Herbivory, phenotypic variation, and reproductive barriers in fucoids. Helena Forslund

Herbivory, phenotypic variation, and reproductive barriers in fucoids Helena Forslund

Helena Forslund, Stockholm University 2012 Cover illustration: Helena Forslund ISBN 978-91-7447-538-8 Printed in Sweden by US-AB, Stockholm 2012 Distributor: Department of Botany, Stockholm University

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Doctoral dissertation Helena Forslund Department of Botany Stockholm University SE-106 91 Stockholm Sweden Herbivory, phenotypic variation, and reproductive barriers in fucoids Abstract. Along the shores of the Northern hemisphere Fucus (Phaeophyceae) species are a prominent presence, providing substrate, shelter, and food for many species. Fucus evanescens, a non-indigenous species (NIS) in Sweden, and F. radicans, a recently described species that so far has only been found inside the species poor Baltic Sea, are the focus of this thesis. Interactions with enemies (e.g. predators, herbivores, parasites) have been shown to play a role in the success of NIS. The low consumption of Fucus evanescens by the generalist gastropod Littorina littorea in Sweden was found to depend on high levels of chemical defense in the introduced population, not the failure of the herbivore to recognize F. evanescens as suitable food. A survey of the relative abundance of F. radicans and F. vesiculosus and the most common associated fauna along the Swedish Bothnian Sea coast showed that F. radicans and F. vesiculosus are equally abundant throughout the range of F. radicans. The most common associated fauna were found to be more abundant on F. radicans compared to F. vesiculosus. In Sweden, where F. radicans had lower levels of defense chemicals than F. vesiculosus, F. radicans was grazed more than F. vesiculosus in bioassays. This could, together with other factors, influence the range of F. radicans. Fucus radicans and F. vesiculosus are closely related, recently separated, and growing sympatrically, therefore, possible reproductive barriers between F. radicans and F. vesiculosus were studied. In Estonia F. radicans and F. vesiculosus reproduces at different times of the year. No such clear reproductive barrier was found between the two species in Sweden where they reproduce at the same time and fertilization success and germling survival were the same for hybrids as for F. vesiculosus. Since the high clonality of F. radicans means that the gentic diversity in F. radicans populations is low I investigated how genetic diversity translates to phenotypic diversity in nine traits. Phlorotannin levels, recovery after desiccation, and recovery after freezing showed inherited variation, while the other six traits showed no variation related to genetic diversity. Phenotypic variation in populations of F. radicans will be higher in populations with higher genetic diversity and this might be beneficial to the community. Keywords Non-indigenous species; Enemy Release Hypothesis; Asexual reproduction; Phlorotannins; Distribution

List of papers This thesis is based on the following papers, which are referred to by their roman numerals in the text: I. Forslund H, Eriksson O, Kautsky L (2012). Grazing and geographic range of the Baltic seaweed Fucus radicans (Phaeophyceae). Marine Biology Research 8:322-330. II. III. Forslund H, Wikström SA, Pavia H (2010). Higher resistance to herbivory in introduced compared to native populations of a seaweed. Oecologia 164:833-840. Forslund H, Kautsky L. Reproduction and reproductive isolation in Fucus radicans (Phaeophyceae). Accepted for publication in Marine Biology Research. IV. Johannesson K, Forslund H, Capetillo NÅ, Kautsky L, Johansson D, Pereyra R, Råberg S (2012). Phenotypic variation in sexually and asexually recruited individuals of the Baltic Sea endemic macroalga Fucus radicans: in the field and after growth in a common-garden. BMC Ecology 12. In paper I I planned and performed the study and experiments as well as wrote the paper and did the statistical analyses. For paper II I planned and performed experiments and studies, wrote most of the paper and did the statistical analyses. For paper III I planned and performed the experiments and studies, wrote the text and performed statistical analyses. For paper IV I planned and performed studies and experiments. Paper I and III is reprinted with permission from Taylor and Francis. Paper II is reprinted with kind permission from Springer Science and Business Media. Paper IV is reprinted with permission from BMC Ecology.

Content Foreword... 10 Introduction... 11 The Fucus species studied... 11 Objectives of the thesis... 13 The associated flora and fauna of seaweed beds... 13 Herbivory and herbivory defense in fucoid algae... 14 Non-indigenous species and marine introductions... 15 Reproduction and reproductive isolation in Fucus species... 17 Effects of genetic diversity on resilience and biodiversity in the Fucus community... 18 Study area... 19 The herbivores... 21 Studies... 21 Surveys of relative abundance of F. radicans and F. vesiculosus and diversity and abundance of the associated fauna... 21 Herbivore defense and phlorotannins... 22 Reproductive effort and reproductive isolation of F. radicans and F. vesiculosus... 24 Genetic diversity and variation in traits in F. radicans... 24 Results and discussion... 25 Acknowledgements... 30 References... 31 Svensk sammanfattning... 42 Tack!... 47 Appendix 1... 49

