Enteromorpha appeared two months a f t e r c l e a r i n g , followed the following month by Ulva. Ulva formed a dense cover over the cleared portion of the rocks, and this cover sometimes lasted for up to two years a f t e r clearing. - 50 -However, i n most cases Ulva thinned out approximately 12 months after clearing and other genera of algae appeared, such as filamentous reds, Fucus, Mastocarpus, and Odonthalia. Itemized r e s u l t s for each rock can be found in Appendix 3. Cultures grown from spores col l e c t e d by both 2 l i t r e samples and by plankton net did not give rise to any Iridaea  cordata within 5 months. There was l i t t l e evidence of any red algae in any of the cultures. Despite the addition of germanium dioxide (50 micrograms/litre) all of the cultures had extensive diatom, and also Enteromorphaf growth. For 10 cultures of net samples and 20 of the water cultures the number of Enteromorpha blades which grew were enumerated. Brockton Point net samples yielded a mean number of Enteromorpha per culture of 1.0 (S. E. +/- 0.3) the Figurehead samples had 3.9 (S. E. +/- 2.5). Water samples for Brockton Point had a mean number of 1.0 (S. E. +/- 0.18) and the Figurehead 14.7 (S. E. +/- 5.46); see also Table 3.1. All the cultures had a few additional species, but these were usually too small to be i d e n t i f i e d and were only classified by general appearance. Green algae were mostly Enteromorpha but other greens, e.g. Ulvaf were present. A few cultures had filamentous greens also. A number of the cultures, especially those that were based on collections in, November had small bladed brown algae that looked like small Laminariaf but could have been Nereocvstis. Red algae present - 51 -consisted of a few filamentous reds and some small red patches which appeared to be red algal spores which had divided but bad yet to produce any uprights; these red patches never developed further. Detailed l i s t i n g of what grew in the cultures can be found in Appendix 2. There was some growth of Iridaea on the rocks, as indicated in Table 3.2. When the f i r s t rocks (I-V) were cleared in June 1986, gametophytes dominated the surrounding Iridaea population so one might expect that the first Iridaea to arise would be predominantly diploid, originating from the diploid carpospores. Plants first became visible on these rocks, and were sampled, in July and August 1987 and were found to be 77 and 80% d i p l o i d , as expected. In November, 1987, 17 months after clearing Rocks I, II, III, and IV all had Iridaea growing on them and ranged from 25-80% d i p l o i d . Iridaea never grew on Rock V over the 25 months i t was monitored. On rocks cleared in November 1986 (Rocks VI-X) had Iridaea reappear in May 1987. Since these rocks were all cleared when diploid tetrasporophytes dominated (65%) the populations it was anticipated that the first plants to appear would be haploid. The plants which regrew were 74% haploid. • The next set of rocks was cleared in January, 1987 when the population was 46% gametophytic. Thus rocks XI-XV, which were cleared at this time would be expected to have - 52 -approximately equal settlement of both d i p l o i d and haploid spores. The first settlement which was sampled (May 1987) shoved an average of 37% gametophytic plants. Plants which settled onto Rock I were collected in July, 1988 when it was possible to make two collections. These two collections were classified by size. The rock had a number of large (>10 cm) blades which proved to be 46% gametophytic, and it also had many small (<5 cm) blades which were 82% gametophytic. A t-test (Zar, 1984) done on the November, 1987 data indicated that there was no significant difference between the rocks that were cleared In the summer vs. those cleared in the winter (p-.375, d.f.=7, t=.74). - 53 -TABLE 3.1 DENSITIES (»/cm*) OF ENTEROMORPHA IN  CULTURES OF FIELD COLLECTED SAMPLES All samples collected July 18, 1986 All cultures enumerated November 1, 1986 Culture coding: N= net samples L= 1 litre water samples BP= Brockton Point FH= Figurehead Culture Density Culture Density N BP 1.0 L BP 1.2 L BP 1.5 N BP 2.0 L BP 1.6 L BP 2.0 N BP . 7 L BP .6 L BP .3 N BP .2 L BP .8 L BP .6 N BP 1.2 L BP .9 L BP .5 Mean density Brockton Point Net samples= 1.0 (Standard Err or= 0.3} 1 litre samples= 1.0 (Standard Error= 0.18) N FH 1.7 L FH 4.2 L FH 19.6 N FH 1.9 L FH 5.0 L FH 5.8 N FH 13.9 L FH 42.0 L FH 49.9 N FH 1.6 L FH 4.3 L FH 3.9 N FH .3 L FH 7.2 L FH 5.5 Mean density Figurehead Net samples= 3.9 (Standard Err or= 2.5) 1 litre samples= 14.7 (Standard Error= 5.5) - 54 -TABLE 3.2 RESULTS OF RESORCINOL TESTING OF IRIDAEA CORDATA  SAMPLED FROM REGROWTH ON CLEARED SUBSTATA All data for rocks cleared at Brockton Point Data expressed as % gametophytic ROCKS I-V CLEARED IN JUNE 1986 ROCKS VI-X CLEARED IN NOVEMBER 1986 ROCKS XI-XV CLEARED IN JANUARY 1987 Pate Sampled Rock # 05-87 07-87 08-87 11-87 12-87 07-88 I 20% 64% II 75% 56% III 0% 60% 20% IV 23% 40% 36% 66% 0% IX 74% 63% 50% 100% 0% 40% X 50% XI 50% 39% 50% 44% 56% XII 23% 21% 29% 25% 25% XIII 42% 60% 50% 75% 58% XIV 50% 60% 40% XV 0% 0% 0% 0% - 55 -DISCUSSION There i s a successional sequence, or a differential settling rate on the rocks examined in this experiment. There i s also some correlation between the r a t i o of plants in the population at time of clearing and the ratio of the ploidy of the first plants which regrow on the cleared substratum. All of the settling patterns observed on the different rocks indicate that there i s an i n i t i a l settlement of diatoms, followed by a settlement of greens, s p e c i f i c a l l y Enteromorpha. There could be several explanations for this pattern. It is possible that it is necessary for the diatoms to settle and alter the conditions on the rocks to allow settlement or germination of different spores. It is also possible, and more l i k e l y , that diatoms are more common in the water column and can grow the f a s t e s t ; as a r e s u l t , diatoms outcompete any spores that settle initially but, over the long term green algae replace the diatoms. Green algae replace diatoms before any other species. This is likely due to the abundance of their spores in the water column. Based on the culture r e s u l t s the abundance of green algae growing on the rocks is not s u r p r i s i n g , since all cultures were almost completely dominated by green algae. This could indicate that chlorophyte spores predominate in the waters sampled or that the spores of these green algae germinate and - 56 -grow faster. In a study of Lithophvllum incrustans populations the number of spores produced per plant was large while the number of recruits was small, indicating that the greatest mortality occurs between spore release and achieving a viable size (Bdyvean and Ford, 1986). Extrapolating on their collected data they found a density of 549,920 conceptacles and 17.6 million spores produced annually per square meter for a Llthophyllum population, which resulted in the growth of 55 one-year-old plants per m2. This is a survival rate of 0.000003 % (Edyvean and Ford, 1986). The mat of Enteromorpha and Ulva that forms on the cleared patches on the experimental rocks is very obvious in the f i e l d . The surrounding undisturbed rocks have much sparser growth of any algae with few greens. These undisturbed rocks are primarily populated by red algae, with less than 50% total cover on each rock. Results of the clearing experiments indicated that it took less than one month for the cleared areas to be recolonized. Diatoms and ulvaceous green algae settled first, in some cases r e s u l t i n g in 100% cover; however, it took two years for cleared areas to appear to return to their preclearance state. Denuded quadrats In the high i n t e r t i d a l in C a l i f o r n i a recovered to t h e i r o r i g i n a l state within 1 month (Murray and L i t t l e r , 1978). The short recovery time of the California population, compared to my cleared areas, was attributed to the rapid recruitment and growth potential of the species present. Murray and - 57 -L i t t l e r (1978) felt their results supported the hypothesis that mid to high i n t e r t i d a l regions, subjected to frequent perturbations, were composed of early successional or opportunistic species. Even with a fast recovery time the regrowth on the cleared surfaces followed a si m i l a r successional sequence to that observed in my areas, with greens followed by browns and then reds, a l l in three months. Rates of succession observed on surfaces I cleared varied with the time of year that they were cleared. After approximately twelve months from the time of summer clearing the dense mat of green algae thinned out and other species could be seen (these data are itemized in Appendix 3). Rock cleared in the summer had no Iridaea cordata present for the f i r s t year i n d i c a t i n g that spores were either not in the water column or were not able to grow on the cleared and subsequently overgrown substrate. Winter-cleared rocks had new Iridaea r e c r u i t s within 4-6 months after clearing. It is possible that Iridaea spores were in the water column but did not grow because fast-growing green algae could outcompete or shade the red algal spores. When Iridaea cordata did appear It was generally only a few blades mixed with the s t i l l present greens. As Iridaea grew larger the amount of green algae on the rocks diminished. While none of my cleared areas was ever initially colonized by late successional species, late successional species appeared much faster on the winter cleared rocks than - 58 -on the summer-cleared ones. In Fahey and Doty's (1949) study summer-cleared transects were f i r s t colonized by Enteromorpha and other relatively short-lived forms, but, winter-cleared transects were f i r s t repopulated by later successional, longer-lived species. Thus, according to both my study and Fahey and Doty (1949), it is possible that in winter the number of late successional algal spores in the water column i s higher relative to the number of early successional spores. The N:2N ratios of Iridaea that settled on the cleared rocks suggest that there may be a correlation between the time of clearing and the ratio of Iridaea that first arises. Spores in the water column appear to be settling onto denuded rock surfaces and remaining viable for months before forming upright blades. In other species of algae, undeveloped gametophytes of