The Life Cycle and Genetic Structure of the Red Alga Furcellaria Lumbricalis on a Salinity Gradient

WALTER AND ANDRÉE DE NOTTBECK FOUNDATION SCIENTIFIC REPORTS No. 33

The life cycle and genetic structure of the red alga Furcellaria lumbricalis on a salinity gradient

KIRSI KOSTAMO

Academic dissertation in Ecology, to be presented, with the permission of the Faculty of Biosciences of the University of Helsinki, for public criticism in the Auditorium 1041, Biocenter 2, University of Helsinki, Viikinkaari 5, Helsinki, on January 25th, 2008, at 12 noon.

HELSINKI 2008 This thesis is based on the following papers, which will be referred to in the text by their Roman numerals:

I. Kostamo, K. & Mäkinen, A. 2006: Observations on the mode and seasonality of reproduction in Furcellaria lumbricalis (Gigartinales, Rhodophyta) populations in the northern Baltic Sea. – Bot. Mar. 49: 304-309.

II Korpelainen, H., Kostamo, K. & Virtanen, V. 2007: Microsatellite marker identiﬁ cation using genome screening and restriction-ligation. – BioTechniques 42: 479-486.

III Kostamo, K., Korpelainen, H., Maggs, C. A. & Provan, J. 2007: Genetic variation among populations of the red alga Furcellaria lumbricalis in northern Europe. – Manuscript.

IV Kostamo, K. & Korpelainen, H. 2007: Clonality and small-scale genetic diversity within populations of the red alga Furcellaria lumbricalis (Rhodophyta) in Ireland and in the northern Baltic Sea. – Manuscript.

Paper I is printed with permission from Walter de Gruyter Publishers and paper II with permission from BioTechniques.

Reviewers: Dr. Elina Leskinen Department of Biological and Environmental Sciences University of Helsinki Finland

Prof. Kerstin Johannesson Tjärnö Marine Biological Laboratory University of Gothenburg Sweden

Opponent: Dr. Veijo Jormalainen Section of Ecology Department of Biology University of Turku Finland

Contributions

The following table shows the main contributions of authors to the original articles or manuscripts. I II III IV Original idea KK, AM HK KK KK, HK Sampling KK VV, KK KK, CM KK Molecular data - KK KK KK Analyses KK HK, KK KK, HK KK MS preparation KK, AM HK, KK KK, HK, CM, JP KK, HK Initials refer to authors of the paper in question: AM = Anita Mäkinen, CM = Christine Maggs, HK = Helena Korpelainen, JP = Jim Provan, KK = Kirsi Kostamo, VV = Viivi Virtanen. 3

The life cycle and genetic structure of the red alga Furcellaria lumbricalis on a salinity gradient

KIRSI KOSTAMO

The life cycle and genetic structure of the red alga Furcellaria lumbricalis on a salinity gradient. – W. & A. de Nottbeck Foundation Sci. Rep. 33: 1-34. ISBN 978-952-99673-4-6 (paperback), ISBN 978-952-10- 4472-4 (PDF).

The life cycle and genetic diversity of the red alga Furcellaria lumbricalis (Hudson) Lamouroux were investigated in 15 populations in northern Europe. The occurrence of different life cycle phases and seasonality of reproduction were studied in four brackish populations in the northern Baltic Sea. Furthermore, a new method, based on genome screening with ISSR markers combined with a restriction-ligation method, was developed to discover microsatellite markers for population genetic analyses. The mitochondrial DNA cox2- 3 spacer sequence and four microsatellite markers were used to examine the genetic diversity and differentiation of red algal populations in northern Europe. In addition, clonality and small-scale genetic structure of one Irish and four Baltic Sea populations were studied with microsatellite markers. It was discovered that at the low salinities of the northern Baltic Sea, only tetrasporophytes and males were present in the populations of F. lumbricalis and that winter was the main season for tetrasporangial production. Furthermore, the population occurring at the lowest salinity (3.6 practical salinity units, psu) did not produce spores. The size of the tetraspores was smaller in the Baltic Sea populations than that in the Irish population, and there were more deformed spores in the Baltic Sea populations than in the Irish populations. Studies with microsatellite markers indicated that clonality is a common phenomenon in the Baltic Sea populations of F. lumbricalis, although the proportion of clonal individuals varied among populations. Some genetic divergence occurred within locations both in Ireland and in the northern Baltic Sea. Even though no carpogonia were detected in the ﬁ eld samples during the study, the microsatellite data indicated that sexual reproduction occurs at least occasionally in the northern Baltic Sea. The genetic diversity of F. lumbricalis was highest in Brit- tany, France. Since no variation was discovered in the mtDNA cox2-3 spacer sequence, which is generally regarded as an informative phylogeographic marker in red algae, it can be assumed that the studied populations probably share the same origin.

Kirsi Kostamo, Department of Applied Biology, Faculty of Agriculture and Forestry, and Department of Biological and Environmental Sciences, Faculty of Biosciences, P.O.B. 27, FI-00014 University of Helsinki, Finland. 4

CONTENTS

1. INTRODUCTION ...... 5 1.1. Plant life-histories and trade-offs ...... 5 1.2. Life histories and reproduction of vred algae ...... 5 1.3. The Baltic Sea ...... 7 1.4. Aims of this study ...... 10

2. MATERIAL AND METHODS ...... 11 2.1. Study species ...... 11 2.2. Description of the studies ...... 13 2.2.1. Life cycle of Furcellaria lumbricalis in the northern part of the Baltic Sea (Paper I) ..... 13 2.2.2. The ploidy and spore production of marine and brackish water population of F. lumbricalis ...... 13 2.2.3. Development of microsatellite markers utilising restriction-ligation method (Paper II) .. 13 2.2.4. Genetic structure of Furcellaria lumbricalis populations in Northern Europe (Paper III) ...... 14 2.2.5. Clonality and small-scale genetic diversity within populations of Furcellaria lumbricalis in Ireland and northern Baltic Sea (Paper IV) ...... 14

3. RESULTS AND DISCUSSION ...... 14 3.1. Development of microsatellite-markers ...... 15 3.2. Life cycle ...... 17 3.3. Genetic structure of populations ...... 22 3.4. Phylogeography ...... 23