Foreword Under the surface of the ocean, where this thesis will take you, an alien world meets us. More people have walked on the surface of the moon than in the deepest trenches in the oceans. I hope and expect that we will be endlessly awed by the mysteries that are uncovered as scientists continue to learn more, but also that we will never learn everything there is to know. My astronomy and astrophysics professor would spend hours explaining a single theory for us, only to end the lecture by saying, Or at least this is what the scientists are saying right now. If you have a better theory I m willing to listen - what we know might change in a year anyway. I would step out from the university building into the night and walk home, looking up at the stars and feeling awed by the thought of both everything we do not know and everything we will never understand. Studying biology only increased this sense of wonder but this time the focus was much closer than the stars: my cells, the bacteria on my skin, and the trees outside of my window - each no less amazing or unknown than the cosmos. Over the last decades we have learned that organisms in the ocean that seem to be among the simplest forms of life, the algae, have intricate systems that can measure the moon s phases, sense light, send warning messages and sense how much the water is moving. We have found that a single genetic individual can be spread over a large area it is as though thousands of copies of you were living in an area so wide that it would take more than a week to bike through. This thesis focuses on a genus of seaweeds the Fucus that are present along temperate shores on the northern hemisphere. Living under water, they exist in an environment that is so foreign to us that it is hard to imagine. They are so different from us that intuition and common sense do not apply to them. They synthesize energy from the sun, they cannot move, and while they might seem like a simple slimy presence along the shores they have intricate systems to sense and react to their environment. 10

Introduction The Fucus species studied Seaweeds of the genus Fucus (Phaeophyceae) are a group of large, perennial brown seaweeds. They are found in temperate and arctic waters on the northern hemisphere. On intertidal rocky shores they usually form a belt with different fucoid species dominating at different shore levels. Pelvetia canaliculata (L.) Decaisne & Thuret and Fucus spirals L. grows in the splash zone, F. vesiculosus L., F. distichus L., F. ceranoides L. and Ascophyllum nodosum L. form a zone at intermediate levels and F. serratus L. and F. evanescens Agardh grow in the lower intertidal zone, rarely exposed to the air (Chapman 1995; Munda 2004; Wahl et al. 2011). The zonation usually depends on both abiotic factors (exposure to air, substrate, dessication) and biotic factors (competition, grazing) (Lubchenco 1980; Kiirikki 1996b; Wahl et al. 2011). Foundation species are species that provide structure, increases the complexity of the habitat, provide shelter and protection from both abiotic and biotic factors to an associated community (Dayton 1972; Roff and Zacharias 2011). Since the Fucus community provides habitat and shelter for many organisms (Colman 1940; Hagerman 1966; Kautsky et al. 1992; Christie et al. 2009; Dijkstra et al. 2011) they can be considered to be foundation species (e.g. Korpinen et al. 2010; Dijkstra et al. 2011). Smaller epiphytic algae and sessile animals live directly on the Fucus thallus. Small arthropods, isopods and gastropods live in and on the Fucus thallus, finding shelter and food there (Colman 1940; Hagerman 1966; Kautsky et al. 1992; Christie et al. 2009). These small animals may graze directly on the adult fucoid, juvenile fucoids, or the surface of the fucoids (Lubchenco 1983; Chapman 1995; Malm et al. 1999). The grazers are also provided with shelter against predators in the fucoid belt. Small fish can not only find an important food source in the Fucus belt, but also shelter from their predators (Hagerman 1966; Kautsky et al. 1992). Fucus evanescens is native in the northern parts of the Atlantic and Pacific Ocean and has been introduced to the southern Scandinavia and the British Isles in the last century (Simmons 1898; Hylmö 1933; Powell 1957; Wikström et al. 2002). Fucus evanescens grows at the lower end of the Fucus belt on rocky shores, but on Iceland where the tidal amplitude 11

is 4 m, they are exposed during low tide (Munda 2004). In Sweden F. evanescens has a strong herbivore defense with higher levels of a defense chemical, phlorotannins, and is less grazed than co-occurring Fucus species. In contrast F. evanescens has lower levels of phlorotannins and is grazed more than co-occurring Fucus species in Iceland (Wikström et al. 2006). Fucus vesiculosus is found in North Atlantic temperate coastal areas. It has a large span of tolerance to environmental factors such as temperature, exposure, and salinity (Lüning 1990; Bäck et al. 1992; Chapman 1995; Nygård and Dring 2008). There are several different morphs of F. vesiculosus, e.g. individuals growing at exposed sites have few vesicles and individuals growing at calm sites have many vesicles (Wærn 1952; Jordan and Vadas 1972). Different morphs have also been noted in the Baltic Sea (Wærn 1952; Kalvas and Kautsky 1993). In the northern parts of the Baltic Sea a smaller morph that was believed to be salinity stressed were found (Wærn 1952; Ruuskanen and Bäck 1999). With a combination of morphological and genetic studies Bergström et al. (2005) managed to ascertain that this morph is in fact a separate species, Fucus radicans Bergström & Kautsky. It was further found that in Sweden, F. radicans is largely clonal, with one genetic clone dominating large parts of the Swedish coast, even extending to Finland, while in Estonia it is mainly sexually reproductive (Bergström et al. 2005; Johannesson et al. 2011). The asexual reproduction is thought to be achieved by adventitious branches becoming detached, forming rhizoids and reattaching to a surface (Tatarenkov et al. 2005). Asexual reproduction is uncommon in fucoids (Serrão et al. 1999; Johannesson et al. 2011) and clonality has only been observed in the Baltic Sea (Bergström et al. 2005; Tatarenkov et al. 2005). 12