many species of Laminar la are able to survive 80 days in the dark (Kain, 1969). Thus, it could be possible for Iridaea spores to l i v e in the shade of opportunistic species u n t i l the l a t t e r die back. It is unfortunate that so few Iridaea grew in the experimental plots since a greater number of plants would have provided a better idea of the N:2N ratio. The number of plants that appeared on the rocks was similar, visually, to the number of plants in the surrounding communities. There was no initially high number of Iridaea on the cleared surfaces that then as reduced to a constant number. It is also possible that an egual number of W and 2N spores settled on the cleared surfaces, but only the dominant - 59 -generation grev up. Hovever, v i t h the c o r r e l a t i o n between the r a t i o of the plants at the time of clearing and the ratio of the plants which arose, this seems unlikely. Knowledge of the numbers of spores released by the different life history stages at different times of the year vould help to evaluate this possibility. The close correlation of the ratio of 2N to N plants on the shore at the time of clearing and the ratio that ultimately arises seems to indicate that Iridaea can take advantage of recently cleared surfaces. However, the length of time that it takes for the plants to form visible uprights is not explained by this study. Hansen (1977) did an in. situ study on growth, maturation and senescence of Iridaea and found that the greatest growth and longest life span were exhibited by winter-spring initiated blades. This corresponds to newly settled tetraspores growing up into gametophytes in the Stanley Park populations, as occurred with the winter cleared rocks which exhibit the first growth of Iridaea of all cleared rocks. Regrowth of Iridaea flaccida on cleared substratum was also examined by Foster (1982). He cleared areas, then excluded grazers from some of these areas, and observed regrowth 54 days after clearing. Scrapings from these cleared areas contained green cells, diatoms, red filaments, and patches of red cells. Small blades of red algae grew up within - 60 -3 months of c l e a r i n g . This growth rate vas much faster than any of the tocks I cleared. The populations Foster was working with were somewhat different than those I examined (see chapter 2 discussion). Foster's Iridaea flaccida populations formed almost 100% cover with few other species present at the same time of year. It is possible that this different population structure dictates that Iridaea flaccida is the most common macroscopic algal spore in the water column; therefore, it faces less competition from other spores for space. This might account for the fast regrowth in Foster's population compared to mine, since Iridaea did not ever form 100% cover at my s i t e s , thus other spore types could be competing for substratum to s e t t l e on. Settlement on the cleared rocks appeared to follow a successlonal sequence. Settlement in intertidal areas has been studied by a number of authors, including Connell, (1985); Hoffman and Ugarte (1985); and Hruby and Norton, (1979). ConnelT (1985) investigated factors which determined structure of intertidal communities. Mortality of propagules which settle was Independent of their settlement densities. The culture work that was completed on spores col l e c t e d from the study s i t e s can be related to work in Scotland (Hruby and Norton, 1979). They found that the most common colonists on glass slides placed in the intertidal were the most abundant propagules in the water. Cultures made from water samples contained primarily green algae, just as the first colonists - 61 -in the cleared s i t e s were greens. The mat of Enteromorpha which formed a f t e r clearing served to prevent other opportunistic spores from settling while enabling spores which are already established to withstand harsh conditions (Hruby and Norton, 1979). Thus, these common early successional species may not have any d i r e c t interaction with the l a t e r successional species but Instead indirectly aid t h e i r success. Green algae which were the first to settle on the cleared substrate are considered opportunistic species which release high numbers of spores released (as spore collection and culture indicated), large dispersal shadows, and fast growth rates ( L i t t l e r and L i t t l e r , 1980). Iridaea spores, by contrast, did not appear in the spore collection and culture. This further supports the concept that Iridaea Is a l a t e r successional species, characterized by low numbers of spores released, a small dispersal shadow, and slower growth rate (Littler and Littler, 1980). A comparison of characteristics of opportunistic and late successional forms i s presented in a table by L i t t l e r and L i t t l e r (1980). The observations and results reported In this chapter cannot be directly related to the issue of whether spores or-perennial crusts establish the Iridaea populations since these two alternatives could not be differentiated by this investigation. - 62 -However, the res u l t s obtained do provide new Insights into the rate of resettlement of Iridaea after disturbances occurring in d i f f e r e n t seasons. To more directly examine the issue of what factors determine the ratios of Iridaea the occurence of apomeiosis will be considered in the next chapter. - 63 -CHAPTER 4 OCCURRENCE OF APOMEIOSIS IN IRIDAEA CORDATA INTRODUCTION In California, Hansen and Doyle (1976) found that Iridaea  cordata populations consisted predominantly of tetrasporophytes. Hansen and Doyle (1976) postulated three possible mechanisms to explain the ratios of mature tetrasporophytes and gametophytes of Iridaea cordata observed in the f i e l d . These were: 1) differential mortality at the spore level such that the carpospores were better able to survive than the tetraspores; 2) the tetrasporophytes are hardier plants than the gametophytes, so the tetrasporophytes were able to arise from perennial crusts and survive well in the conditions of that area; and 3) that apomeiosis is occurring and the tetrasporanglal mother cells do not undergo ^ meiosis but instead re-form 2N tetrasporophytes. This chapter will address the last hypothesis. If apomeiosis occurred In the majority of tetrasporangia, then the population would be continually dominated by diploid plants. Those tetraspores that did undergo normal meiosis would germinate into gametophytes and cycle according to a typical Polysiphonia-type life history. These events would - 64 -r e s u l t in a population dominated by diploid plants with a few haploid plants. Though apomeiosis is unlikely to be a causal factor in populations cycling between N and 2N plants it is worthwhile considering in populations which display diploid dominance. It is possible that diploid plants cycle apomeiotically and the haploid plants cycle by some other parthenogenic means. I only examined the possibility of apomeiosis in this study. Apomeiosis is nuclear division without a reduction in chromosome number. In the case of Iridaea cordata apomeiosis results in 2N plants (tetrasporophytes) arising from tetraspores instead of the usual N carpospores. In a life cycle unaffected by apomeiosis tetraspores arise from meiosis In the sporangium and give rise to IN gametophytes. Apomeiosis has been found to occur in a number of plants, both vascular and nonvascular. For example, the vascular plant Elvmus  r e c t i s e t u s (New Zealand wheatgrass) was found to undergo apomeiosis by Hair (1956). In cultured algae, apomeiosis has been postulated to occur frequently. For example West and Norris (1966) cultured Calllthamnlon sp. and found that tetraspores gave rise to tetrasporophytes, indicating the occurrence of apomeiosis. In culture studies of Rhodochorton  concrescens West (1970) found that successive generations were diploid and he was unable to culture haploid i n d i v i d u a l s . Although he was not able to obtain chromosome counts in the - 65 -cultured tissue, he f e l t that the non-sexual life history resulted from apomeiotic tetrasporophytes. In Dermatolithon lltorale (Sunsen) Hamel et Lemoine (Rhodophyta, Corallinaceae) bispores often replace tetraspores in the l i f e history. These bispores can be uninucleate or blnucleate, the occurrence of uninucleate bispores being attributed to apomeiosis (Sunsen, 1982). In a study of Padlna laponica Allender (1977) proposed that apomeiosis was responsible for cycling of diploid-dominated populations. He apparently did not actually test for apomeiosis as this was a hypothesis resulting from an ecological study of this species in Hawaiian populations. Apomeiosis has also been proposed to occur in recycling the diploid generation of Callithamnion corvmbosum (Whittick, 1978). Failure to undergo meiosis is not the only abnormality observed for tetraspores. In Gracllarla tetraspores have been observed undergoing meiosis but not undergoing cytokinesis. This results in large spores containing all four meiotic products and can result in plants arising with both male and female reproductive structures (van der Meer, 1977). Since this should not affect the population structure in terms of ploidy it will not be considered here. - 66 -The only reproductive structures found on Hildenbrandla are tetrasporangla. These tetrasporangla release tetraspores, which in culture give r i s e to 2N tetrasporangial t h a l l i (Fletcher, 1983). Apomeisis has also been postulated to occur in populations of L o m e n t a r i a orcadensis. In these populations game tanglal plants are unknown and chromosome studies indicate that i t i s apomeiosis that is forming the tetrasporangial plants (Foran and Guiry, 1983). Kim (1976), while investigating development of tetrasporangial sori and cystocarps, found that some tetrasporophytes produced spores that germinated back into tetrasporophytes in Iridaea cordata. No frequency of apomeiosis was reported by Kim (1976). Chromosome counts for Jr j daea cprdflta var. splendens were attempted, but no results were obtained. The most noteworthy study was by Magne (1986) who examined rates of apomeiosis in some members of the Rhodophyta. Antithamnionella sarniensis, a red alga with a Polysiphonla-like life history, was found to release tetraspores that give rise to tetrasporophytes (Magne, 1987). Different strains of this plant were examined and proportions of tetrasporophytes that arose from tetraspores ranged from 0-100% with many