4. CONCLUSIONS ...... 24

5. FUTURE WORK ...... 24

6. ACKNOWLEDGEMENTS ...... 25

7. REFERENCES ...... 26 5

1. INTRODUCTION reproducing asexually may have higher fi t- ness values than do sexually producing 1.1. Plant life-histories and trade-offs individuals, although this may vary over time (Bengtsson & Ceplitis 2000). The bal- An alternation between haploid and diploid ance between the two modes of population nuclear phases is a necessary consequence of regeneration is one of the most important eukaryotic sexuality. Variation in the relative life-history characteristics of plants due to timing of meiosis and syngamy allows its effects on the demography (Abrahamson organisms to vary widely in size at each phase 1980, Eriksson 1986), population genetic and in the duration of these two phases. In structure (McLellan et al. 1997, Chung & the diplonts (higher plants, vertebrates), the Epperson 1999, Ceplitis 2001), dispersal haploid phase is limited to one or a few cells, (Stöcklin 1999, Winkler & Fischer 2001) and it undergoes little or no development. and meta-population processes (Piquot et In the haplonts (bryophytes), the diploid al. 1998, Gabriel & Bürger 2000, Stöcklin phase is limited, and vegetative growth & Winkler 2004). occurs primarily during the haploid phase. However, many algae, ferns, bryophytes, and fungi have a biphasic or triphasic life history, 1.2. Life histories and reproduction of during which both the diploid and haploid red algae phases undergo substantial development (Bell 1994). The red algae (Rhodophyta) are a distinct Diplohaploidy has probably developed eukaryotic lineage, which lacks chlorophyll to reduce the cost of sex: If the duration of b and c but contain allophycocyanin, the life cycle phases is equal, then the diplo- phycocyanin and phycoerythrin in the form haplonts are required to produce sexual of phycobilisomes on unstacked thylakoids. structures half as often as either haplonts or Furthermore, their plastids are bound by diplonts (Richerd et al. 1993, 1994). Recent two membranes and they produce fl oridean studies support the concept that diplohaplon- starch that is deposited in the cytoplasm. tic organisms may exploit their environment All red algae lack fl agella and centrioles at more effi ciently through habitat differentia- all stages of their life history (Gabrielson tion of different ploidy phases, although this et al. 1990, Graham and Wilcox 2000). It is less evident in the case of isomorphic life has been estimated that 27 % of all known histories (Kain 1984, DeWreede & Green species of marine macrophytes are red 1990, Bolton & Joska 1993, Phillips 1994, algae (Dring 1982). Although a number Dyck & DeWreede 1995, Gonzáles & Me- of unresolved issues remain in red algal neses 1996, Lindgren & Åberg 1996, Pi- taxonomy, a phylogenetic consensus at riz 1996, Hughes & Otto 1999). However, and above the ordinal level has started to wherever the cost of sex is high, it can be emerge after the use of molecular methods assumed that the evolution of asexual repro- became more common in phycological duction is favoured (Mabel & Otto 1998). studies (Saunders & Hommersand 2004, Most plant species have the capacity for Yoon et al. 2006). vegetative reproduction in addition to sexu- The life histories of the red algae are in- al reproduction (Salisbury 1942, Klimeš et terpreted as biphasic or triphasic (Hommer- al. 1997). In some habitats, individual plants sand & Fredericq 1990). In general, the sex- 6 ual system of the red algae consists of three an ineffi cient fertilization in the absence of phases (Polysiphonia-type): a haploid sexu- motile gametes (Hommersand & Fredericq al phase (the gametophyte), a diploid phase 1990). The retention of the carposporophyte that develops directly on the female thallus and its nourishment by the gametophyte are (the carposporophyte) and a free-living dip- essential components of this adaptation, loid phase bearing meiosporangia (the tet- which enables the carposporophyte to uti- rasporophyte) (Fig. 1). Most modifi ed life lize the nutrients provided by the gameto- histories encountered in the fi eld and labo- phyte. Furthermore, there is a trend toward ratory are thought to be derived secondar- zygote amplifi cation (Hawkes 1990), cor- ily from the triphasic life history. However, responding to the splitting of one biparental the recruitment of each stage depends on the embryo into many genetically identical em- survival, fertility and migration success, as bryos that share the same genotype. Thus, well as on the effi ciency of selection during a mating between male and female game- the previous life history phases. tophytes descending from one or more tet- It has been hypothesised that selection rasporophytic individuals sharing the same has favoured the evolution of a triphasic life genotype is analogous to selfi ng. Further- history in red algae as a compensation for more, in studies on the red alga Gracilaria gracilis (Stackh.) Steentoft, Irvine & Farn- ham (syn. Gracilaria verrucosa (Huds.) Pa- penf.), it has been discovered that approxi- mately 5 % of individuals displayed ”rare sexual phenotypes”, which share the common feature that they effectively allow the population to skip the haploid macroscopic phase of the algal life cycle to some degree (Destombe et al. 1989). Similar phenom- ena have been observed in other red algal species as well (Tokida & Yamamoto 1965, Bird et al. 1977, West et al. 2001), although the germination of male and/or female spores and the development of haploid reproductive organs on the diploid plant were not stable characteristics of an individual over time. However, it has been discovered that in the red alga Gracilaria gracilis the survival of the gametophyte and tetrasporophyte stages was more important for population persistence and growth than for the fertility aspects (Engel et al. 2004), which indicates that the survival of adults is much Figure 1. The sexual life cycle of the red alga Fur- more important for population dynamics cellaria lumbricalis consists of three phases: the than is reproductive success. tetrasporophyte, the gametophyte and the carpospo- Clonal propagation is quite common in rophyte. the Bangiophycidae, occurring via ramets 7 or thallus fragments, and it has the advan- ered in several macroalgal species in the tage of allowing continuous occupation of hyposaline Baltic Sea, including the brown space (Hawkes 1990). Asexual reproduction alga Fucus vesiculosus L. (Tatarenkov et and exclusively asexual algal populations al. 2005, Bergström et al. 2005) and the are often assumed to be found in marginal red algae Ceramium tenuicorne (Kützing) environments (Wærn 1952, Bierzychudek Wærn (Gabrielsen et al. 2002, Bergström 1985, Lewis 1985, Destombe et al. 1989, et al. 2003). The asexual population regen- 1993, Wallentinus 1991, Karlsson et al. eration of red algae can also occur through 1992, Gaggiotti 1994, Bergström et al. tetraspore-to-tetraspore-cycling, where the 2005, Johannesson & André 2006), where diploid tetrasporophyte produces diploid the reduced resource distribution and abil- spores, thus avoiding the haploid part of the ity to produce clonal descendants adapted life history (West 1970, Maggs 1988, 1998) to a particular resource array allow the (Fig. 2). Some red algae are also capable asexually reproducing populations to ex- of asexual reproduction via monospores or ist (Case & Taper 1986). The occurrence other dispersal propagules, although this of asexual reproduction has been discov- phenomenon is more common in the primi- tive groups (Hawkes 1990). Also, spore and germling coalescence may infl uence the demography and genetic structure of red algal populations (Maggs & Cheney 1990, Santelices et al. 1996, 1999, 2003). The coalescence of two or more spores or germlings produces chimeric individuals which subsequently develop into single organisms. Furthermore, since it is not known to what degree coalescence occurs and because secondary pit connections are usually formed through the cell walls of two coalescing individuals, it is possible that genomic material or compounds infl u- encing gene expression can be exchanged between the different parts of the chimeric individual.