Objectives of the thesis The main objective of my thesis was to study how different Fucus species interacts with other organisms, e.g. herbivores and competitors, and how these interactions affect the distribution of Fucus species. I also wanted to study the reproduction and reproductive barriers between F. radicans and F. vesiculosus and the link between genetic diversity and phenotypic variation in F. radicans. Specific objectives of the thesis were: To study the Non-Indigenous Species (NIS) F. evanescens in Sweden and how interactions with a common grazer could potentially affect the introduction to Sweden. To gain more knowledge about the recently described F. radicans by studying the distribution, reproduction, associated flora and fauna, and herbivore defense of F. radicans. To find and study reproductive barriers between F. radicans and F. vesiculosus. To investigate how genetic diversity of F. radicans transfers to phenotypic variation and to investigate how the clonality of F. radicans could affect the heterogeneity of the Fucus community in the Baltic Sea. The associated flora and fauna of seaweed beds A rich community of algae and animals usually lives in and on the larger algae that structure the rocky temperate shores. These associated flora and fauna species find substrate, shelter, and food in the seaweeds (Hagerman 1966; Christie et al. 2009). The epiphytic algae and animals that live on seaweeds can have a large effect on them by shading and nutrition competition among other things (reviewed in Wahl 1989). The mobile fauna can both benefit the seaweeds by grazing epiphytic organisms (Råberg and Kautsky 2007) and affect them negatively by direct grazing on the seaweeds (e.g. Lubchenco 1983; Engkvist et al. 2000). A single kelp individual can host more than 100 species and 90 000 individuals and fucoid belts can host hundreds of species with animal densities of 100 000 individuals m -2 (Christie et al. 2009). Many factors, such as genetic diversity, size, complexity, and species composition of 13

the host will affect the diversity and abundance of the associated community (Hauser et al. 2006; Christie et al. 2009; Tomas et al. 2011). A larger seaweed or patch of seaweeds will have room for more individuals and probably be more heterogeneous. A more complex, more branched, seaweed or a seaweed with different parts like kelp holdfast, stipe, and blade gives better shelter and will constitute a more heterogeneous habitat than a simple seaweed, and will be likely to host a more abundant and diverse community (Hauser et al. 2006; Christie et al. 2009; Hansen et al. 2011). A more genetically diverse habitat is also believed to correlate with a more heterogeneous habitat, which in turn is believed to host a more diverse associated community (Johnson and Agrawal 2005; Tomas et al. 2011). Since the foundation species is an important factor in deciding the diversity and abundance of the associated community (Dayton 1972) and since F. radicans differs from F. vesiculosus in morphology and genetics (Bergström et al. 2005; Tatarenkov et al. 2005) I wanted to investigare if F. radicans has a different associated community compared to F. vesiculosus. Herbivory and herbivory defense in fucoid algae Herbivores are animals that consume primary producers. Marine herbivores, such as fish, isopods, gastropods, arthropods and other small invertebrates are usually generalist in contrast to many terrestrial animals that are highly specialized (Hay and Steinberg 1992). Herbivores can either just graze on the surface of a seaweed (Norton et al. 1990), suck the juices out of seaweeds (Hagerman 1966), or graze down whole stands of seaweeds (Engkvist et al. 2000). Common grazers on fucoid algae are gastropods, isopods, and amphipods (Hagerman 1966). Since grazing can have such a large effect it could be expected that algae would have some type of mechanism to cope with or defend against loss of resources to herbivores. Some species are adapted to tolerate grazing by compensatory growth and avoidance of the meristem being grazed (Cerda et al. 2009). There could also be either structural defenses or chemical defenses to deter grazing. Structural defense such as calcifications are common in some seaweeds (Paul and Hay 1986), but studies of fucoids suggest that they have no structural defense (Rohde et al. 2004). Chemical defenses entail the production of a substance that 14