intermediate values; however, the percentage was believed to be constant within a strain (Magne 1987). - 67 -Magne (1987) further examined the phenomenon of apomeiosis as it is influenced by environmental factors. Results of this study indicated that day length and energy levels had no effect but that temperature may have had a slight effect, and he concluded that apomeiosis is a genetically controlled characteristic. This portion of my study was designed to establish whether apomeiosis is occurring in populations of Iridaea cordata in Vancouver, British Columbia. Apomeiosis will be considered from the perspective of whether i t occurs s u f f i c i e n t l y to be a significant factor in structuring the N/2N ratio of a population. If apomeiosis is occurring in these populations it will manifest itself in that spores released from tetrasporangla will germinate and grow into blades that are diploid, instead of haploid. These diploid blades would be expected to produce a negative response to the resorcinol test, while blades that resulted from normal meiosis would have a positive response. - 68 -MATERIALS AND METHODS Reproductive plants of Iridaea cordata were collected from the Figurehead study s i t e in September, 1986. Plants were considered to be reproductive i f they had obvious tetrasporangia or obvious carposporangia. Males were looked for but not found, therefore were not included in the reproductive category. A minimum of 10 plants of each type was collected, placed in plastic bags, and stored overnight in a r e f r i g e r a t o r to desiccate the plants s l i g h t l y . Excessive desiccation resulted in much reduced spore release upon remoistening. After r e f r i g e r a t i o n the plants were rinsed in a 10% bleach solution to clean off the blades, then rinsed in distilled water, cut into pieces and placed in 1/2 strength Provasoll »s Enriched Seawater Medium (McLachlan, 1973). Deep glass culture dishes were used with approximately 250 ml of media per dish. The seawater was coll e c t e d at the study s i t e s , f i l t e r e d through Whatman GF/C glass fiber filters 2-4 times, followed by filtration through a 0.45 micrometer Gelman or Sartorius filter. Cut pieces were removed the following day if spores had been released, otherwise the pieces were left for another day. Released spores showed up as small mounds easily visible in the culture dishes. Spores were initially grown in a chamber with a 8/16 light/dark regime and set at 10°C. When the spores showed no sign of germination the chamber was reset to a 12/12 light/ dark regime and 14°C. Under these conditions the spores - 69 -germinated and started to grow uprights. These plants were, however, all killed by a chamber malfunction 3 months later, so a new experiment was set up based on plants collected in December 1986. Cultures based on the December plants also died, again due to culture chamber difficulties. Finally plants were collected in Febuary 1981, and these spores grew successfully. It was, however, necessary to make a few adjustments to the c u l t u r i n g procedure. Since Iridaea often grows in areas of wave action the cultures were placed on a rocking platform to agitate the medium. This proved quite successful. The most successful method was a combination of a long daylight regime (16/8 light/dark at 15°C) and bubbling air into the cultures. In a l l instances the medium was changed every week, and germanium dioxide was (50 micrograms/1itre) also added weekly to reduce any diatom growth. Once the blades were large enough (2-4 mm length) they were harvested and subjected to the resorcinol test. Fresh blades were tested because air-dried young blades gave no results. Between 22 and 50 plants were harvested for the test. Critical to this study was the release of spores in the laboratory from collected thalli. A study of the conditions under which Iridaea would release spores was completed. This study Involved examining whether spores were released in - 70 -darkness. It had been reported that spore release was correlated with sunrise (Mumford et al., 1980) and therefore spores might not release without exposure to light. Plants were collected in November 1986 at 2:00 a.m.. All plants were wrapped in newspaper, placed in p l a s t i c bags and l e f t in the r e f r i g e r a t o r overnight. The plants were removed from the bags in darkness the following night. The plants were then cut into approximately 5x5 cm squares and half were placed in petri dishes covered with aluminum f o i l while the other plants were put i n uncovered dishes. Pieces of the same plants were placed in both light and dark dishes to remove individual v a r i a b i l i t y . All of the glass petri dishes were filled with P.B.S. using f i l t e r e d seawater collected from the s i t e . Both gametophytes and tetrasporophytes were used, and 40 cultures were set up. There were 10 tetrasporophytes In the dark and 10 in the light, and the same for the gametophytes. - 71 -RESULTS Some observations were made of the cultures while experimenting with spore release. Plants that were collected in the winter months showed abundant release of tetraspores but poor carpospore release while plants that were collected in the summer months released more carpospores. Tetraspores grew best at 15°C and at 16/8 light/dark regime. Carpospores were never cultured successfully. Any carpospores that were released for culture in the winter months died very quickly while grown under the same conditions as the tetraspores. Any carpospores released in summer months also died, but from other causes (culture chamber malfunction). Due to difficulties encountered with c u l t u r i n g there i s not a complete set of data on germling tetrasporophytes (grown from carpospores). Data that were collected were all based on cultures grown from tetraspores which should all have tested positive with the resorcinol te s t . The r e s u l t s can be seen in Table 5.1. In over half the cultures examined there was no evidence for apomeiosis. Light is not necessar ily the only trigger of spore release. All the cultures that were set up, whether maintained in complete darkness or in light, released spores when put in seawater afer a period of partial desiccation. - 72 -Carpospores were released In very low numbers from the gametophytes collected during the winter months. There was some release, indicating the plants were mature, but nowhere near the spore release observed in the summer months. Likewise the tetrasporophytes collected during the summer did not release as many spores as during the winter. In both the field and the laboratory a senescence pattern was observed. The l i f e history stage which is dominant in the intertidal degenerates quickly after releasing spores, while the less common type does not. For example, by late July and August, when gametophytes are dominant, the larger haploid (>20 cm) plants begin to break down and appear torn and full of holes. When collected and used for spore release In the laboratory these plants deteriorated rapidly and had to be discarded after one or two days. Tetrasporophyte plants of similar size collected at the same time continued to release spores and looked healthy for one to two weeks after collection if kept in a flow-through seawater tank. The opposite was true for plants collected in the winter. The spores that were released by the less common life history stage, e.g. tetraspores in late summer, did not grow well in culture. They guickly lost their colour and died. The spores that were released at the same time but by the dominant plant type did much better in culture and eventually formed uprights. - 73 -Table 4.1 RESULTS OF TESTING CULTURES OF TETRASPORES FOR APOMEIOSIS (H t e s t i n g + = number of plants i n d i c a t i n g meiosis occurred) (# tes t i n g - = number of plants i n d i c a t i n g no meiosis occurred) Culture tf testing + tf test i n g - % apomeiosis T-l 50 0 0 T-2 48 2 4 T-3 46 4 8 T-4 50 0 0 T-5 50 0 0 T-6 50 0 0 T-7 47 3 6 T-8 46 4 8 T-9 49 1 2 T-10 50 0 0 T-ll 50 0 0 T-12 49 1 2 Mean % apomeiosis =2.5 Standard Deviation =3.2 Standard Error = 0.9 - 74 -DISCUSSION The r e s u l t s of the apomeiosis experiment show that i t i s unlikely that apomeiosis is a major factor in establishing the ratio of d i p l o i d to haploid plants of Iridaea in the I n t e r t i d a l zone. If apomeiosis occurs in 2% of the cultured spores and to a similar degree in the wild populations, i t i s not s i g n i f i c a n t in determining N:2N ratios of the Iridaea populations. Hansen and Doyle (1976), the authors who proposed that apomeiosis was a determinant of the populations of Iridaea they examined, were working on populations which were primarily 2N. Thus, the occurrence of apomeiosis in their populations could account for the apparent cycling of 2N plants observed. Love and Connor (1982) working on New Zealand wheatgrass correlated the occurrence of apomeiosis with disturbance. In marine sites it is possible that some disturbance could result in the Iridaea population becoming apomeiotic for a period of time, but r e s u l t s from culture experiments indicate that l i t t l e , i f any, occurs. The observations on the apparent spore release of the different life history stages imply that the dominant plant type releases its spores in large numbers during its reproductive season and then dies. These spores divide and - 75 -grow i f a suitable substratum i s available and then grow up into the appropriate alternate life history stage since no strong evidence was found to support the occurrence of apomeiosis. Plants that were not dominant released their spores much more slowly and did not senesce as quickly. This might account for the continual presence of a low number of the nondominant form In the field instead of a complete dominance of the alternate life history form. For example, in the winter the tetrasporophytes are dominant and these d i p l o i d plants disperse haploid spores which grow up In the summer to form a population dominated by haploid gametophytes. Also in the winter a smaller proportion (30%) of the Iridaea population is composed of haploid gametophytes which slowly release d i p l o i d spores all season and It is these spores which grow up to form the summer population of tetrasporophytes. The observations of decreased spore release by gametophytes in the winter months can possibly be accounted for by the results of another experiment. Light