1.3. The Baltic Sea

Different factors, including the characteristics of the physical environment (salinity, light quality and availability, sedimentation Figure 2. The asexual population regeneration of rate), and, to some degree also interactions red algae can occur, e.g., through thallus fragmen- with other organisms, infl uence the indivi- tation and reattachment or tetraspore-to-tetraspore duals and populations inhabiting the Baltic cycling. Sea. The genetic structure of populations 8 is also infl uenced by the unique history passively tolerating an increased cell vol- of the area, which provides the basis for ume or by reducing the concentration of all ecological and population genetic osmotically active solutes in the cell (Kirst processes. Despite the unique character of 1990, Lobban & Harrison 1994). As a re- the present Baltic Sea, many characteristics sult, algae may suffer from decreased per- of its fl ora and fauna are still unknown. formance due to ineffi cient cellular metab- Many species occurring along the Baltic Sea olism, changes in cellular ultra structure, or salinity gradient show at least some degree ion metabolite defi ciency in the cell (Kirst of physiological adaptation to the brackish 1990). Impairment of the reproductive sys- water conditions (Smith 1964, 1977, tem due to reduced performance of gametes Rietema 1991, 1993, 1995, Kristiansen or unsuccessful fertilization may also infl u- et al. 1994, Düwel 2001). In addition, a ence the distribution of algal species at low number of species show marked genetic salinities (Raven 1999, Serrão et al. 1999). differentiation between the Baltic and North However, the effects of low salinity are Sea populations (Väinölä & Hvilsom 1991, probably strongest along the salinity gradi- Luttikhuizen et al. 2003, Olsen et al. 2004, ent, causing the exclusion of genotypes that Johannesson & André 2006), although, are not able to acclimate to low prevailing in many cases the differences may be seawater salinity. introduced through the invasion of separate Another factor infl uencing all organisms evolutionary lineages rather than being the inhabiting the Baltic Sea is eutrophication. result of local adaptation (Röhner et al. In the photic part of the benthos, its effects 1997, Väinölä 2003, Nikula et al. 2007). can be seen as increased levels of nutrients During the last glacial maximum, the that are available for all photosynthetic or- whole Baltic Sea area was covered by a ganisms. This often results in an increased continental ice sheet (Andersen & Borns amount of phytoplankton in the water col- 1994). The melting of the ice sheet was umn (Niemi 1975, Hällfors et al. 1981), re- followed by several freshwater and ma- duction in the amount of light in the water rine phases. The present marine connection column, and a consequential infl uence on through the Danish straits opened up about the distribution of species and their life his- 8 000 years ago, providing a colonization tories. In red algae, adaptation to the chang- route to marine organisms occurring in the ing irradiation environment occurs through Atlantic Ocean (Björck 1995). The rapid changes in the photosynthetic pigmentation decrease in salinity in the narrow and shal- (Kirk 1983, Ramus 1983). Solar radiation low Danish straits has had a great infl uence infl uences the amount of photosynthetic on the fl ora and fauna of the Baltic Sea. The pigmentation, with short-term changes in- overall salinity of the water basin is infl u- duced by UV light (Figueroa et al. 1997). enced by infrequent infl ows of water from Photosynthetic properties, such as com- the Atlantic Ocean as well as by freshwater pensation and saturating irradiances, have river run-off, the latter infl uencing salin- been suggested to regulate the zonation of ity on a more local scale (Backhaus 1996, macroalgae on rocky shores (Lüning 1981) Gustafsson 2001). (Fig. 3), so that deeper-growing algae are All marine species exhibit strong hy- generally more sensitive to light (due to poosmotic stress at low salinity. Mac- lower compensation irradiances), which is roalgae respond to hypoosmotic stress by considered to be an adaptation to low light 9 levels at greater depths. Furthermore, this (Sagert et al. 1997). The maximum penetra- adaptation does not disappear after a long tion of UV light in the water column varies period of cultivation in the laboratory, which from a few cm to more than 20 m (Karentz means that it is most likely of a genetic ori- & Lutze 1990, Kirk 1994), depending on gin both for the upper light limit (Hanelt et the particular wavelength and on the con- al. 1997) and for the lower light limit (Kirst centration of dissolved organic material in and Wiencke 1995). In the red alga Chond- water. Sensitivity to UV light stress is one rus crispus Stackhouse, sensitivity to pho- of the factors infl uencing the zonation pat- toinhibition correspons to the zonation of terns of algae on rocky shores (Maegewa et the algae on the shore, with deeper-grow- al. 1993, Bischof et al. 1998, 2000). ing algae showing a greater depression of Along with reduced light availability, fl uorescence yield and slower recovery an eutrophication-induced increase in the

Figure 3. Underwater vegetation zonation in the northern Baltic Sea. Along with prevailing seawater salinity, the amount of photosynthetically active radiation (PAR), the amount of UV light and sedimentation rate infl uence the occurrence of algal belts on each shore. 10 sedimentation rate can change macroalgal ores (Hemmi & Jormalainen 2004), while distribution in the littoral zone of the Bal- a change in the predation pressure on her- tic Sea. It has been discovered in an ear- bivores may result in habitat choices over- lier study (Eriksson & Johansson 2005) riding feeding preferences (Hay et al. 1987, that species that depend on short periods of 1989). Thus, changes in one trophic level spore dispersal are signifi cantly favoured by can have an impact on all other trophic lev- experimental sediment removal, while spe- els within the littoral community, including cies that mainly disperse by fragmentation the species composition and biomass of the or have long continuous periods of spore algal fl ora. release are more tolerant to sedimentation. Dispersal by fragments probably increases the likelihood of fi nding suitable patches of 1.4. Aims of this study substrate in a temporally unstable sediment environment, because loose thalli fragments The aim of my thesis was to study the survive in the water for longer periods than life cycle and genetic structure of the do free spores and the release of fragments populations of the red alga Furcellaria will not be confi ned to one short period of lumbricalis in the Baltic Sea. In paper time (Eriksson & Johansson 2005). Thus, I, we studied the reproduction mode of sedimentation has the potential to select F. lumbricalis in four populations along against dependence on spores for popula- the coast of Finland in order to discover tion regeneration in a temporally unpredict- whether asexual reproduction becomes able sediment environment in the deep end more prominent in marginal environments. of the red algal belt (Eriksson & Johansson Furthermore, some unpublished data 2005). In colonization experiments, it has on ploidy and spore production in two been discovered that population regenera- marine populations and two brackish water tion of the Furcellaria lumbricalis (Huds.) populations are included in section 3.2. in Lamour. in the northern Baltic Sea occurs order to further test the hypothesis that the only by thallus fragments that attach to the role of asexuality is more pronounced in the substrate with secondary rhizoids (Johans- hyposaline Baltic Sea. In the second paper, son 2002) (Fig. 2) and that colonization is we developed a new method to discover a relatively uncommon event when com- microsatellite markers, and then we utilized pared to other algal species. the new methodology to develop marker The increase in the amount of nutrients sets for a number of bryophyte and algal also promotes the growth of fi lamentous species, including F. lumbricalis. In paper algae that can overgrow and overshadow III, we studied the genetic structure of red even larger perennial algal species (Kan- algal populations in northern Europe with gas et al. 1982, Wallentinus 1984, Rönn- mtDNA sequencing and microsatellites, berg et al. 1992). Nutrient enrichment can and in paper IV, we studied the degree also change the interactions between mac- of clonality and the fi ne-scale genetic roalgae and other organisms. For example, structure within one marine and four a change in the nutritional quality of algae brackish populations of F. lumbricalis with can make them more suitable for herbiv- microsatellite markers. 11

2. MATERIAL AND METHODS female and the male gametophyte) (Austin 1960 a, b). The third life cycle stage, the 2.1. Study species diploid carposporophyte, resides on the female gametophyte (Fig. 1). Furcellaria lumbricalis is a perennial red F. lumbricalis is well known among the alga, occurring in cold waters of the North larger red algae for its tolerance of low sa- Atlantic and Arctic Oceans (Holmsgaard linity (Bird et al. 1991). The four practical et al. 1981, Bird et al. 1983). The species salinity unit (4 psu) isohaline in the Gulf of is monotypic. The distribution of F. Bothnia and Gulf of Finland marks the in- lumbricalis ranges in Europe from northern nermost distribution of F. lumbricalis in the Spain to the Arctic, including the British Baltic Sea (Zenkevitch 1963, Bergström Isles, the Faeroe Islands and the Baltic Sea. & Bergström 1999). However, an experi- In North America, the major population of mental assessment on the effects of salinity F. lumbricalis grows in the southern Gulf of on growth showed an increase in biomass St. Lawrence, and it has spread sparingly to to be maximal at 20 psu under favourable adjacent outer coasts since the 1930’s (Bell conditions of light and temperature (Bird & MacFarlane 1933). It is likely that the et al. 1979). Fertile Furcellaria individuals populations in southern Europe and North have not be detected from the lowest salini- America are introduced, because they are ties, but they can be found from the south- disjunct and limited (Bird et al. 1991). ern part of the Baltic Sea, on the south-east Furcellaria lumbricalis is a subtidal al- coast of Sweden and around the island of gal species that grows in sheltered to mod- erately exposed habitats (Schwenke 1971). It grows in marine habitats in tidal pools from the upper eulittoral zone all the way down to depths of 28 m. In the Baltic Sea, F. lumbricalis grows in the bladder wrack belt and even in shady spots under Fucus vesiculosus. Below the bladder wrack belt, F. lumbricalis forms the red algal belt at depths of 2-10 m depending on water tur- bidity, competition and suitable substratum (Rosenvinge 1917, Wærn 1952, Taylor 1975, Kornfeldt 1979, Holmsgaard et al. 1981, Mäkinen et al. 1988) (Fig. 3). The individuals of F. lumbricalis form dense tufts on rocky surfaces and provide growing surface and shelter for many different invertebrates. Figure 4. Fifteen Furcellaria lumbricalis popula- Furcellaria lumbricalis has a complex tions from the Baltic Sea and the Atlantic Ocean haploid-diploid life cycle typical of red were included in the study. The seawater salinity seaweeds, including two free-living stages in the Atlantic Ocean is 35 practical salinity units of different ploidy levels (a diploid stage, (psu), while the Baltic Sea populations inhabit sa- the tetrasporophyte and a haploid stage, the linities between 3.6-30 psu. 12