makes the algae an undesirable food source. The defense substance can be either toxic or make the algae less nutritious (Hay 1996). The fucoids contains phlorotannins (Ragan and Glombitza 1986), a group of chemicals that has many functions such as protection from UV light (Swanson and Druehl 2002), wound healing (Fulcher and McCully 1971; Lüder and Clayton 2004), cell wall formation (Schoenwaelder and Clayton 1999), polyspermy block (Scoenwaelder and Clayton 1998), and adhesion (Vreeland et al. 1998). They also function as herbivore defense (Geiselman and Conell 1981; Amsler and Fairhead 2006), but there are contradicting results and suggestions that other factors, such as other chemicals (Deal et al. 2003; Kubanek et al. 2004) and habitat value (Jormalainen et al. 2001) will be more important for herbivore choice. In this thesis herbivory defense and phlorotannin content in F. radicans, F. vesiculosus and F. evanescens was studied to compare differences between species (paper I), populations (paper II), and to investigate if phenotypic variation in phlorotannin content was related to the genetic diversity of F. radicans (paper IV). Non-indigenous species and marine introductions With the increasing mobility of humans and increasing transports of good, including live organisms, the number of species that are introduced into new habitats are increasing. The introduction of species are now considered a large part of global change (Vitousek et al. 1996; Ricciardi 2007) and is changing communities by homogenizing them and by causing extinctions and decline of species (Lodge 1993; Mack et al. 2000). Both the ecological and economic costs of non-indigenous species (NIS) are high (Pimentel et al. 2005). In marine communities accidental transport with ships is believed to be the most common means of introductions. Ships could introduce species either trough ballast water that can contain larvae, plankton, or planktonic life stages of a number of organisms or through organisms becoming attached to the hull or other equipment on the ships. Aquaculture is another common cause of introductions and has lead both to intentional and unintentional introductions. The aquaria trade, where organisms sold for aquaria have been released and become established are another common way of introduction of marine species (Shaffelke et al. 2006; Williams and Smith 2007). 15

There are several theories about factors that either facilitate or prevent introduction both in regards to the introduced organism and the recipient community. The biotic resistance theory states that a more diverse recipient community will be harder for an introduced species to establish in (Maron and Vilà 2001). The enemy release hypothesis (ERH) states that the introduced species will not have as many enemies (herbivores, predators, parasites etc) in the recipient community since the enemies there will be unable to recognize it as a host since they don t share an evolutionary history. This will give the introduced species a competitive advantage over native competing species (Elton 1958; Keane and Crawley 2002). The loss of enemies should allow the introduced species to allocate more resources to growth, reproduction, and competition, an idea that gave rise to the Evolution of Increased Competitive Ability (EICA) hypothesis (Blossey and Nötzold 1995). The ERH was developed studying plant herbivore systems that are known for highly specialized enemies, while specialist enemies are very rare in marine systems (Hay and Steinberg 1992). Even so the ERH states that generalist enemies should also follow the general pattern suggested and prefer native hosts over an introduced host (Keane and Crawley 2002). Many studies so far on invasions of seaweeds shows that they are consumed less than native co-occuring seaweeds (Gianguzza et al. 2002; Levin et al. 2002; Britton-Simmons 2004; Smith et al. 2004; Sumi and Scheibling 2005). However, these studies have not studied resistance to herbivory and thus cannot separate an exemplification of the ERH and the NIS having a high level of resistance to herbivory. Despite the low dispersal ability of Fucus species F. serratus has been introduced to Iceland and to Eastern North America, probably from southern Norway (Robinson 1903; Dale 1982; Coyer at al. 2006) and Fucus evanescens has been introduced from the North Atlantic to southern Sweden and the southern Baltic Sea (Simmons 1898; Hylmö 1933; Powell 1957; Wikström et al. 2002). Previous studies have shown that common Swedish grazers, such as Idotea granulosa Rathke and Littorina obtusata L., prefer other co-occurring Fucus species over F. evanescens while in Iceland it is preferred over other co-occurring Fucus species. These studies also showed that the phlorotannin levels of F. evanescens in Sweden were higher than the other Swedish fucoids, and in Iceland the levels were lower (Wikström et al. 2006). These results 16

cannot discern between F. evanescens being avoided by Swedish herbivores because of the novelty as predicted by the ERH or because of the higher levels of phlorotannins. To be able to separate these two possible causes for the observed patterns of herbivory I made a study where Swedish grazer were allowed to choose between F. evanescens from Iceland and F. evanescens from Sweden (paper II). If herbivore preference was determined by novelty the two populations would be expected to be grazed in equal and low amounts. If on the other hand the chemical defense was the cause for the observed patterns it could be expected that the Swedish grazers would consume more of F. evanescens from Iceland that has lower levels of phlorotannins. Reproduction and reproductive isolation in Fucus species Fucus species can be either dioecious with a thallus being either male or female, or hermaphroditic with each thallus having receptacles that contains both egg and sperm. Receptacles are formed at the apex of braches and eggs and/or sperms forms in small vesicles, conceptacles, in the receptacles. Eggs are formed in groups of up to eight eggs called oogonia that are released from the receptacle before it breaks down into individual eggs (Vernet and Harper 1980). The eggs vary in size between 48 x10-5 and 16x10-5 mm 3 (Steen and Rueness 2004), are negatively buoyant so that they sink when they are released, and photosynthetic (McLachlan and Bidwell 1978). The sperm from in bundles of 64 called antheridia (Vernet and Harper 1980). The sperm has one flagellum and an orange eye-spot that allows them to swim away from the light, towards the bottom where the eggs are (Manton and Clarke 1956). Eggs and sperm mature at different time of the year for different species and even different populations (Berger et al. 2001; Steen and Rueness 2004). Gametes are synchronously released, cued by the moon and tidal phase (Brawley 1992; Andersson et al. 1994). Gametes are released late in the evening under calm conditions (Serrão et al. 1996b). Eggs releases pheromones that attract sperm and the egg membrane has recognition receptors that are species specific (Muller and Gassmann 1978) but hybrids are still found (Coyer et al. 2002; Billard et al. 2005). At lower salinities Fucus reproduction becomes problematic. The polyspermy block of the eggs stops functioning with the results of several sperm entering the egg, a condition that is lethal. The swimming of the sperm also becomes very erratic, and this probably leads to them not 17