Intensity has been found to affect the fertility and morphology of gametophytes in the Laminariales (Luning, 1980; Hsiao and Druehl, 1971). The different light qualities occurring at d i f f e r e n t times of the year may a f f e c t the f e r t i l i t y of Iridaea gametophytes in the field, resulting in the decreased spore release during the winter months. Cultures may not have done well due to overcrowding of spores. Vadas (1972) found that Nereocystis luetkeana grew at - 76 -maximum rates between 5-15°C and under 16/8 light/dark cycle. This i s s i m i l a r to what I found to be optimal for Iridaea gametophytes. Vadas (1972) also found that Nereocystis luetkeana growth rates were greatly reduced by increased plant density. The spores of Iridaea were released in static culture so the spores all settled into clumps beneath each sporangium. This resulted in high densities of the spores in each clump. In the f i e l d , although the spores are likely to be scattered by wave action and the densities reduced, they probably retain a patchy distribution. The growth of the spores in culture was extremely slow. Blades that were tested had been growing for 5 months and the largest were approximately 5 mm in length. In the field Iridaea populations change very quickly, and in semi-closed culture individual plants have been observed to increase by 9.5 % fw/day for larger blades (Waaland, 1978). This indicates that Iridaea does not do well in either static or small volume aerated cultures; it needs either large volumes of water with aeration or a flow-through system. The experiment performed to test whether spore release occurs in the darJc as well as in the l i g h t was informative, since no work has been done on isolating the factors which cause spore release i n Iridaea. Mumford et al. (1980) proposed that tetraspores and carpospores are released following the plants' exposure to light after a period of darkness. My results, however, indicated that exposure to l i g h t i s not - 77 -e s s e n t i a l t o the r e l e a s e o f e i t h e r c a r p o s p o r e s or t e t r a s p o r e s . R e s u l t s o f my e x p e r i m e n t i n d i c a t e t h a t r e v e t t i n g a f t e r a p e r i o d o f d e s i c c a t i o n i s an i m p o r t a n t f a c t o r i n t r i g g e r i n g spore release. It i s possible that: 1) the light may a c t as a t r i g g e r when the plants are not exposed to air during the tidal cycle, or ii) plants may only release spores during low tide cycles when they e x p e r i e n c e p e r i o d s o f d e s i c c a t i o n . T h i s does n o t , h o v e v e r , take into account s u b t i d a l p o p u l a t i o n s o f Iridaea. This chapter examined whether apomeiosis is significant in determining the N:2N ratio of Iridaea populations, and found it was not significant. The n e x t c h a p t e r v i l l a g a i n d e a l v i t h a f a c t o r t h a t may d i c t a t e the o b s e r v e d r a t i o s o f Iridaea over the course of a year. Using image analysis some morphological parameters of the two Isomorphic forms of Iridaea will be examined in the next chapter. Any differences between these two forms may be significant in e s t a b l i s h i n g a morphological or physiological reason for diploid plants dominating in the winter and haploid plants dominating in the summer. Since the morphological characteristics examined includes the number of reproductive structures per plant some indication of the potential number of spores being released by the two plant types can be e s t i m a t e d . - 78 -CHAPTER 5_ QUANTITATIVE CHARACTERISTICS OF  DIFFERENT REPRODUCTIVE STAGES OF IRIDAEA CORDATA INTRODUCTION In the previous four chapters different aspects of populations of Iridaea cordata have been considered. The structure of the population over time, the population structure for different size classes, the speed with which individuals resettle on cleared surfaces, and the occurrence of apomeiosis in the population have all been discussed in previous chapters. In this chapter I examine c h a r a c t e r i s t i c s of the plants themselves with the object of establishing possible causes for the results observed in previous chapters as well as considering the questions posed in this chapter. The purpose of these observations was to compare morphological characteristics of two Isomorphic forms of Iridaea cordata. By examining the density and total number of carposporangia and tetrasporangla on each life history phase one can ascertain whether these life history stages have identical physical characteristics and have the same apparent ability to produce spores. - 79 -Only limited vork has been done comparing c h a r a c t e r i s t i c s of isomorphic life history phases. Allender (1977) compared the two isomorphic forms of Padina laponica. He found that the sporophyte generation dominated all fringe reef populations at all seasons. No reference was made as to how he determined the phase of these plants. Allender concluded that the sporophyte dominance was primarily due to its greater resistance to water motion. The gametophytes grew faster than the sporophytes only at temperatures below ambient sea water temperature at the sites considered, and only with very high illumination (>75% incident sunlight). Also, the gametophytes grew faster than the sporophytes at all tested levels of water motion. When the gametophytes were artificially structurally weakened they became susceptible to destruction. Thus, Allenders study Indicated that there are physiological differences between the two life history stages of Padina laponica with the sporophyte being a hardier plant than the gametophyte. There also existed differences in the grovth rates of the sporophytes and gametophytes, and this difference varied v i t h physical conditions. In many populations of algae tetrasporophytes have been found to dominate the northern fringes of the geographical range. This is often attributed to the tetrasporophyte's ability to withstand some environmental condition that the gametophytes can not (Dixon, 1960; Edwards, 1973; Garbary, 1976; Allender, 1977; Gulry and Cunningham, 1984). This supports the contention that there are some physiological or - 80 -physical difference between the two plant types; however, no testing has yet been done on these plants to establish what the difference may be. A study was conducted on isomorphic forms of Iridaea  laminarloides and L- clllata in South America examining the ecological differences between the two forms (Hannach and Santelices, 1985). This study considered whether there are any ecological differences between the two isomorphic forms that could then be correlated to physiological or physical differences. Included in this study is a review of all reports of morphological differences between different life history stages of isomorphic species. The information available Is sparse and Inconclusive. Some of the features measured include relative size of the gametophyte, branching patterns, basal filament systems, spore diameter and chemical differences. It has been demonstrated in my research and in other papers (Pickmere et al., 1973; McCandles et al., 1975; Dyck et al., 1985) that differences exist in the biochemistry of the d i f f e r e n t l i f e history stages of Iridaea cordata and related species and genera. The difference In the type of carrageenan produced has f a c i l i t a t e d the i d e n t i f i c a t i o n of the r e l a t i v e number of gametophytes and tetrasporophytes In nonreproductlve populations. The alternate generations in heteromorphic plants have been studied more extensively. The theory that there i s some - 81 -advantage to a heteromozphic alternation of generations has been considered by L i t t l e r and L i t t l e r (1980), Lubchenco and Cubit (1980) and Dethiez (1981). Most hetezomozphtc plants alternate between a reduced or czustose form and a bladed or upright form. The reduced or crustose form has been found to dominate populations when herbivory rates are high, and thus may be advantageous in su r v i v a l of the species during periods of intense herbivory. In experiments conducted by Lubchenco and Cubit (1980) when hezblvozes were excluded fzom azeas dominated by the reduced form the bladed fozms azose and dominated. The reduced forms tend to be characterized by a tough thallus with a very slow growth rate. The bladed form by contzast has a much more rapid growth rate and i s chazactezized by a thinner thallus more susceptible to herbivory. By alt e r n a t i n g between these two forms the algae are able to take advantage of what would othezwlse be mutually exclusive chazactez1stics- a dense hezbivoze-zesistant thallus, and a fast-growing, fast-reproducing form. It i s possible that the isomorphic plants also have differences which make a l t e r n a t i n g between the two phases advantageous; howevez these differences may not be so obvious as in the hetezomozphic plants. In my study two categories of plants were collected, high and low i n t e r t i d a l . The high i n t e r t i d a l plants may be under more physical stress than the lower intertidal plants due to prolonged exposure to the atmospheze. For Iridaea cozdata, Quadiz et al. (1979) have shown that submerged plants are better able to carry on photosynthesis than emerged ones. - 82 -The gametophyte stage has a rough surface when reproductive as the carposporangla form wart-like structures In the thalli. The tetrasporophyte stage has less obvious reproductive structures since the tetrasporangla are smaller and not raised off the surface of the plant. The r e s u l t i n g contrast in density and colour of the reproductive structures against the rest of the thallus enables such structures to be examined using Kontron Image Analysis. The results of my analysis give rough estimates of the reproductive e f f o r t of the plants but, more s i g n i f i c a n t l y , they provide noteworthy comparisons of the tetrasporophytes over time, compare characteristics of tetrasporophytes and gametophytes, and examine varlability in physical characteristics at different tidal heights. - 83 -MATERIALS AND METHODS Reproductive Izidaea cozdata were c o l l e c t e d from s i t e s i n Stanley Pazk, Vancouver, B r i t i s h Columbia. Tetrasporophytes were c o l l e c t e d fzom both Bzockton P o i n t and the Figurehead i n January 1 9 8 7 , August 1 9 8 7 , and January 1988. Gametophytes were collected in August 1987 from the