Gotland (Svedelius 1901, Levring 1940), The Furcellaria individuals discovered where the salinity is above 7 psu (Kautsky from the tidal pools grew only in locations & Kautsky 1989). where they stayed permanently submerged Altogether 15 marine and brackish wa- and formed often mixed cultures with the ter populations of the red alga F. lumbrica- red alga Polyides rotundus (Huds.) Grev. lis were investigated in this study (Table 1, The population in Tjärnö, on the west coast Fig. 4). Marine populations, which grew in of Sweden, was found in somewhat lower saline water of 35 psu, include populations salinity (15-30 psu). The sampled Baltic from Roscoff (France), Donegal (Republic Sea populations occur in brackish water of Ireland), Castle Island and Dorn (North- environments where salinities range from ern Ireland) and Lofoten (Norway). Sam- 3.6-8 psu. Samples from the Baltic Sea pling the marine populations was carried populations were collected from bladder out in tidal pools because no Furcellaria wrack belt and from red algal belt using were discovered from the deeper parts of SCUBA. the sublittoral, despite an extensive search.

Table 1. Sampling sites of the Furcellaria lumbricalis populations, seawater salinity, sample sizes, and the numbers of different genotypes detected by a microsatellite analysis. Haploid individuals were excluded from the genetic analyses.

Locality Geographic region Seawater salinity Sampled location (psu)

1. Roscoff Brittany, France 35 Tidal pool 2. Fanad Head Co. Donegal, 35 Subtidal Republic of Ireland Tidal pools 3. Dorn Strangford Lough, Northern Ireland 35 Tidal inlet 4. Castle Island Strangford Lough, Northern Ireland 35 Large tidal pool 5. Andenes North Sea, Norway 35 Subtidal 6. Tjärnö Skagerrak Sea, Sweden 15-30 Subtidal 7. Palanga Baltic proper, Lithuania 8 Subtidal 8. Askö Baltic proper, Sweden 6 Subtidal 9. Utö Archipelago Sea, Finland 6 Subtidal 10. Seili Archipelago Sea, Finland 5.4 Subtidal 11. Pori Gulf of Bothnia, Finland 6 Subtidal 12. Närpiö Gulf of Bothnia, Finland 4.9 Subtidal 13. Tvärminne Gulf of Finland, Finland 5.3 Subtidal 14. Helsinki Gulf of Finland, Finland 4.6 Subtidal 15. Ulko-Tammio Gulf of Finland, Finland 3.6 Subtidal 13

2.2. Description of the studies 2.2.2. The ploidy and spore production of marine and brackish water population 2.2.1. Life cycle of Furcellaria lumbricalis of F. lumbricalis in the northern part of the Baltic Sea The ploidy of individuals inhabiting marine (Paper I) and brackish water habitats were studied The life cycle of Furcellaria lumbricalis based on morphological characters, i.e., was studied by monthly sampling (n = 30/ the presence and absence of reproductive sampling site) for one year along the coast of structures. The spore production was Finland at four locations spanning 500 km studied by measuring the size and shape along a salinity gradient from 3.6 to 5.4 psu of tetraspores in two marine populations (Table 1). Sampled individuals were studied and two brackish water populations of with a light microscope to identify the life F. lumbricalis. Frequencies of different cycle phase. A two-way ANOVA was used spore size classes were calculated and to determine whether there were differences plotted. A logarithmic transformation was in the number of reproducing individuals performed for the dataset and a one-way among sampling sites and months. Tukey’s ANOVA was used to determine whether HSD test was used as a post hoc test for there were differences between the Irish multiple pairwise comparisons. A logistic and Baltic Sea populations in spore sizes. regression analysis was used to study Finally, a linear regression analysis was whether environmental factors infl uence used to determine whether the shape of the production of spores in populations the Baltic Sea spores infl uenced the size inhabiting different environments. Further, of the spores. Unfortunately, the growing in order to investigate the effects of experiments failed despite the fact that environmental factors (salinity, seawater they were attempted several times with, temperature, water transparency, day length) e.g., different growing media and light on the intensity of reproduction, a logit conditions. Therefore, the viability of the transformation was fi rst conducted for the marine versus Baltic Sea spores could not variable describing reproductive intensity be estimated. and then a linear regression analysis was performed for the dataset. Furthermore, 2.2.3. Development of microsatellite principal component analyses (PCA) were markers utilising restriction-ligation used to study whether there are some method (Paper II) combinations of environmental variables A new method based on genome screening that explain the environmental variability with intersequence simple repeat (ISSR) across the sampling sites and times. Axes markers and restriction-ligation was which explained a signifi cant proportion developed to fi nd microsatellite sites of the environmental variability were then from an organism’s genome. Traditional correlated with reproduction intensity. methods for microsatellite development Finally, because Component 1 of the PCA include pooling the DNA, DNA digestion correlated to the reproduction intensity, the and ligation with different restriction and relationships between the regression slopes ligation enzymes, the forming of single- of individual sampling sites were examined stranded DNA library and the cloning of with ANCOVA. double stranded DNA (e.g., Zane et al. 14

2002). The major disadvantages of the et al. 2005), resulting in the identifi cation of traditional methods are that they are both marine glacial refugia in the Atlantic Ocean time-consuming and expensive (Zane et al. (Provan et al. 2005) and distinguishing 2002, Squirrel et al. 2003). The new method the level of genetic differentiation among developed in this study includes genome populations inhabiting the Baltic Sea screening for microsatellite regions using and the Atlantic Ocean (Gabrielsen et al. ISSR primers, cloning the acquired DNA, 2002). Genetic fi ngerprinting with four which has a microsatellite region in both polymorphic microsatellite markers was ends of the sequence, and sequencing the used to obtain information about the genetic DNA sequences acquired from positive population structure among algal populations bacterial colonies. The fi rst specifi c in the Atlantic Ocean and the Baltic Sea. primer for each microsatellite locus was Descriptive statistics of the within- and then developed based on the information between-locality genetic diversity (Nei obtained from the fi rst sequencing. Then a 1978), the estimator FST, deviations from two-stranded DNA sequence (adapter) with Hardy-Weinberg equilibrium, presence a known sequence was ligated on restricted of linkage disequilibrium, polymorphism DNA. After this, a PCR amplifi cation was information content (PIC) value (Botstein performed using the specifi c microsatellite et al. 1980), analyses of molecular variance primer and a primer designed based on the (AMOVA), assignment tests and the adapter sequence. The PCR amplifi cation isolation by distance were calculated from product was sequenced, and the second the microsatellite dataset. specifi c microsatellite primer was designed based on the information obtained from 2.2.5. Clonality and small-scale genetic the second sequencing. The new method diversity within populations of Furcellaria is simpler than the traditional method, lumbricalis in Ireland and northern Baltic and it reduces the costs and laboratory Sea (Paper IV) requirements for marker development. The degree of clonality and small- Altogether, 21 species of bryophytes, three scale genetic structure of Furcellaria species of algae and racoon dog were used lumbricalis were studied within one Irish as material for this study when developing and in four Baltic Sea populations with methodology and marker sets. four microsatellite markers. In the Irish population, algal samples were collected 2.2.4. Genetic structure of Furcellaria from one subtidal subpopulation (12 lumbricalis populations in Northern individuals) and three small tidal pool Europe (Paper III) subpopulations (10 individuals from each The phylogeography and genetic diversity tidal pool). In the Baltic Sea, altogether 30 were studied in 15 populations of the red individuals were collected from each of four alga Furcellaria lumbricalis in northern locations, including 10 algal individuals Europe using the mitochondrial cox2-3 inhabiting the maximum growing depth (red spacer and four microsatellite markers. The algal belt), 10 algal individuals inhabiting mtDNA cox2-3 spacer has previously been the optimum growing depth (red algal belt) used in several red algal studies (Zuccarello and 10 algal individuals inhabiting the et al. 1999, Gabrielsen et al. 2002, Provan minimum growing depth (bladder wrack 15 belt) at each location. Descriptive statistics the fi rst sequencing, and further 41 % after of the within-locality genetic diversity (Nei sequence walking (II). The new method-