swimming away from the light (Serrão et al. 1996a; Serrão et al. 1999). Thus sexual reproduction is most likely the life stage that limits the spread to lower salinities, since Fucus thalli that comes from low salinity areas can survive in salinities as low as 2 psu or even freshwater for weeks (personal observation). Fucus radicans, and to some extent Baltic Sea F. vesiculosus, also reproduces asexually by adventitious branches that fall off forming rhizoids and reattaching (Tatarenkov et al. 2005). It is possible that this mechanism is not as sensitive as sexual reproduction to low salinities and thus could be an advantage in the low salinities of the Baltic Sea. Reproductive barriers are to be expected when two closely related species live in sympatry as is the case of F. radicans and F. vesiculosus in the Baltic Sea. Reproductive barriers are mechanisms that prevent gene flow between two species or populations, preventing them from interbreeding (Niklas 1997; Coyne and Orr 2004). Reproductive barriers will provide information not only about the mechanisms that keeps sympatric species isolated but also about mechanisms that could have had a role in speciation (Coyne and Orr 2004). Little was known about the reproduction of F. radicans, other than that it is unique since the Swedish populations reproduces asexually to a large extent which results in a high clonality. Thus the aim was to study the reproductive effort, time of reproduction, and searching for possible reproductive barriers between F. radicans and F. vesiculosus both in Sweden and Estonia. Effects of genetic diversity on resilience and biodiversity in the Fucus community Theoretically it is assumed that a community or ecosystem with high diversity will be more resilient and adaptable since a system that has many species will have species with different responses to disturbances, and the likelihood of at least a few of these species surviving or adapting to the disturbance would be higher compared to a system with just one or two species (McNaughton 1977; Chapin 2000; Elmqvist et al. 2003). There are studies that have shown that communities that have higher species richness have a higher resilience (Steneck et al. 2002). There are also studies that show that a more genetically diverse population can withstand disturbance better (Hughes and Stachowicz 2004; Reusch et al. 18

2005; Gamfeldt and Källström 2007) and will host a more diverse community (Booth and Grime 2003; Crutsinger et al. 2006). However, there are also examples of very diverse systems that are still sensitive to disturbance and have a low resilience (Bellwood et al. 2003). Since F. radicans is clonal it has a low genetic diversity within the population (Johannesson et al. 2011). In contrast F. vesiculosus has a high genetic diversity although it is genetically differentiated at small scales (Tatarenkov et al. 2007). For the low genetic variation of F. radicans to affect the resilience and biodiversity of the Fucus community it needs to be manifested in phenotypic traits. To investigate how the genetic diversity in F. radicans affects the phenotypic variation we measured the variation in nine phenotypic traits for three groups consisting of clonal thalli and one group of genetically unique thalli. Study area The studies on F. radicans and F. vesiculosus (paper I, III, and IV) were conducted within the Baltic Sea, i.e. the Bothnian Sea and northern Baltic Proper. The Baltic Sea is atidal, but still has water level changes of up to 1 m that can last several days due to weather conditions. The salinity varies from 15 psu at the entrance and 2 psu in the north (Bernes 2005). In combination with the short history of the Baltic Sea under present conditions (Voipio 1981; Björck 1995; Winsor et al. 2001) this probably explains why the Baltic Sea is species poor with a combination of marine and freshwater species (Remane and Schlieper 1971; Snoeijs 1999). Due to the low salinity many species in the Baltic Sea functions at or close to their physiological limits (e.g Westerbom et al. 2002; Bergström et al. 2003) and many populations are genetically differentiated from and have a lower genetic diversity than populations outside the Baltic Sea (Johannesson and André 2006). For the study on F. evanescens (paper II) samples were collected from the western parts of Iceland and the west coast of Sweden. Iceland is a true marine area with tides of ~4 m and a salinity of 35 psu. On the Swedish west coast the salinity varies and is usually within the range of 15-30 psu. The tides on the west coast are small, with an amplitude of only ~0.2 m and they are often obscured by changes in water level due to high or low pressures and strong winds. These fluctuations can last for several days to weeks at a time. 19