Figurehead. The January 1987 and August 1987 collections were sampled from two intertidal heights classified as low (approximately 0.2m) and high (approximately 0.7m) above chart datum. High contrast photographs of the plants were obtained using Kodak FX 5060 film. One photograph was taken of the complete thallus for use in calculating the surface area of the blade. A second photograph was a close-up of an area of the blade which was reproductive, and was used in calculating the density and surface area of r e p r o d u c t i v e s t r u c t u r e s . The photographs were analysed using the Kontron SEM-IPS image processing system. This system can be used to analyze input from still photographs or negatives u s i n g a video camera. The Kontron system is capable of enhancing the image, modifying it for analysis, and analyzing it, provided a difference exists in the shades of grey of the objects to be measured (e.g. tetrasporangia). - 84 -The photographs used were analyzed on the Kontron to obtain surface area of the entire blade, the number of reproductive structures per unit area and, in one case, the amount of surface area of the plant which was reproductive. In an attempt to further quantify the reproductive effort of the different reproductive stages of Iridaea cordata, the mounds of spores that were released in the laboratory were counted after spore release was induced (March 1987). Since the spores were not subjected to any water motion they were laid down In visible clumps on the bottom of the dishes. In the case of Iridaea the size of the tetraspores and the carpospores is the same (20 micrometers) so, as a first approximation, it was assumed that similarly sized clumps represented approximately the same number of spores. An estimate of the number of spores released per surface area of reproductive tissue was estimated by R.E. DeWreede, and by his permission, i s used to further examine the reproductive output of Iridaea cordata in this study. He estimated the spore output by setting up a series of spore release cultures. The reproductive plants were cut to fit in the base of a culture dish. The plants were left in plastic bags overnight before being added to filtered seawater In the culture dishes the next day. After the plants were removed the number of clumps of spores, number of spores In the clumps and number of loose spores were evaluated under the microscope. In addition, a count was made of the density of tetrasporangial "bumps". - 85 -RESULTS The t e t r a s p o r o p h y t e s had a s i g n i f i c a n t l y higher d e n s i t y of r e p r o d u c t i v e s t r u c t u r e s than the gametophytes (Table 5.2). The highest density for a tetrasporophyte was 151 (N=i6, S.E.= 16.7) reproductive s t r u c t u r e s per square centimeter while the hi g h e s t f o r the gametophytes was 42 (N= 10, S.E.= 3.5). However, in terms of size, the gametophytic blades were much l a r g e r than the t e t r a s p o r o p h y t e reaching a maximum mean of 605 cm* (N=10, S.E.= 76.1) for high intertidal plants in August 1987, whereas the tetrasporophytes maximum mean was 452 cm* (N=9, S.E.= 92.2) for low Intertidal plants at the same sampling period. The low intertidal tetrasporophytes grew larger d u r i n g the summer than i n the w i n t e r , although they are the dominant ploldy in the winter. No measurements were made on the haploid plants d u r i n g the winter months due to d i f f i c u l t i e s with the procedures. The d e n s i t y of the r e p r o d u c t i v e s t r u c t u r e s In the te t r a s p o r o p h y t e s was s i g n i f i c a n t l y higher (136 (S.E.= 5.8)-January 1987; 152 (S.E.= 16.7)- January 1988) in the winter months than in the summer (83 (S.E.= 11.7)- August, 1987) as seen In Table 5.3. No comparable measure was made for the gametophytes due to procedural difficulties. The total number of reproductive structures per plant was estimated by calculating surface area and density of - 86 -reproductive structures. The average number of reproductive structures per plant in the high intertidal vas statistically the same for both the tetrasporophytes and the gametophytes during t h e i r respective periods of dominance. That is, the gametophytes had a mean total per plant of 26,752 in August and the tetrasporophytes had a mean t o t a l per plant of 26,853 i n January. During the summer the average total number of reproductive structures increased to 36,367 for the tetrasporophytes even though the density of reproductive structures decreased for this time. Results of the Kontron analysis are shown in Table 5.1 and the raw data are in Appendix 4. The surface areas of the plants were higher in the summer than in the winter. The tetrasporophytes grew much larger in the summer than in the winter reaching a mean of 194 cm" in January 1987, and 151 cms January, 1988, while in the summer (August, 1987), the tetrasporophytes mean surface area vas 394 cm 2. By contrast the carposporophytlc gametophytes grew to 580 cm3 in the summer (August, 1987). S t a t i s t i c a l analysis of the tetrasporophyte summer vs. vinter and the tetrasporophyte vs. gametophyte summer data can he found in Tables 5.2 and 5.3. In August, 1987 samples were collected d i v i d i n g the two plant types into high and low i n t e r t i d a l . For the dominant haploid gametophytes no significant difference vas apparent between zones for the density of reproductive structures, the surface area or the total number of reproductive structures per - 87 -plant. Tetrasporophytes did exhibit some difference between the two tidal heights, though most differences were not statistically significant. The results are in Table 5.1. Lower intertidal plants had a larger surface area (451 cm2), a greater density of tetrasporangla (89/cm2), and thus more tetrasporangla per plant (36,367). Statistical analysis of the i n t e r t i d a l comparison i s given in Table 5.4 and 5.5. A preliminary investigation was completed on spore release from both tetrasporophytes and carposporophytes. The f i r s t study (March, 1987) indicated that more tetrasporangia per cm 2 released spores than carposporangla (see Table 5.6). This conclusion was based on observing the number of clusters of spores released by a specific area of thallus. However, a later experiment (September, 1987) indicated that both tetrasporangla and carposporangia released the same number of spore clusters per cm2 of thallus (see Table 5.7). This September study also considered the number of spores released, and the ratio of spores released in clusters to spores released singly. Carposporangia release 32% of their spores in clusters, whereas tetrasporangla released 7% in c l u s t e r s . Overall carposporangia released 18 times as many spores as tetrasporangia for a given area of thallus. When considered with the results from the Kontron analysis, it appears that when gametophytes dominate the population, carposporangia are releasing more spores per cm2 of thallus than are tetrasporangia. In the winter months, while - 88 -the 2N plants dominate the population, they are releasing more spores per cm* than the N plants. - 89 -TABLE 5,1 DENSITY OF REPRODUCTIVE STRUCTURES AND SURFACE AREA  OF IRIDAEA CORDATA COLLECTED AT BROCKTON POINT H.I.= High Intertidal L.I.= Low Intertidal (Standard Error in Brackets) Mean Density Qf Mean Surface Area Reproductive Structures of Blade (#/cm*) (cm3) TQta.X R e p r o d u c t i v e S t r u c t u r e s ( # / b l a d e ) January, 1987 Tetrasporophyte N=37 H.I.- 148 (6) N=35 L.I.- 121 (5) 169 (22) 220 (29) 26,184 (3,829) 26,853 (3,974) Total- 136 (6) 194 (19) 26,598 (2,756) August, 1987 Tetrasporophyte N=2 H.I- 59 (4) N=9 L.I.- 89 (14) 164 (61) 452 (92) 9223 (2,970) 36,367 (6,305) Total- 83 (12) 394 (83) 30,938 (6,130) CarpQSPQXQPhytes N=10 H.I.- 42 (4) 605 (76) 26,753 (4,512) N=10 L.I.- 40 (3) 553 (55) 21,836 (2,995) Total- 41 (3) 580 (49) 24,190 (2,803) January, 1988 Tetrasporophyte N=16 151.6 (16.7) - 90 -TABLE 5.2 Results of s t a t i s t i c a l analysis (t-tests) for the data on carposporophytes vs. tetrasporophytes for August, 1987. (TRS= total number of reproductive structures per plant) (Density refers to density of carposporangia and tetrasporangia) Parameter D.F. T - y a l u e p - v a l u e s i g n i f i c a n c e Surface Area 27 1.987 .025<p<.05 YES Density 28 -4.337 p<.0005 YES TRS 27 -1.105 .Kp<.375 NO - 91 -Results of statistical analysis (t-tests) for the data on tetrasporophytes for January, 1987 vs. August, 1987. Density and TRS as in Table 5.3 Parameter D.F. T - v a l u e P - v a l u e Significance Surface Area 45 -3.667 p<.005 YES Density 81 3.378 .0005<p<.005 YES TRS 43 -.589 . K p < . 3 7 5 No TABLE 5.4 Results of statistical analysis (t-tests) for the data on tetrasporophytes for high vs. low intertidal plants in January, 1987. Density and TRS as in Table 5.3 Parameter D,F, T-yaJue P-value Significance Surface Area 70 1.479 .05<p<l NO Density 70 -2.259 .01<p<.025 YES TRS 70 -0.104 p>.4 NO - 93 -TABLE 5.5 Results of statistical analysis (t-tests) for the data on carposporophytes for high vs. lov intertidal plants in August, 1987. Density and TRS as in Table 5.3 Pa rame te r P . F , T - v a l u e P-value Significance Surface Area 17 .503 .Kp<.375 NO Density 17 .670 . K p < . 3 7 5 NO TRS 17 .932 .Kp<.375 NO - 94 -Table 5.6 To compare the reproductive output of carposporangia and tetrasporangia, the following are counts of the numbers of clusters of spores released per cm* of thallus (March, 1987). TETRASPORE CLUSTERS Culture D e n s i t y (#/cm*) T-A 1.6 T-B 0.5 T-C 1.2 T-D 1. 