1978), the estimator FST, deviations from the ology utilises sequences containing mi- Hardy-Weinberg equilibrium, presence of crosatellite repeat motifs on both ends of linkage disequilibrium, degree of clonality, the studied sequence, which obviously in- occurrence of rare alleles, assignment of creases the likelihood of successful marker individuals to different populations and development. Additionally, the new meth- analyses of molecular variance (AMOVA) odology makes it also possible to estimate were calculated. the frequency of microsatellites in the genome of the studied species already at an early stage of the marker development, 3. RESULTS AND DISCUSSION which gives valuable information during the fi rst few steps of the marker develop- 3.1. Development of microsatellite ment. The total number of microsatellites markers developed while testing the new methodology equalled 191 markers, of which 95 % Microsatellite repeat sequences are were found to be polymorphic (II) (Kor- known to be ubiquitous in prokaryotic and pelainen et al. 2007, Kostamo et al. 2007). eukaryotic genomes and present both in Altogether eight microsatellite loci were coding and noncoding regions. However, originally discovered from the genome of the distribution of microsatellites is not Furcellaria lumbricalis. However, one of homogeneous within a single genome, the microsatellite loci (F110R) was linked because of different constraints of coding vs. to another microsatellite locus (F110L), noncoding sequences, historical processes and only the more variable locus (F110L) (Arcot et al. 1995, Wilder & Hollocher 2001), was used in this study. Furthermore, three and the possible different functional roles of microsatellite loci were monomorphic. different repeats (Valle 1993, Hammock & Sequencing of microsatellite-rich areas Young 2004). The frequency of microsatellite in red algae never resulted in fi nding micro- sequences also varies across taxa, in terms of satellites within the sequenced ISSR ampli- both absolute numbers of microsatellite loci fi cation product between two microsatellite and repeat preference (Hancock 1999). In repeat sequences (II). A similar phenom- Bangiophycidae, microsatellite marker sets enon was also observed in the green alga have earlier been developed for Gracilaria Ulva intestinalis L., in which all developed gracilis (Luo et al. 1999) and G. chilensis markers resulted from sequence walking, Bird, McLachlan & Oliveira (Guillemin et although only four markers were developed al. 2005). for this species (II) (Kostamo et al. 2007). It has previously been estimated that as This was not the case in bryophytes, where many as 83 % of the loci originally includ- microsatellite-rich areas were often discov- ed in the process of microsatellite devel- ered, and 81 % of microsatellite markers opment are lost during the different phases were developed directly after the sequenc- of the process (Squirrel et al. 2003). How- ing of the ISSR-amplifi cation bands (II). ever, when utilising the new protocol, on It is thus possible that the great diffi culties average 54 % of ISSR sequences resulted associated earlier with the development of directly in microsatellite development after microsatellite markers for red algae and 16 other seaweeds, are caused by the fewer ia chilensis, six developed microsatel- microsatellite regions present in the red al- lite markers had 2-4 alleles at each locus gal genome. (Guillemin et al. 2005), which means that It has previously been discovered that the PIC-values were lower than those we AG/CT repeats are the most common re- found in F. lumbricalis, although also the peat type in plants, (Morgante et al. 2002). allele frequencies within a locus infl uence Therefore, the use of ISSR primers that the PIC values. However, the dataset for G. contain an AG/CT or AC/TG repeat in the chilensis was tested with only 40 individu- screening of the genome probably results als from different populations along the in more ISSR bands for sequencing and in- Chilean coast, while the dataset for F. lum- creases the likelihood of discovering suit- bricalis consisted of 185 individuals from able markers when compared to screening 15 populations in Northern Europe. An in- with other types of ISSR primers. There- crease in the sample size and an increase fore, the AG/CT repeat was the most com- in the geographic range of sampling most mon ISSR repeat type used in our study (II). likely also increased the number of discov- Overall, AG/CT repeats outnumbered the ered alleles per locus, and thus affected the other repeat types discovered on several al- PIC values. gal and moss species (29.2 %) (II).The sec- When comparing the acquired sequence ond most common repeat type was AC/TG data to GenBank databases, only four out (11.8 %) (II). In the red alga Furcellaria of the 191 microsatellite loci (0.02 %) ap- lumbricalis, six out of eight microsatellite peared to be physically linked to a known repeat sequences were AG/CT repeats and gene area (Table 2) (Korpelainen et al. the remaining two microsatellite sequences 2007). All four linked loci were found in were AC/TG repeats (III). bryophyte species. One of the microsatel- Typically, the increase in the number of lites was located in the middle of the cod- alleles per microsatellite locus translates ing area, while in the remainder of the cases into an increase in the polymorphism infor- the region was located just before or after mation content (PIC) value (Botstein et al. a gene. All of the linked microsatellite loci 1980). PIC values are commonly used in were polymorphic. Furthermore, it is likely studies of cultured vascular plants (Dávila that even a higher number of the microsat- et al. 1999, Richards et al. 2004, Oliveira ellite loci discovered are linked with some et al. 2005, Palmieri et al. 2005) and oc- gene, but it is not possible to associate se- casionally of wild plants (Erickson et al. quences containing microsatellites to a spe- 2004). In an earlier microsatellite marker cifi c genomic region due to the lack of se- set developed for the red alga Gracilar- quence data in GenBank. 17

Table 2. Microsatellite linkage to active gene areas was discovered in four bryophyte markers out of the 191 microsatellite markers developed for 21 bryophyte species, 3 algal species and the racoon dog. The sequences of the developed microsatellite markers were compared to the GenBank database in order to discover whether the markers were physically close to a genomic area containing a gene.

Species Locus Polymorphic Linkage area in Species GenBank genome (GenBank) Accession number Hylocomium HYSP1 Yes Located in the IGS Bryophyta X80212 splendens area before the 5S (several

[CA]6 gene species) Polytrichum POJU5 Yes Large subunit Bryophyta DQ629273 juniperinum rDNA gene in the (several

[AG]6 chloroplast species) Rhizomnium RHPU1 Yes Rac-like GTP Bryophyta AF146341 punctatum binding protein (Physcomitrella

[TG]7 (rac3) precursor patens) RNA Rhytidiadelphus RHSQ8 Yes mRNA for putative Asteraceae AJ880395 squarrosus peroxidase (pod (Zinnia

[CAA]6 gene) elegans)

3.2. Life cycle the Baltic Sea. Numerical dominance of gametophytes is common on a whole- There was a difference between the marine habitat basis and/or on an annual basis and brackish water populations in the in red algae (Mathieson & Burns 1975, number of different life cycle phases (Table Craigie & Pringle 1978, Mathieson 1982, 3). In three populations studied in the Bhattacharya 1985, Dyck et al. 1985, northern Baltic Sea, only tetrasporophytes Hannach & Santelices 1985, May 1986, and spermatophytes were discovered Lazo et al. 1989, DeWreede & Green (I). In the fourth population, occurring 1990, Bolton & Joska 1993, Scrosati et al. at the lowest salinity (3.6 psu), all algae 1994, Dyck & DeWreede 1995, González examined were vegetative. Austin (1960 a, & Meneses 1996, Lindgren & Åberg 1996, b) has earlier postulated that the life cycle Piriz 1996, Zamorano & Westermeier phase ratio of F. lumbricalis in the northern 1996, Scrosati 1998), while the numerical Atlantic Ocean is 2:1:1 (tetrasporophytes : dominace of tetrasporophytes seems to be male gametophytes : female gametophytes). restricted to smaller spatial or temporal However, this was not the case in either scales and has been reported only for a the Irish or the Baltic Sea populations few species (Hansen & Doyle 1976, Dyck studied. Furthermore, all the populations et al. 1985, Lazo et al. 1989, DeWreede were strongly tetrasporophyte-biased in & Green 1990, Bolton & Joska 1993, 18

Phillips 1994, Dyck & DeWreede 1995). processes, such as survival, reproduction or It has been estimated that in some other recruitment rates, can inﬂ uence the ploidy long-lived perennial red algal species frequencies of populations (Engel et al. even slight changes in population dynamic 2001, Engel et al. 2004, Fierst et al. 2005).