The low salinity of the Swedish west coast decreases the species diversity compared with areas with higher salinities. The species diversity is not only lower in Sweden compared to Iceland the species composition in Iceland and Sweden also differs in part due to different salinities and part due to other factors that limits the range of species. There are differences both in the grazer and seaweed community composition (Munda 2004; Wikström 2006). Figure 1. Collections of algae for paper II were made on Iceland (A) and the Swedish west coast (B) and the bioassays were made at Tjärnö Marine Laboratory (B). Experiments in paper I, III, and IV were made at the Askö Laboratory (C). Algae for the study in paper IV were collected in Finland (D). Fucus radicans range is marked in grey. 20

The herbivores In the studies of herbivore defense in paper I and IV, Idotea balthica Pallas was used. In paper I I also used Gammarus spp. Idotea balthica is an isopod that is common in the fucoid community (Salemaa 1979; Wikström and Kautsky 2007). It is known to consume (Naylor 1955; Ravanko 1969) and even prefer Fucus species over other species (Jormalainen et al. 2001). Even though it is small it can occur in great densities and are then known to be able to graze a large part of Fucus thalli and can have a great negative effect to the point that it can severely decimate a population (Engkvist et al. 2000). Gammarus spp. generally prefers to consume filamentous algae and microscopic algae that grow on the Fucus thalli (Ravanko 1969), but they can also graze directly on the fucoids (Pavia et al. 1999; Kotta et al. 2006). In paper II the gastropod Littorina littorea L. was used as herbivore. Littorina littorea is a common herbivore on the west coast of Sweden (Wikström et al. 2006), but it is not present on Iceland where F. evanescens is native (Johannesson 1988). Gastropods can generally consume most algae, but L. littorea has been shown to consume Fucus species readily and it is generally sensitive to phlorotannins (e.g. Geiselman and McConnell 1981; Lubchenco 1983; Wikström et al. 2006). Studies Surveys of relative abundance of F. radicans and F. vesiculosus and diversity and abundance of the associated fauna Surveys of the distribution pattern of F. radicans and F. vesiculosus along the Swedish coast of the Bothnian Sea and their associated flora and fauna were performed in August 2007 (paper I) and 2008 (Fig. 2). In 2008 a survey of Estonia was also made (Fig. 2). I wanted to investigate if F. radicans became more common as the salinity dropped in the northern parts of the Bothnian Sea and if this change in cover were matched by a decrease in cover of F. vesiculosus. 21

The relative cover of F. radicans and F. vesiculosus were recorded at 16 sites along the Swedish coast (paper I). I also wanted to investigate the associated flora and fauna. In the first survey, in August September 2007 (paper I) the abundance of the most common grazers, Idotea spp., Gammarus spp., and Theodoxus fluviatilis L., were collected by placing a mesh bag (mesh size < 1mm) over a Fucus thallus. In the second, unpublished, study I collected F. radicans and F. vesiculosus at six sites in Sweden and six sites in Estonia in August 2008 (Fig 2). At each site six pairs of F. radicans and F. vesiculosus were collected at 0.5 to 4 meters depth. The two thalli making up a pair were growing within 1 m of each other to avoid confounding differences in abiotic factors such as exposure, depth, and bottom characteristics from affecting the taxon composition, richness and density between the two Fucus species. A mesh bag (mesh size < 1mm) was gently placed over the thallus being sampled and closed before detaching the thallus and transporting it to the laboratory within two days. Samples were frozen until they were sorted. Organisms larger than 1 mm were identified to the lowest possible taxonomic level. Animals were counted and the Fucus thalli were dried and weighed. Herbivore defense and phlorotannins Since the most common herbivores, Idotea spp. and Gammarus spp., were found at higher abundances on F. radicans than F. vesiculosus (paper I) bioassays were performed to investigate if this could be linked to herbivory defense. A comparison between F. evanescens from Iceland where it is native and from Sweden where it is introduced were made to evaluate if a Swedish grazer, L. littorea, prefers other Fucus species over F. evanescens (Wikström et al. 2006) because of high levels of phlorotannins or the novelty of it (paper II). To eliminate the effects of structure and morphology on herbivore choice a bioassay where freeze dried and pulverized algae were incorporated in agar and presented to L. littorea were also made. In paper IV I studied if grazing by Idotea spp. and phlorotannin content differs depending on genotype, to investigate if genetic differences manifests as differences in these traits. 22

In all bioassays one branch from one thallus were used in the control treatment and another from the same thallus in the grazing treatment. The branches were weighed before and after the experiments were grazers were allowed the choice between species (paper I) or populations (paper II). In the experiment in paper IV with genetic individuals there was no-choice experiments performed. Phlorotannin content was measured (see the respective papers for methods) in all the bioassays. Figure 2. Sites in the 2008 survey of F. radicans and F. vesiculosus. 23