7 T-E 1.0 T-F 1.2 T-AA 2.8 T-BB 0.5 T-CC 1.2 T-DD 1.0 T-EE 2.1 T-FF 0.5 CARPOSPORE CLUSTERS Culture D e n s i t y (»/cms) C-A 0.02 C-B 0.09 C-C 0.02 C-D 0.05 C-E 0.74 C-F 0.23 C-AA 0.02 C-BB 0 C-CC 0.11 C-DD 0.15 C-EE 0.05 C-FF 0.05 Tetraspore (T) mean = 1.65 Standard Deviation = 1.18 Standard Error - 0.34 (N=12) Carpospore (C) mean = 0.54 Standard Deviation = 1.36 Standard Error = 0.39 (N=12) - 95 -TABLE 5.7 Results of spore counts for carpospore and tetraspore release i n September, 1987 Tetraspores Carpospores Mean # spore clus t e r s per cm* Mean # spores per cluster Mean # loose spores per cms Mean # spores in clusters cm~ s 1.1 235 3644 267 1.3 334 23866 11105 Spores released in clusters 7% 32% Mean # spores released cm~a of thallus 3909 46075 - 96 -DISCUSSION The most unexpected result to arise from this study was the variation in the density of reproductive structures over the course of one year. The tetrasporophytes, which are the dominant ploidy in the winter months, have a significantly higher density of reproductive structures in the winter (144/cms) than the summer (83/cm3). Unfortunately due to logistical difficulties there could be no such comparison of the carposporophytes. The reason for this difference in the tetrasporophytes could be related to seasonal growth rates since during the summer the number of reproductive structures per unit area decreases but the surface area of the plants increases, so the net number of reproductive structures i s s t a t i s t i c a l l y the same in the summer (30,983/plant) and winter (26,589/plant). The surface area of the plants changes significantly from summer to winter, with the winter plants having less surface area than the summer ones. The results of this Investigation indicated that physical differences related to reproduction do exist between the diploid and haploid plants of the isomorphic alga Iridaea  cordata and within a single life history stage for different times of the year. From the l i m i t e d information available on comparisons of isomorphic forms of algae it is evident that there Is not always a discernable difference between the - 97 -plants. For example, Littler et al. (1987) Indicated that for the red alga Polycavernosa debilis (Forsskal) Frederlcq and J. Norris, there was no significant difference when considering the isomorphic life history phases from a physiological and ecological perspective. Littler et al. measured net photosynthesis, calorific content, structural makeup, and resistance to predation. They felt that the adaptive differences between the life history stages are either very subtle or are genetically controlled. Hannach and Santelices (1985) also found a seasonal change in the size of Irjd^ea^ laminarloldes and L- clliata. In both species there was a decrease in the size of the tetrasporophytes in the southern hemisphere winter. Their study indicated that cystocarpic plants were significantly larger in the summer than in the winter. These results correspond with mine in that my results Indicate that tetrasporophytes are larger in the summer than the winter and gametophytes are larger than tetrasporophytes in the summer. It is possible that the density of sporangia Increases when the surface area of the plant decreases, maintaining a constant number of sporangia per plant. Winter water Is much colder (9°C) than summer (15°C) and winters have much shorter days and less sun. This would all Intuitively imply that the plants are less productive due to reduced photosynthesis and slower metabolism in winter; thus the surface area of the - 98 -plants is decreased and the reproductive structures form closer together. The density of carposporangial structures of the gametophyte during its dominant time of the year is much lower (40/cms) than the tetrasporangial structures (144/cms) for the tetrasporophytes' dominant time of the year. However, due to the larger surface area of the gametophytes the two ploidies have the same net number of reproductive structures per plant (26,000/plant). Despite having the same number of reproductive structures per plant the two stages do not necessarily release the same number of spores per sporangium. These plants may allot the same effort to reproduction based on the numbers of reproductive structures, but the actual success of the plants is dependent on the number of spores which actually release, settle, germinate and form a new plant. My results indicate that gametophytes are consistantly larger than tetrasporophytes. This is contrary to results presented by West (1968) and Bold and Wynne (1978) both of whom found that gametophytes were smaller than tetrasporophytes for other species of algae. West (1968) examined Acrochaetlum  pectinatum (Rhodophyta) and Bold and Wynne (1978) reported on Sphflcelflrifl furcigera (Phaeophyta). Though some figures for spore release are presented in this chapter they represent a far from complete picture of the seasonal spore release of isomorphic algae. In order to - 99 -achieve a complete overview of this phenomenon it would be necessary to sample at different times of the year and at di f f e r e n t times of the day for both ploidies. Gelidium  robustum was i d e n t i f i e d as having a maximum spore output in August and September with a gradual decrease in total spore output u n t i l January and February (Guzman et a l . , 1972). Carpospozlc plants were also found to release a consistently higher number of spores than tetrasporic plants in this same study. Carpospore output from red algae has been estimated to range from 2 (Polvsiphonia lanosa) to 760 (Antithamnion  plumula) per gram fresh weight (Boney, 1960). Diurnal p e r i o d i c i t y of spore shedding has also been observed in red algae. Gelidium shed a maximum number of spores during the night, while Gelidiopsis peaked during the day (Umamaheswara and Kallaperumal, 1987). Mlcrograzers also play a significant role in releasing spores from Iridaea (Buschmann and Santelices, 1987). Mlcrograzers were found to feed preferentially on reproductive tissues, and in doing so released large numbers of spores. Thus, sampling spore release at one time of the year, or one time of the day, and without considering the impact of micrograzers, is not representative of the entire pattern of spore output. The results of the spore release experiment are only preliminary; however, they do indicate a possible relationship between spore output of each plant type, and time of the year. Recalling that tetrasporophytes dominate winter populations and gametophytes dominate In summer, the data collected suggest - 100 -that the dominant plant type for a given time of year releases more spores than the alternate form. This may r e s u l t from a more e f f i c i e n t use of available resources by the 2N plants in the winter and N plants in the summer, resulting in higher production of spores. The data on spore release also show that the number of sporangia i s not in d i c a t i v e of the number of spores being released since the tetrasporophytes maintain the same number of sporangia year round. Hannach and Santelices (1985) examined differential susceptibility of the different life history stages of Iridaea to herbivory. They found that susceptibility to grazing depends on frond s i z e and the species of grazer. In the case of Iridaea laminariodes. grazed by Collisella ceciliana, gametophytes were consumed at a significantly higher rate than sporophytes; however, no difference was noted for larger herbivores. Differential susceptibility to herbivory could be a difference between the gametophytes and the tetrasporophytes in the Stanley Park populations, but, I did not investigate this issue. A limited examination was done of the difference between the high and low intertidal plants. The r e s u l t s indicated that there was no statistically significant difference between the high and low i n t e r t i d a l gametophytes in terms of density of carposporophytes, surface area and total number of reproductive structures per plant. - 101 -There was a cons istantly higher surface area for the lower i n t e r t i d a l plants in the case of the tetrasporophytes, though it did not prove to be s t a t i s t i c a l l y s i g n i f i c a n t . This could be because the d i p l o i d plants are less r e s i s t a n t to desiccation than the haploid plants, so the diploids do not grow as large as the haplolds under warm summer conditions. The higher i n t e r t i d a l plants are more affected than the lower ones by this lack of resistance to warm weather and desiccation or do not have as much time in the water to photosynthesize, thus they do not grow as large as the lower intertidal ones. Higher intertidal plants suffer more desiccation stress than the lower plants (Taylor and Hay, 1984). The density of tetrasporangia on the tetrasporophytes is significantly different between high and low i n t e r t i d a l plants. It must be noted at this point that the high intertidal tetrasporophytes that were collected were difficult to identify, and upon checking with resorcinol testing, eight out of the ten of the plants sampled proved to be gametophytic. Thus the comparison of high vs. low intertidal for the 2N plants must only be interpereted as indicative of what may be occurring. It can be concluded from my work that there are some physical differences between gametophytes and tetrasporophytes of Iridaea cordata. These physical differences may be indicative of basic physical and physiological differences between the two life history stages which result in one life history stage being better adapted to a set of environmental conditions. If th i s i s true then i t might account for the - 102 -seasonal switch-over in dominant ploidy of Iridaea populations. Tetrasporophytic plants grow larger in the summer than in the winter, even though they dominate the populations in winter, indicating better growth conditions in the summer. - 103 -CHAPTER 6 I GENERAL DISCUSSION The data I have presented bear on one aspect of algal population structure, the ratio of haploid to diploid plants of Iridaea cordata. The first hypothesis considered was: d i p l o i d and haploid plants are expected to occur in equal proportions in a given population. This hypothesis vas rejected. The populations vere found to alternate seasonally between diploid and haploid dominance. The rest of my study was an effort to isolate some factors which determine this ratio. The second hypothesis considered the populations in more detail by examining population structure at the level of different size classes; summarily: d i f f e r e n t s i z e classes are expected to have different proportions of d i p l o i d and haploid plants than the population as a whole, if the population is not equally composed of N and 2N plants. The examination of the individual s i z e classes did not provide any obvious evidence for the progression of a dominance in ploidy from one size class to the next; as a r e s u l t the second hypothesis was rejected. It i s possible that the plants of d i f f e r e n t s i z e classes are established by d i f f e r e n t grovth rates, and not by d i f f e r e n t spore i n o c u l i or d i f f e r e n t times of i n i t i a t i o n from perennial crusts. If this were the case it would be expected - 104 -that all size classes would have the same d i p l o i d to haploid