Table 3. Numbers of different life cycle phases in the marine and Baltic Sea populations of Furcellaria lumbricalis. N = number of sampled individuals, 2n = diploid individuals, n = haploid individuals, I = Ireland, NI = Northern Ireland, FI = Finland.

Location N 2n n n Vegetative individuals Donegal (I) 89 8 10 2 69 Giant’s Causeway (NI) 16 10 0 0 6 Castle Island (NI) 28 15 4 2 7 Dorn (NI) 43 5 7 10 21 Pori (FI) 456 112 17 0 327 Hanko (FI) 518 40 23 0 455 Helsinki (FI) 520 80 12 0 428 Kotka (FI) 388 0 0 0 388

A large portion of the algae in both habi- seasonality of tetrasporangial discharge tats did not reproduce at all during the study of spores have been reported from other period. In a nine-year study spanning all parts of Northern Europe (Austin 1960b) seasons, reproductive individuals of Chon- and North America (Bird 1977). However, dracanthus pectinatus (Daw.) L. Aguilar & smaller sori with only a few tetrasporangia R. Aguilar were absent from the popula- were observed during spring at higher sa- tion only on two occasions (Pacheco-Ruíz linities, and some individual sporangia & Zertuche-González 1999), although the were detected in the population at 5 psu sa- intensity of reproductive effort ﬂ uctuated. linity even during the summer months. It In an earlier study Bird (1976) discovered appears that the timing and intensity of re- that in the red alga Gracilaria, the ploidy production in the northern Baltic Sea might of vegetative individuals reﬂ ects the ploidy be regulated by a combination of factors, frequencies of reproducing haploid and dip- including seawater salinity, seawater trans- loid individuals. Thus, most of the vegeta- parency, temperature and photoperiod. tive individuals would be tetrasporophytes The tetraspores are formed in tetrads in the northern Baltic Sea. where there are two semi-circle-shaped Winter months are the main season for spores and two square-shaped spores de- tetrasporangial production in the Northern veloping simultaneously (Fig.5). In the ma- Baltic Sea, when sori with masses of spo- rine populations, the spores attain a round rangia are present. Similar results on the or slightly oval shape quickly after they are 19

A. B.

Figure 5 A-D. – A. Maturing tetrasporophytic sorus of Furcellaria lumbricalis. – B. Cross-section of thallus consisting of medulla ﬁ laments, tetrasporophytic sori and cortex. – C. Released tetrasporangia consisting of four tetraspores. – D. An individual tetraspore from a marine population.

released into seawater. However, the Baltic ses. It was discovered that the Baltic Sea Sea populations produced tetraspores with tetraspores were signifi cantly smaller in different shapes (round, oval, square, tri- size than the Irish tetraspores (MS = 44.2, angular, semicircle, indefi nite), and, there- F = 1649.4, df = 1, p<0.000) (Fig. 6). Fur- fore, instead of spore size, the area of all thermore, there were signifi cant differences tetraspores were calculated for the analy- among individuals within the Baltic Sea 20 populations of F. lumbricalis in spore sizes alga Fucus vesiculosus, low osmolalities (Table 4). It also appears that the shape of (70-125 mmol/kg; 125 mmol/kg equals ≈ the spore signifi cantly affects its size (B = 4.3 ‰) cause an increase in the volume of -1.7×10-8, R²=0.049, p<0.000), round tet- both the eggs and sperm (Wrigth & Reed raspores being larger in size. Macroalgae 1990, Serrão et al. 1996). Most unfertilised respond to hypoosmotic stress by passively eggs burst at low salinities soon after their increasing the cell volume or by reducing release. Furthermore, the sperm have a the concentration of osmotically active rounder shape in brackish water, and this solutes in the cell (Kirst 1990, Lobban & appears to cause the displacement of the Harrison 1994). As a result, the changes in sperm’s eyespot, accounting possibly for the cellular ultrastructure or ion metabo- the lack of negative phototaxis of sperm lite defi ciency in the cell may decrease the in brackish water. However, a similar in- performance of an individual reproductive crease in the size of the tetraspores was not cell or an algal individual (Kirst 1990). In observed in F. lumbricalis populations in the Baltic Sea populations of the brown the northern Baltic Sea. Thus, the irregu-

Figure 6. Tetraspore size class frequencies* in the Irish (M) and Baltic Sea (BS) populations. The Baltic Sea spores were signiﬁ cantly smaller than the spores originating from the marine populations.

*Size classes were < 40 × 10-9 m2, < 60 × 10-9 m2, < 80 × 10-9 m2, < 100 × 10-9 m2, < 200 × 10-9 m2, < 400 × 10-9 m2, < 600 × 10-9 m2, < 800 × 10-9 m2, < 1 × 10-6 m2, < 2 × 10-6 m2 , < 4 × 10-6 m2 . 21 lar shape and smaller size of tetraspores in role of spore production is not as important the Baltic Sea populations compared to the for populations inhabiting low salinities as Irish populations might indicate that the for populations inhabiting higher salinities.

Table 4. One-way ANOVA on the effects of population, individual and population × individual on the size of tetraspores in Furcellaria lumbricalis. A logarithmic transformation was performed for the dataset.

Factor df MS F p Model 22 0.28 12.93 <0.000 Population 1 1.79 84.22 <0.000 Individual 15 0.25 11.82 <0.000 Population × Individual 6 0.10 4.77 <0.000 Error term 880 0.02