Reproductive effort and reproductive isolation of F. radicans and F. vesiculosus Fucus radicans is clonal (Bergström et al. 2005) and the adventitious branches of F. radicans produces rhizoids and reattach more than those of F. vesiculosus (Tatarenkov et al. 2005). The difference in levels of clonality and rate of successful asexual reproduction could be caused either by factors that affects the reproduction or by a difference in reproductive allocation between F. radicans and F. vesiculosus. Therefore reproductive effort of F. radicans and F. vesiculosus in Sweden and Estonia was studied in paper III. Reproductive effort was measured as eggs released per receptacle, receptacles per dry weight algae and adventitious branches per wet weight algae. Fucus radicans and F. vesiculosus are closely related and recently diverged (Pereyra et al. 2009). Since they also live in sympatry throughout the range of F. radicans it could be expected that some hybrids would be found, and although there are intermediate forms (pers obs.) no individuals that are identified as genetic hybrids have been found (pers comm. with Kerstin Johannesson and Ricardo Pereyra). Since no hybrids has been found and genetic studies shows that F. radicans and F. vesiculosus are reproductively isolated (Pereyra et al. 2009) reproductive barriers should be in place and I made a few studies to try and find out which if any they were. First I measured the time of reproduction by observing at what time the receptacles were mature and the period of egg release. Second, I made artificial crosses between Swedish F. radicans and F. vesiculosus and measured the fertilization success and survival after two weeks. Genetic diversity and variation in traits in F. radicans In paper IV the genetic diversity and how this affects the phenotypic variation in nine traits were studied to investigate if the low genetic diversity of F. radicans could be expected to affect the heterogeneity of the Baltic Sea Fucus community. Three clones with ten replicate thalli and one group of ten unique genetic individuals were studied and the variation in traits was compared between the clones and the group of unique individuals. 24

The traits studied were recovery after freezing, recovery after desiccation, photochemical yield under ambient conditions, phlorotannin content, grazing, growth rate, thallus width, distance between dichotomies, and water content after desiccation. For details on the methods used to measure these see paper IV. Results and discussion The results from the studies in paper I, III, and IV indicates not only the previously known morphological, genetic, and reproductive differences between F. radicans and F. vesiculosus (Bergström et al. 2005) but also differences in the associated community, in herbivore defense, and reproductive allocation and timing. Further, I found differences in reproduction and chemical composition between the two studied populations of F. radicans i.e. between the Swedish Bothnian Sea and Estonia. Salinity (Khfaji and Norton 1979), grazing (Lubchenco 1982; Worm and Chapman 1998), and competition (Lubchenco 1980; Chapman 1990) all have the potential to affect the distribution of seaweeds. Since F. radicans is only found in low salinities I investigated if there was a gradient in the abundance of F. radicans in response to salinity and if this gradient could be mirrored in reverse for the potential competitor F. vesiculosus. My survey in paper I showed that there were no such gradient, but that the two species were equally and randomly distributed along the Swedish Bothnian Sea coast. Since we know of no abiotic factor that limit the range of F. radicans this raises the question of why F. radicans is not found further south. A possible explanation for the distribution of F. radicans could be that, as I show in paper I, a common grazer, I. baltica, is both more abundant on F. radicans and consumes more of F. radicans compared to F. vesiculosus, possibly because F. radicans has lower levels of phlorotannins. Another reason could be that F. vesiculosus is a stronger competitor, but there have been no studies that compare the competitive abilities of F. radicans and F. vesiculosus directly. The growth rate was found to be the same for F. radicans and F. vesiculosus in a common garden experiment (paper I). However since F. vesiculosus thalli are significantly larger there could be shading (Choi 2005) and whiplash effects (Kiirikki 1996a) as well as competition for space (Lubchenco 1980). Since F. radicans is a newly evolved species 25

(Pereyra et al. 2009) another explanation for the current range of F. radicans could be that it is still spreading further south. Since fucoid algae usually don t spread far or fast (Arrontes 1993; Serrão et al. 1997) this process could take a long time. The most common grazers, I. baltica and Gammarus spp., in the Baltic Sea were more common on F. radicans compared to F. vesiculosus in Sweden and F. radicans were also grazed more than F. vesiculosus in Sweden (paper I). However, since F. radicans and F. vesiculosus in Sweden hosts the same amounts of taxa, but F. radicans is smaller (Fig. 3 and paper I Fig. 3), this means that a specified biomass of F. radicans will contain more species than the same biomass of F. vesiculosus. Combined with the results in paper I that also suggest that a specified biomass of F. radicans will contain a higher abundance of animals compared to the same biomass of F. vesiculosus this suggests that it would be interesting to measure the average biomass of F. radicans and F. vesiculosus for a specified area. This would indicate if the abundance and diversity of the associated community could be expected to depend on the relative abundance of F. radicans and F. vesiculosus. This was the first study of the associated community of F. radicans in Estonia and I found that there was no difference between F. radicans and F. vesiculosus in abundance and taxon richness. However both F. radicans and F. vesiculosus were host to species that were not found on the other Fucus species (Table 1). The data needs further analysis to understand more details about what species makes these differences. However, these preliminary results indicate that the identity of the host species is important both in Sweden and Estonia and that a site where both F. radicans and F. vesiculosus grows will likely host a more diverse and abundant associated community than a site with only one of the two Fucus species. 26