r a t i o for any given time. Size class analysis established that the patterns of dominance observed in the populations as a whole existed in a l l siz e classes. It is not a progression from one size class to the next. The factor, or factors, which establish the population structure affect the plants of all size classes. The next topic considered was the plants' characteristics when settling onto cleared substrata. Specifically, the t h i r d hypothesis was: spore settlement onto cleared surfaces reflects the ratio of diploid to haploid plants in the population at the time of c l e a r i n g . This hypothesis was accepted. The rock clearing experiment was intended to establish whether the ratio of plants settling on the cleared substrate Is a reflection of the ratio of Iridaea in the whole population at the time of c l e a r i n g . It was found in most cases that the ratio of the plants which regrew on the cleared rocks was very close to the ratio of plants in the population at the time of cle a r i n g . This ratio was often d i f f e r e n t from the r a t i o of the population at the various sampling times. Iridaea which grew on the rocks cleared in summer did not form any v i s i b l e uprights u n t i l 11 months after clearing. Winter-cleared rocks had a reappearance of Iridaea 4-6 months after clearing. In all instances the correlation between the plants which reappeared and the population ratios at the time of clearing - 105 -indicate that the spores settled on the rocks shortly after clearing and remained viable for months after settlement before differentiating into recognizable blades. Rocks in the field may have spores settling annually and arising immediately because the surface of the rock will already be receptive to spores, having not been as completely cleared as in these experiments. The contrasting argument is that Iridaea which arose did so only because the space was cleared and normally this space would not have been available for settlement. My study did not examine this argument. In the areas of Stanley Park where Iridaea cordata grew there appears to be much open space available. Since space remains open it is possible that space is not the limiting factor in this population. There could also be a microscopic population of bacteria, diatoms, and other protists, which interferes with spore settlement. If this were the case it is likely that the populations are arising from perennial crusts, not from annual spore recruitment. Since Iridaea populations are dominated by diploid plants for a portion of the year, and apomeiosis has been proposed to influence population structure of Iridaea cordata (Hansen and Doyle, 1976), the t h i r d hypothesis was: apomeiosis occurs in populations of Iridaea cordata exhibiting diploid dominance. This hypothesis was rejected. - 106 -Results of the apomeiosis exper iment indicated that apomeiosis is not a significant factor in structuring the populations. Indications of apomeiosis were found in 2% of the plants cultured. This showed that, as for many other algae (reported in Chapter 4), apomeiosis probably occurs in Iridaea  cordata but it can be discounted as a significant influence on the structuring of Iridaea populations in the study s i t e s examined. Since the populations observed were consistent in their alternation of diploid and haploid phase over the period they were sampled, with the proportions of d i p l o i d and haploid plants remaining constant, it does not appear that one ploldy has a consistent advantage over the other. It is possible that the diploid phase has some advantage over the haploid phase in the winter months and that the opposite is true for the summer. The final hypothesis deals with physical differences between the two phases. The final hypothesis is that: isomorphic plants exhibit no difference in the number of reproductive structures per plant, surface area, or density of reproductive structures. This hypothesis was rejected. The examination of Iridaea by Kontron Image Analysis revealed that both diploid and haploid plants have the same net number of sporangia on their surfaces. In September, however, the gametophytes, which are the dominant plants at this time, released 46,075 spores per cmz and the tetrasporophytes - 107 -released 3,909 spores per cm* of thallus. Thus, the dominant plant type may consistently be releasing more spores than the nondominant type. Therefore, the number of reproductive structures is not representative of the number of spores released. A more detailed examination of the number of spores released by individual sporangia, and of the number of sporangia per unit area, i s needed before any d e f i n i t i v e statement can be made about the number of spores which are released. A comparison of winter and summer i s also necessary. According to observations made from cultures, the spores of the dominant ploidy of plants do much better in culture than the spores of the alternate ploidy. Thus, it is possible that in the field spores from the dominant phase survive better. As discussed in the Introduction, many authors have examined Iridaea populations. Their studies examined populations at specific times of the year (Dyck et al., 1985; May, 1986), or considered only reproductive plants (Hansen and Doyle, 1916; Adams, 1979). By contrast, my study presents a more complete view of two Iridaea cordata populations. This was achieved by sampling year round, sampling different size classes and sampling reproductive and nonreproducti.ve plants. Some factors which may be a t t r i b u t i n g to seasonal population structure were also investigated as were some physical and reproductive characteristics of Iridaea cordata. - 108 -The observations made and data col l e c t e d on these populations of Iridaea cordata cannot be assumed to hold true for all populations. The phenomenon of seasonal alternation of gametophytes and tetrasporophytes in an isomorphic red alga has been documented here for Iridaea cordata. This alternation may occur in other bladed isomorphic algae but, without long term and detailed population study, i t remains conjecture. This study has served as a stepping stone in understanding one aspect of the population structure of red algae, s p e c i f i c a l l y , the seasonal variation in haploid and d i p l o i d l i f e history phases. - 109 -REFERENCES ABBOTT, I. A., 1971. On the species Iridaea (Rhodophyta) from the Pacific coast of North America. Syesis 4:51-72 ABBOTT, I.A., 1972. 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Prentice-Hall, Inc., Englewood Cliffs, N.J. pp 150-152. - 118 -APPENDIX #1 -119-APPENDIX #1 RAW DATA FROM RESORCINOL TESTING  BROCKTON POINT AND THE FIGUREHEAD  IRIDAEA CORDATA POPULATIONS (Numbers in brackets indicate standard error) Rep= Reproductive (not included in calculation "All") BROCKTON POINT Transect % gametophytic JUNE, 1986 <5cm 5-15 >20 All 51 77 77 6 8 ( 7 ) FIGUREHEAD Transect <5cm 5-15 >20 All % gametophytic 72 59 45 59(6) <5cm 5-15 >20 All 61 50 56 55(2) <5cm 5-15 >20 All 54 62 77 64(5) <5cm 5-15 >20 All 66 60 61 62(1) <5cm 5-15 >20 All 55 78 81 71(6) <5cm 5-15 >20 All 80 42 22 48(14) <5cm 5-15 >20 All 69 69 80 73(3) July 1986 <5cm 5-15 >20 All Rep <5cm 5-15 >20 All 51 59 73 61(5) 100 65 62 59 62(1) <5cm 5-15 >20 All Rep <5cm 5-15 >20 All 6 7 70 93 77(7) 81 77 94 93 88(5) - 120 -Rep 3 <5cm 5-15 >20 All Rep 4 <5cm 5-15 >20 All Rep August, 1986 1 <5cm 5-15 >20 All Rep 2 <5cm 5-15 >20 All Rep 3 <5cm 5-15 >20 All Rep 4 <5cm 5-15 >20 All Rep November, 1986 1 <5cm 5-15 >20 All Rep 2 <5cm 5-15 >20 All Rep 88 Rep 88 63 3 <5cm 68 59 5-15 54 43 >20 50 55(5) All 57(5) 74 Rep 94 70 4 <5cm 49 77 5-15 41 67 >20 71 71(3) All 54(7) 68 Rep 89 65 53 57 58(3) 66 <5cm 5-15 >20 All 77 73 100 83(7) 64 64 74 67(3) 62 61 63 48 57(4) 71 <5cm 5-15 >20 All Rep <5cm 5-15 >20 All 77 52 95 74(10) 91 77 83 95 85(4) 83 68 54 68(7) 82 <5cm 5-15 >20 All Rep 77 74 77 76(1) 84 44 1 <5cm 82 40 5-15 33 26 >20 83 37(4) All 66(14) 39 Rep 65 62 2 <5cm 62 43 5-15 27 14 >20 75 40(11) All 55(12) 24 Rep 32 - 121 -3 <5cm 60 3 <5cm 70 5-15 32 5-15 76 >20 25 >20 67 All 39(9) All 71(2) Rep 41 Rep 41 4 <5cm 59 4 <5cm • 73 5-15 25 5-15 44 >20 23 >20 74 All 36(10) All 64(8) Rep 33 Rep 67 December, 1986 1 <5cm 37 1 <5cm 38 5-15 51 5-15 38 >20 32 >20 19 All 40(5) All 32(5) Rep 21 Rep 43 2 <5cm 24 2 <5cm 42 5-15 40 5-15 27 >20 54 >20 29 All 39(7) All 33(4) Rep 27 Rep 33 3 <5cm 21 3 <5cm 46 5-15 60 5-15 35 >20 48(9) >20 30 All 43 All 37(4) Rep 20 Rep 40 4 <5cm 11 4 <5cm 40 5-15 63 5-15 29 >20 62 >20 53 All 45(14) All 41(6) Rep 23 Rep 33 January, 1987 1 <5cm 51 1 <5cm 32 5-15 47 5-15 46 >20 27 >20 33 All 42(6) All 38(4) Rep 30 Rep 33 2 <5cm 56 2 <5cm 74 5-15 69 5-15 27 >20 36 >20 34 All 53(8) All 45(12) Rep 17 Rep 24 3 <5cm 69 3 <5cm 62 5-15 64 5-15 19 >20 20 >20 36 - 122 -A l l Rep 4 <5cm 5-15 >20 All Rep May, 198 7 1 <5cm 5-15 >20 All 2 <5cm 5-15 >20 All 3 <5cm 5-15 >20 All 4 <5cm 5-15 >20 All June, 1987 1 <5cm 5-15 >20 All 2 <5cm 5-15 >20 All 3 <5cm 5-15 >20 All 4 <5cm 5-15 >20 All 50(13) 38 52 43 22 39(7) 25 75 55 83 71(7) 87 36 83 68(13) 57 52 85 65(8) 50 70 79 66(7) 53 46 81 60(9) 80 41 63 61(9) 56 55 57 56(1) 52 47 63 54(4) All Rep <5cm 5-15 >20 All Rep <5cm 5-15 >20 All <5cm 5-15 >20 All <5cm 5-15 >20 All <5cm 5-15 >20 All <5cm 5-15 >20 All <5cm 5-15 >20 All <5cm 5-15 >20 All <5cm 5-15 >20 All 39(10) 28 52 36 28 39(6) 28 51 70 94 71 71 58 95 75(9) 88 84 65 78 95 79(7) 61 54 100 72(12) 59 64 93 72(9) 67 43 90 67(11) July, 1987 - 123 -1 <5cm 64 5-15 35 >20 71 All 57(9) Rep 95 2 <5cm 50 5-15 44 >20 52 All 49(2) Rep 79 3 <5cm 24 5-15 40 >20 82 All 48(14) Rep 82 4 <5cm 53 5-15 46 >20 42 All 47(3) Rep 64 August, 1987 1 <5cm 52 5-15 70 >20 39 All 54(7) Rep 94 2 <5cm 73 5-15 60 >20 14 All 49(15) Rep 60 3 <5cm 68 5-15 62 >20 16 All 48(13) Rep 61 4 <5cm 63 5-15 73 >20 29 All 55(11) Rep 70 September, 1987 <5cm 5-15 >20 All Rep <5cm 5-15 >20 All Rep <5cm 5-15 >20 All Rep <5cm 5-15 >20 All Rep <5cm 5-15 >20 All Rep <5cm 5-15 >20 All Rep <5cm 5-15 >20 All Rep <5cm 5-15 >20 All Rep 32 74 91 66(14) 74 38 68 92 66(13) 63 75 82 73(5) 100 41 41 57 46(4) 55 50 55 53(1) 69 48 70 62(6) 86 39 74 67(11) 97 c 69 32 58 53(9) 94 <5cm 5-15 >20 61 54 42 <5cm 5-15 >20 40 21 - 124 -A l l 52(5) All 31(5) 3 <5cm 39 5-15 23 >20 15 All 26(6) 4 <5cm 41 >20 27 All 34(4) November, 1987 1/2 <5cm 32 5-15 35 >20 12 All 26(6) December, 1987 1/2 <5cm 44 5-15 22 >20 A l l 33(6) NOTE: 1/2 indicates transect 1 and 2 combined - 125 -APPENDIX »2 -126-APPENDIX » 2 RESULTS QF CUL.TVRING WATER SAMPLES All Brockton Point samples collected November 30, 1986 All cultures examined January 5, 1987 All Figurehead samples collected December 1, 1986 All cultures examined January 5, 1987 Culture coding; N= net samples L= 1 l i t r e water samples A-E= identification code BP= Brockton Point FH= Figurehead Culture Algae observed N A BP Diatoms, Enteromorphaf Red blotches (Iridaea?), Bladed brovn (Laminaria?). Branched green, Filamentous red N B BP Diatoms, Enteromorpha, (very little growth) N D BP Diatoms, Enteromorpha (little), Red blotches (Iridaea?) Bladed brown (Laminaria?) N E BP Diatoms, Enteromorpha, Red blotches (Iridaea?), Bladed brown (Laminaria?). Filamentous red N A FH Diatoms, Enteromorpha, Red blotches (Iridaea?)r Bladed brown (Laminaria?)r Filamentous green N B FH Enteromorpha. Red "Iridaea" blotches, Bladed brown (Laminaria?)r Bladed red (Polyneura) N C FH Diatoms, Enteromorpha N D FH Diatoms, Enteromorpha, Filamentous red, Brown blade (Laminaria?) N E FH Diatoms, Enteromorpha, Bladed red, Bladed brown (Laminaria?). Red "Iridaea" blotches Note: There was noticeably less growth in the net samples than in the water samples collected without a net. L A BP Diatoms, Enteromorpha. Filamentous red, Bladed brown (Laminaria?), Red "Iridaea" blotches - 127 -( L i t t l e growth in thi s culture) L B BP Diatoms, Enteromorpha, Red Blotches (many!), Brown blades (Laminaria?), Red blades (Iridaea) L C BP Enteromorpha, Red "Iridaea" blotches L D BP Diatoms, Enteromorpha, Red "Iridaea" blotches (many!), Bladed browns (Laminaria?)f Filamentous red L E BP Enteromorpha, Red "Iridaea" blotches (many!) Red blades, Brown blades (Laminaria?) L A FH Enteromorpha, Filamentous red, Brown blades (Laminaria?) L B FH Enteromorpha, Bladed red, Bladed brown (Laminaria?) Red "Jrjdaea" blotches L C FH Diatoms, Enteromorpha, Red "Iridaea" blotches, Bladed brown (Lamianria?), Filamentous red L D FH Diatoms, Enteromorpha. Red "Iridaea" blotches L E FH Diatoms, Enteromorpha. Red "Iridaea" blotches, Bladed brown (Lamianria?), Filamentous red - 128 -APPENDIX »3 -129-APPENDIX #3 OBSERVATIONS OF CLEARED ROCKS BROCKTON POINT Note- whenever possible detailed descriptions are made. << = 1-20% cover < = 20-40% cover * = 40-60% cover > = 60-75% cover >> = 75-100% cover ROCK I COVER August, 1986 Diatom film > Enteromorpha < Viva « December, 1986 Diatom film << Enteromorpha * Ulva * Polyneura << A n t i t f t a m n j o n << August, 1987 Enteromorpha < Ulva >> Mastocarpus << September, 1987 Ulva * Giaartina < Odonthalia << November, 1987 Ulva > QdpntftaJia. << Microcladia << Iridaea Note: approximately 30 less than 1 cm (length) Iridaea July, 1988 Ulva < Odonthalia << Microcladia << - 130 ~ RQCK II DATE  COVER August, 1986 December, 1986 August, 1987 September, 1987 November, 1987 July, 1988 DATE COVER August, 1986 December, 1986 August, 1987 September, 1987 November, 198 7 SPECIES Diatom film Enteromorpha Enteromorpha Ulva Polyneura  Antithamnion Enteromorpha  Ulva Ulva Antithamnion Giqartina Ulva Odonthalia mcroclatiia Iridaea  Ulva Odonthalia ROCK in SPECIES Enteromorpha Enteromorpha  Ulva Polynuera Antithamnion Enteromorpha  Ulva Alaria Iridaea Ulva Ulva Polyneura Antithamnion Neoaahardiella Saraassum - 131 -PERCENT < > * * < » > < > > < PERCENT < < * » » « << << << IziQaea July, 1988 Ulva < Sazaassum < Odonthalia > Mi czocladia < izjdaea < ROCK IV DATE SPECIES PERCENT COVER August, 1986 Entezomozpha » December, 1986 Entezomozpha << Ulva « Antithamnion << Iridaea << August, 1987 Ulva < Antithamnion << Mastocazpus << Iridaea < September, 1987 Ulva << Izidaea < Novembez, 1987 Ulva * Antithamnion << Polyneuza << Sazaassum << Izidaea < July, 1988 Ulva * Odonthalia < Izidaea < ROCK V DATE SPECIES PERCENT COVER August, 1986 Entezomozpha >> December, 1986 Entezomozpha < Ulva * Polyneuza < August, 1987 Entezomozpha < Ulva > - 132 -Mastocaxpus < Fucus << September, 1987 Ulva » Mastocaxpus << Fucus << November, 1987 Ulva << P p l y n e u r a << J u l y , 1988 Ulva < Entexomoxpha < Fucus < Mastocaxpus < ROCK VI DATE SPECIES PERCENT COVER August, 1987 Entexomoxpha * Ulva * September, 1987 Entexomoxpha * Ulva * Novembex, 1987 Ulva < MtlthamnlQn << Laminaxla << ROCK VII DATE SPECIES PERCENT COVER August, 1987 EnteXQmQXPha < Ulva < G i g a r t i n a < I r i d a e a << September, 1987 Enteromorpha < Ulva >> Novembex, 1987 Ulva * Iridaea « KPCK IX - 133 -DATE SPECIES PERCENT COVER August, 1987 Enteromorpha * Ulva * Fucus << Iridaea « September, 1987 Enteromorpha * Ulva * Fucus << GUgaxtlna << November, 1987 EntexomQXPha << Ulva » F»cus « Antithamnion << NeQaghaxd.le.lla << Mastocarpus << Polyneura << July, 1988 Enteromorpha << Ulva » Fucus « Antithamnion << Mastocarpus << ROCK X DATE SPECIES PERCENT COVER August, 1987 Ulva » Fucus << September, 1987 Enteromorpha * Ulva > Fucus << November, 1987 Ulva » Antithamnion << Polyneura << Rock XII DATE SPECIES PERCENT COVER August, 1987 Enteromorpha * Ulva * Mastocarpus < - 134 -Iridaea < September, 1987 Enteromorpha < Ulva * Iridaea < November, 1987 Ulva << Mastocarpus < Neoacthardiella << Iridaea * Rock XIU DATE SPECIES PERCENT COVER August, 1987 Enteromorpha * Ulva * Prionitls << Iridaea September, 1987 Ulva » Iridaea < November, 1987 Ulva < Mastocarpus < Polvneura << Iridaea < Rock XIV PATE SPECIES PERCENT COVER August, 1987 Enteromorpha * Ulva * Mastocarpus << Iridaea << September, 1987 Ulva * Mastocarpus < I r i d a e a << November, 1987 Ulva » Neoaahardlella << Iridaea « Rock XV - 135 -PATE SPECIES PERCENT COVER August, 1987 Ulva > Fucus << Antithamnion << September, 1987 Ulva * Fucus << Mastocarpus < Iridaea < November, 1987 Ulya > Fucus < Mastocarpus < Polyneura << Antithamnion << Iridaea < - 136 -APPENDIX #4 -137-APPENDIX #4 RAW DATA FROM KONTRON IMAGE ANALYSIS S.A.= Surface Area (cms) # R.S.= Reproductive structures per u n i t area (#/cma) S.A x #J?.S.= Number o f r e p r o d u c t i v e s t r u c t u r e s p e r plant January, 1981 T e t r a s p o r p p f t y t e s Brockton Point 0.2 m collection (low intertidal) Plant Surface area (S.A.) » R. S. S.A.x #R.S. 1 619 173 107,448 2 176 40 7,024 3 238 127 30,264 4 405 84 33, 784 5 199 103 20,357 6 191 118 22,503 7 448 106 47,509 8 415 137 56,620 9 257 139 35,622 10 402 93 37,386 11 488 82 39,772 12 328 146 47,931 13 335 81 27,167 14 359 55 19,767 15 241 117 28,100 16 771 122 94,001 17 77 67 5,105 18 123 217 26,673 19 142 163 23,026 20 137 154 20,968 - 138 -21 74 157 11,511 22 104 116 12,047 23 115 216 24,883 24 49 122 5,924 25 90 150 13,495 26 49 111 5,401 27 48 75 3,605 28 45 110 4,904 29 81 100 8,075 30 99 145 14,255 31 81 83 6, 760 32 124 116 14,310 33 129 170 21.916 34 159 127 20,164 35 96 190 18,172 Mean Surface Area = 220 Standard Deviation = 174 Standard Error = 26 Mean Density of Reproductive Structures = 121 Standard Deviation = 45 Standard Error = 5 Mean Number of Reproductive Structures per P l a n t = 26,184 Standard Deviation = 22,655 Standard Error = 3,829 BrQcHton P o i n t 0.7m collection (high intertidal) Plant Surface area (S.A.) » R. S. S.A.x tf R % S, 1 249 162 40,306 2 182 236 42,905 3 296 148 43,778 4 579 45 26,064 5 559 161 90,063 6 313 246 77,096 7 283 246 69,569 8 224 164 36,738 9 207 80 16,584 10 190 153 28,975 11 236 248 58,460 12 330 120 39,447 13 325 205 66,360 14 22 222 47,197 15 199 162 32,187 16 297 246 72,791 17 183 122 22,265 - 139 -18 165 134 22,054 19 24 79 1,892 20 62 86 5,360 21 74 143 10,515 22 105 153 15,967 23 52 154 7,934 24 55 142 7, 759 25 63 207 13,039 26 82 135 2, 788 27 21 135 2,788 28 58 107 6,242 29 26 79 2,028 30 43 165 7,064 31 137 110 14,865 32 42 115 4, 784 33 146 161 23,481 34 102 134 13,628 35 37 96 3,502 36 52 95 4,924 37 73 124 9,061 Mean Surface Area =170 Standard Deviation = 136 Standard Error = 22 Mean Density of Reproductive Structures = 150 Standard Deviation = 54 Standard Error = 6 Mean Number of Reproductive Structures per Plant = 26,853 Standard Deviation = 24,177 Standard Error = 3,975 August, 1987 Caroosoorooh vtes Brockton PQint 0.7m collection (high intertidal) Plant Surface area. (S . A , ) » R. s . s.A.x »R.S. 1 690 51 35,195 2 595 58 34,498 3 457 45 20,543 4 259 34 8,789 5 618 25 15,443 6 714 50 35,715 7 1193 47 56,071 8 402 36 14,472 9 444 25 11,110 10 673 53 35,690 - 140 -M e a n S u r f a c e A r e a = 605 S t a n d a r d D e v i a t i o n = 241 S t a n d a r d Error = 76 M e a n Density of Reproductive Structures = 42 Standard Deviation = 11 Standard Error = 4 Mean Number of Reproductive Structures per Plant = 26,753 Standard Deviation = 14,268 Standard Error = 4,512 Brockton Point 0.2m collection (low intertidal) Plant Surface area (S.A.) tf R. S« S r A r X tf R t S , 1 2 3 4 5 7 8 9 10 864 295 394 535 493 505 425 703 760 40 59 28 29 26 45 40 35 48 34,560 17,405 11,026 15,509 12,808 22,721 17,012 24,591 36,461 Mean Surface Area = 553 Standard Deviation =175 Standard Error = 55 Mean Density of Reproductive Structures = 40 Standard Deviation = 10 Standard Error = 3 Mean Number of Reproductive Structures per Plant = 21,836 Standard Deviation = 8,985 Standard Error = 2,995 A u g u s t , 1987 T e t r a s p o r o p h y t e s - 141 -NOTE Ten plants were sampled for the high intertidal tetrasporophytes but upon resorcinol t e s t i n g the plants proved to he gametophytic so th i s data was eliminated. B r o c k t o n Point 0.7m collection (high intertidal) Plant Surface area IS.A.) » R. S. S.A.x tf R i S, 1 250 54 13,425 2 78 64 5,022 Mean Surface Area = 164 Standard Deviation = 86 Standard Error = 61 Mean Density of Reproductive Structures = 59 Standard Deviation = 5 Standard Error = 4 Mean Number of Reproductive Structures per Plant = 9,223 Standard Deviation = 4,202 Standard Error = 2,971 Brockton Point 0.2m collection (low intertidal) Plant Surface area (S.A.) i R, S- S,Afx R^ • S. 1 163 86 14,001 2 413 99 40,981 3 521 52 26,925 4 294 176 51,691 5 341 124 42,169 6 206 101 20,837 7 1008 71 71,568 8 670 34 22,763 9 54 Mean Surface Area = 452 Standard Deviation = 261 Standard Error = 29 - 142 -Mean Density of Reproductive Structures = 89 Standard Deviation = 41 Standard Error =14 Mean Number of Reproductive Structures per Plant = 36,367 Standard Deviation = 17,834 Standard Error = 6,305 BzocKtQn Point g e n e r a l collection Plant » R.s, 1 121 2 78 3 80 4 70 5 168 6 273 7 61 8 158 9 150 10 64 11 225 12 150 13 165 14 222 15 185 16 256 Mean Density of Reproductive Structures = 151 Standard Deviation = 67 Standard Error =17 - 143 -

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