Furthermore, some clonality was ob- ters of the Baltic Sea, including the brown served in the Baltic Sea populations of F. alga Fucus vesiculosus (Tatarenkov et al. lumbricalis in Lithuania and Finland (III, 2005, Bergström et al. 2005) and the red al- IV). The degree of clonality was about 13.5 gae Ceramium tenuicorne (Gabrielsen et al. % in four of the studied Baltic Sea popula- 2002, Bergström et al. 2003). In F. vesicu- tions (n = 30/population) (IV). However, losus, reproduction may be limited at times only two clonal individuals were discov- in the brackish populations by a low rate ered from the six marine populations in of gamete release, low fertilization success the Atlantic Ocean (III, IV), even though and polyspermy (Serrão et al. 1996, 1999), sampling in one of the locations included and some of the northernmost populations also different subpopulations inhabiting the are able to regenerate through adventitious subtidal and small tidal pools (IV). branches that reattach to the substratum The hypothesis that asexual reproduc- (Tatarenkov et al. 2005). In the red alga C. tion via thallus fragmentation and/or tet- tenuicorne, population regeneration occurs raspore-to-tetraspore cycling is more im- in the northern Baltic mainly by vegetative portant in the population regeneration of F. reproduction, such as fragmentation and lumbricalis in the brackish water than in a rhizoidal reattachment, although sexual re- marine environment is highly likely, based production is at least occasionally possible on the following collected data: ploidy ra- down to salinities of around 4 psu. tios are biased towards tetrasporophyte It can thus be hypothesised that popu- dominance, the size and shape of spores lations occurring at low salinities (4.9-5.4 are different compared to those in marine psu) in the northern Baltic Sea regenerate populations, and there also exists a higher by thallus fragmentation and possibly by proportion of clonal individuals within asexual tetraspore-to-tetraspore cycling populations. The occurrence of asexual re- without meiosis. Furthermore, the popula- production has been discovered in several tion occurring at a salinity of 3.6 psu prob- macroalgal species in the hyposaline wa- ably survives solely by thallus fragmenta- 22 tion and reattachment. This hypothesis is mon, and asexual reproduction tends to be also supported by observations made by prevalent in the marginal parts of the spe- Johansson (2002) in colonisation experi- cies’ distributions (Dixon 1965, Hawkes ments, which show that the regeneration of 1990, Maggs 1998). As mentioned previ- the red algal species F. lumbricalis, Polysi- ously, the occurrence of asexual reproduc- phonia fucoides (Huds.) Grev. and Rho- tion has been discovered in several mac- domela confervoides (Huds.) Silva occurs roalgal species in the hyposaline waters in the northern Baltic Sea mainly by frag- of the Baltic Sea (Gabrielsen et al. 2002, ments, which are attached to the substratum Bergström et al. 2003, 2005, Tatarenkov by the byssus threads of the mussel Mytilus et al. 2005). However, when studying the trossulus L. small-scale genetic structure of one Irish and four Baltic Sea populations of F. lumbricalis, we discovered that most popula- 3.3. Genetic structure of populations tions and subpopulations are in or close to the Hardy-Weinberg equilibrium. This Based on microsatellite markers, it means that sexual reproduction occurs was discovered that there is no genetic both in Ireland and in the Baltic Sea popu- differentiation among the Furcellaria lations, despite the fact that female game- lumbricalis populations in Northern tophytes have not been discovered in the Europe (III). However, even the Baltic northern Baltic Sea. However, it is possible Sea populations growing in extremely low that females exist but they do not annually salinities showed a fair amount of genetic produce carposporangia. Since the lifespan variability (III). These results agree with an of algae is several years, sudden environ- earlier study (Valatka et al. 2000) using the mental changes, e.g., irregularly occurring RAPD method, which reported a high genetic salinity pulses, may change the environ- diversity within the species in the Baltic ment to be more suitable for carpospore Sea. Asexual populations often harbour a production. The excess of heterozygosity wealth of genetic variation (Ellstrand & in the Baltic Sea populations revealed by Roose 1987, Herbert 1987, Suomalainen et the means of ﬁ xation indices suggests that al. 1987, Asker & Jerling 1992, Bengtsson a mild effect of heterozygosity advantage 2003), coming from new mutations, as may be present. Selection for high levels well as from remnant sexuality and/or of heterozygosity at the geographic limit multiple origins. Only a few sexual events has been discovered in the aquatic phan- per generation are sufﬁ cient to prevent the erogam Zostera marina (Billingham et al. formation of an asexual genomic pattern 2003, Hämmerli & Reusch 2003) but also (typically monomorphisms at several loci), in common garden experiments and recip- which would demonstrate the presence rocal transplant experiments (Backman of a predominantly asexual lifecycle 1991, Hämmerli & Reusch 2002). (Bengtsson 2003). Thus, populations with a A further study showed that in the At- predominantly asexual mode of population lantic Ocean, there were only slight differ- regeneration can display almost any pattern ences in subpopulation pairwise FST-values of genotypic variation. within the studied algal population consist- In red algae, a combination of sexual ing of one subtidal and four tidal pool pop- and asexual modes of reproduction is com- ulations (IV). Some subpopulation struc- 23

turing, based on FST-values, dendrogram size or an extreme colonisation-expansion and AMOVA, was discovered in the four F. scenario (Nikula & Väinölä 2003). lumbricalis populations studied in the Bal- The genetic divergence based on mi- tic Sea as well (IV). It is possible that en- crosatellite markers was relatively low, vironmental factors, such as eutrophication especially considering that some of the in the form of, e.g., an increased sedimen- populations included in the study are from tation rate, can inhibit the regeneration of extreme habitats at the edge of the species’ species or populations that depend on short distribution range (III). The highest genet- periods of spore dispersal, while species or ic diversity was discovered from Brittany, populations that mainly disperse by frag- France (III). Furthermore, based on the mentation or have long continuous periods pairwise FST-values, the Norwegian popu- of spore release may gain an advantage lation was clearly differentiated from the (Eriksson & Johansson 2005). rest of the European populations, resulting from isolation during the LGM, a recent populations bottleneck (Väinölä 1992) or 3.4. Phylogeography an isolation-by-distance effect (Watts & Thorpe 2006, Gómez et al. 2007). Earlier, When examining the mitochondrial cox2- it has been suggested that marine glacial 3 spacer in F. lumbricalis in Northern refugia have been present in Southern Europe, no variation in the spacer sequence France (van Oppen et al. 1995a, b, Stam among different geographic populations et al. 2000, Coyer et al. 2003, Gysels et was discovered (III). Previously, Provan al. 2004, Provan et al. 2005, Hoarau et al. et al. (2005) found 13 polymorphic sites 2007) and in northern Scandinavia (van in the cox2-3 sequence when studying the Oppen et al. 1995a, b). Our results clearly phylogeography of the red alga Palmaria support these fi ndings. Furthermore, link- palmata in the North Atlantic. Furthermore, age disequilibrium was more common in Gabrielsen et al. (2002) discovered that the Baltic Sea subpopulations than in the in the red alga Ceramium tenuicorne, the Atlantic Ocean subpopulations (IV). This proportion of intraspecifi c variation in the may be due to random factors infl uenc- cox2-3 spacer sequence was up to 4 % in ing the molecular markers used. However, populations inhabiting the Baltic Sea and high levels of linkage disequilibrium are the southern coast of Norway. However, also expected in recently bottlenecked and in the brown alga Fucus distichus (L.) expanded populations, appearing, e.g., Powell., only one mtDNA haplotype was in areas glaciated during the last ice age found throughout a 2 800-km distance (Hansson & Westerberg 2002). ). It has in the northeast Atlantic (Coyer et al. been earlier suggested that F. lumbricalis 2006), possibly indicating a recent originates from Europe, since its popula- bottleneck or a founder effect (Grant & tions in Northern America are disjunct and Bowen 1998). Furthermore, in the lagoon limited (Bird et al. 1991). The lack of dif- cockle, Cerastoderma glaucum Poiret, ferentiation in the mtDNA sequence along all of the Baltic Sea populations were with a lack of structure in the dendrogram monomorphic based on the mtDNA COI based on the microsatellite data describing gene and allozymes, thus indicating either population differentiation clearly suggests a relatively small post-glacial population that the species has spread into its modern 24 day distribution area from a single or very the populations probably share the same few locations, the Roscoff region in France origin. However, a fair amount of genetic being the most probable area of origin. It variability was discovered in all studied is possible that only a limited amount of populations of F. lumbricalis with micro- divergence has occurred after the last gla- satellite markers. When quantifying the ciation, during which the species survived amount of genetic variability, the highest in one or two glacial refugia. Furthermore, amount of genetic diversity was discov- the high salinity tolerance and capability ered in Brittany, France, which may result of asexual population regeneration may from historical factors, i.e., the species prevent genotype exclusion even in mar- probably resided in the area during the ginal populations. last glaciation. The population in northern Norway was genetically differentiated from all other populations in Northern Eu- 4. CONCLUSIONS rope, probably due to isolation during the last glacial maximum, a recent population The populations of Furcellaria lumbricalis bottleneck or isolation-by-distance effect. in the brackish Baltic Sea are facing diffi cult However, neutral molecular markers prob- environmental conditions, including low ably do not reveal a realistic picture of the prevailing seawater salinity, along with functional genetic diversity within the spe- different factors related to eutrophication, cies and therefore studies on the diversity i.e., reduced amount of light in the water of functional genes and the magnitude of column, increased sedimentation rate variability in their functions are needed. and changes in the biological interactions between different functional faunal and fl oral groups. Thus, it is likely that 5. FUTURE WORK algae respond to the stress caused by the unfavourable environment by altering We have investigated so far the amount their life history parameters, provided of neutral genetic variation among and that there is enough genetic variability in within red algal populations. However, the genome. The increase in the level of neutral genetic variation does not present within-population clonality and the lack a valid picture of the adaptive capabilities of some life cycle phases observed in the of algae in a changing environment. Baltic Sea populations, when compared to Therefore, we have recently initiated a the Atlantic Ocean populations, indicate study on the genetic variation of genes that the overall role of asexual population involved in salinity stress tolerance in the regeneration through thallus fragmentation red alga Furcellaria lumbricalis and in pH and reattachment and/or tetraspore-to- tolerance of an angiosperm water weed, tetraspore cycling is more important in Elodea canadensis, in a project funded the brackish water populations than in the by the Academy of Finland 2007-2010. marine populations. The general aim of the project is to link There were no differences in the the adaptive variation found within the mtDNA cox 2-3 spacer sequence among populations with an environmental factor. the studied populations, indicating that The objectives of this project are: 25