Figure 3. Preliminary data from the second survey showed that A) there was no difference in number of taxa per thallus, B) that in Sweden more animals were found on Fucus vesiculosus than on F. radicans but that there were no difference in animal abundance in Estonia, and C) that Swedish F. vesiculosus were heavier than F. radicans but that there were no difference in dry weight between the two species in Estonia. Error bars shows a 95% confidence interval. 27

Since biodiversity at different levels (ecosystem, community, population) have been shown to be beneficial for the resilience and function of communities (Hughes and Stachowicz 2004; Hooper et al. 2005; Stachowicz et al. 2007; Hughes et al. 2008) this finding has an important implication for the management of the Baltic Sea. It is important to separate the two Fucus species in inventories and since we don t know what factors that determines if F. radicans or/and F. vesiculosus are present at a site and what induces and makes the vegetative reproduction of F. radicans possible, further studies are needed. In the studies of phlorotannin as herbivore defense (paper I and II) I found that the grazers consumed more of the algae that had the lowest levels of phlorotannins. The Swedish F. radicans were grazed significantly more by I. baltica and Gammarus spp. and had significantly lower levels of phlorotannins compared to Swedish F. vesiculosus (paper I). This further confirms previous studies that show the effect phlorotannin has as herbivore defense (Geiselman and McConnell 1981; Amsler and Fairhead 2006; Wikström et al. 2006). The Icelandic F. evanescens was grazed significantly more by L. littorea, both as live tissue and incorporated in agar, than the Swedish F. evanescens that had significantly higher levels of phlorotannins (paper II). This shows that it is the herbivory defense measured as phlorotannin levels in this study, not novelty of the species to the area that influences the consumption by L. littorea in this system. These results show that assuming that a NIS (Non-Indigenous Species) will have the same characteristics when it is introduced as where it is native is not always correct, and it has been pointed out that studies of NIS will benefit from studying the NIS both in the native and recipient community (Hierro et al. 2005) or other Fucus species. In Estonia F. radicans reproduces in August and September, while F. vesiculosus reproduces in May and June (paper III). The release of eggs is usually synchronous, but a few eggs can be released later than the peak, thus there could be a small overlap in reproduction. However, this difference in time would function as a reproductive barrier that can explain how two so closely related species that grows in sympatry could form and remain isolated. In Sweden F. vesiculosus in the southern part of the Baltic Sea reproduce at two times (Berger et al. 2001) and it is 28

possible that the difference in time of reproductive period could be beneficial in avoiding competition with filamentous algae (Berger et al 2001; Berger et al. 2004; Kraufvelin et al. 2007). In Sweden there was no difference between F. radicans and F. vesiculosus in time of reproduction at the scale studied here. However, recent studies indicates that difference in reproductive timing at the scales of hours could function as reproductive barriers (Monteiro et al. 2012) and studies at finer scales are necessary to rule out reproductive barriers at the scales of hours. Hybridizing F. radicans and F. vesiculosus in the laboratory showed that the fertilization success and survival during the first two weeks after fertilization for hybrids were the same as that for F. vesiculosus zygotes and germlings. This means that no reproductive barriers between F. radicans and F. vesiculosus were found in Sweden. The long term survival and reproductive abilities of the hybrids needs to be investigated to be able to understand if the reason the hybrids in paper III did not survive is because of them being hybrids or because the environmental conditions in the laboratory were unfavorable. Swedish F. radicans produces significantly more adventitious branches than Estonian F. radicans and both Swedish and Estonian F. vesiculosus and significantly fewer receptacles than Estonian F. radicans and F. vesiculosus (paper III). These differences in reproductive allocation indicate that Swedish F. radicans has an asexual reproductive mode, further confirming that F. radicans reproduces mainly asexually (Bergström et al. 2005; Tatarenkov et al. 2005). In the last study (paper IV) I compared the phenotypic variation in nine traits for three groups of F. radicans clones and one group of unique F. radicans individuals to see if a population that has low genetic diversity will have low phenotypic variation. Variation in phlorotannin content, recovery after desiccation, and recovery after freezing was found to depend on genetic variation while the other traits were independent of genetic variation. However, phenotypic variation within the clones was only 68% of the variation within the group of unique individuals. Phenotypic variation could be assumed to confer benefits to the population, and if the species is a habitat for other species, for the associated community as well (Hughes and Stachowicz 2004). The results indicate that the genetic diversity of F. radicans will be 29

manifested as phenotypic variation and that sites where one or a few clones dominate will have a more homogenous Fucus population than those with higher genetic diversity. In this thesis I found that F. evanescens has different chemical compositions in the studied regions and that F. radicans has different chemical compositions, reproductive periods, and reproductive modes in the studied regions. These results further confirms that we cannot assume that one species has the same characteristic over larger regions, and these possible variations has to be taken into account when making assumptions about the ecology and ecophysiology of one species and its interactions over larger regions. Acknowledgements Thanks to Lena Kautsky and Ove Eriksson for comments on the thesis. I also want to thank Jonne Kotta for help with the survey in Estonia. Thanks to Meg Peresich for proof reading parts of the thesis. 30

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