1) To identify stress-responsive genes. 6. ACKNOWLEDGEMENTS 2) To analyse population genetic variation in stress-responsive genes. I would never have managed without the 3) To investigate and compare the evolution help and support of several people. First of neutral and adaptive genomic regions. of all, I would like to thank my supervisors 4) To test the relative importance of pheno- Dr. Helena Korpelainen and Dr. Anita typic plasticity and contemporary evolu- Mäkinen, for their support during the work. tion in plants exposed to changing envi- Helena, the discussions we have had and ronments. the fact that you have always found time to 5) To develop faster and more cost-effec- listen to my problems – both work related tive methods to identify and analyse genes and other – have been really important involved in specifi c, adaptive charac- to me during the years we have worked teristics. together. Furthermore, your ideas about the 6) To develop prognostic tools for moni- new methodology have provided me with toring populations. challenging tasks in the laboratory and I am very happy that we can continue to work At the moment, the genomic regions together in the future, too. Nitsu, you gave containing stress-responsive genes are me the idea for this work and I have to say under search. Thereafter, we will identify it has been really interesting – all the places the genes and study variation within I have seen and all the people I have met. these genes. We are using an approach I would also like to thank Professor involving the exposure of a brackish Christine Maggs and Dr. Jim Provan at water algal individual to marine water. the Queen’s University of Belfast for their After this, the resulting transcribed RNA cooperation. Chris, it was really exciting will be extracted, and complementary to come to your group and discuss algal DNA will be produced for the extracted matters with you – for once I did not have RNA. The Amplifi ed Fragment Length to explain what red algae are or do. Jim, Polymorphism method (AFLP) will then be working with you gave me some valuable used to analyse the differences between a experience which I, hopefully, can use in control sample, incubated in plain brackish the future. water, and an algal sample exposed to I would like to acknowledge Juhani Vait- marine water treatment. Differences in tinen, the Finnish Coast Guard, the Hel- AFLP profi les will be used as indicators of sinki Environment Centre, the Tvärminne genomic regions that are activated during Zoological Station and the Archipelago the exposure of brackish water algae to Research Institute staff for their assistance marine water. After sequencing these in sampling, which in some occasions oc- regions, the full-lengths of the genes from curred in very strange places and in very the transcription-derived fragments will unfavourable conditions. I would also like be reconstructed, and specifi c primers will to thank the Jenny and Antti Wihuri Foun- be developed for the characterised genes dation, Kone Foundation and all the other to allow direct sequencing and population funding bodies that gave me support during genetic sequence analysis to discover single this Ph.D. work. nuclear polymorphisms (SNP) or other The girls in our research group – Hanna, sequence polymorphisms. Maria Po, Maria Pi, Johanna and Tea, sim- 26 ply were there and were ready to take me Arcot, S. S., Wang, Z., Weber, J. L., Deininger, P. L. to coffee breaks even if I was sometimes & Batzer, M. A. 1995: Alu repeats: a source for (or most of the time?) a little bit light- the genesis of primate microsatelites. – Genom- headed or moody. Special thanks to Viivi, ics 29: 136-144. who made me realize that it was time to Asker, S. E. & Jerling, L. 1992: Apomixis in Plants. fi nish the thesis and listened to me during – 299 pp. CRC Press, Boca Raton. Austin, A. P. 1960a: Life history and reproduction of the whole fi nishing process. Special thanks Furcellaria fastigiata (L.) Lam. 1. The haploid also to Marjo who has helped me in the lab plants and the development of the carposporo- and been a good friend. Anne M., thanks phyte. – Annals Bot. N.S. 24: 257-276. for collecting some of the samples. I would Austin, A. P. 1960b: Life history and reproduction like to thank Kent Tankersley for help in in Furcellaria fastigiata (L.). Lam. 2. The tet- prepairing the manuscript. rasporophyte and reduction division in the tet- I would like to thank my friends for their rasporangium. – Annals Bot. N.S. 24: 296-312. support during the years I have been work- Backhaus, J. O. 1996: Climate-sensitivity of Euro- ing on this project – I know that my mind pean marginal seas, derived from the interpre- sometimes tends to wander and it can be tation of modelling studies. – J. Mar. Syst. 7: diffi cult to keep up with me during those 361-382. moments. But it has been really good to Backman, T. W. H. 1991: Genotypic and phenotypic variability of Zostera marina on the West Coast have friends who take me out for bar-hop- of North America. – Can. J. Bot. 69: 1361- ping or to other activities and help me to 1371. forget the stress I have been under. Bell, G. 1994: The comparative biology of the al- And fi nally, thanks to my family for their ternation of generations. – In: Kirkpatrick, M. support and help during the work. Cold win- (ed.), Lectures in mathematics in the life sci- ter days can be really cold when you have ences: theories for the evolution haploid-diploid to stand next to the frozen Baltic Sea, it is life cycles. Am. Math. Soc. Providence, R.I., 25: snowing, you can barely see the bubbles and 1-26. you know it is going to take another hour Bell, H. P. & MacFarlane, C. I. 1933: The marine until you can get back to the car. algae of the Maritime Provinces of Canada. I. List of species with their distributions and prev- alence. – Can. J. Res. 9: 265-279. Bengtsson, B. O. 2003: Genetic variation in organ- 7. REFERENCES isms with sexual and asexual reproduction. – J. Evol. Biol. 16: 189-199. Abrahamson, W. G. 1980: Demography and veg- Bengtsson, B. O. & Ceplitis, A. 2000: The balance etative reproduction. – In: Solbrig, O. T. (ed.), between sexual and asexual reproduction in Demography and evolution in plant populations, plants living in variable environments. – J. Evol. pp. 89-106. Blackwell Scientifi c Publications, Biol. 13: 415-422. Oxford. Bergström, L. & Bergström U. 1999: Species diver- Andersen, B. G. & Borns, H. W. Jr. 1994: The Ice sity and distribution of aquatic macrophytes in Age World: an Introduction to Quaternary His- the Northern Quark, Baltic Sea. – Nord. J. Bot. tory and Research with Emphasis on North 19: 375-383. America and Northern Europe During the Last Bergström, L., Bruno, E., Eklund, B. & Kautsky, L. 2.5 Million Years. – 200 pp. Scandinavia Uni- 2003: Reproductive studies of Ceramium tenu- versity Press, Oslo. icorne near its inner limit in the brackish Baltic Sea. – Bot. Mar. 46: 125-131. 27

Bergström, L., Tatarenkov, A., Johannesson, K., Bischof, K., Hanelt, D. & Wiencke, C. 2000: Effects Jönsson, R. B. & Kautsky, L. 2005: Genetic and of ultraviolet radiation on photosynthesis and morphological identifi cation of Fucus radicans related enzyme reactions of marine macroalgae. sp. nov. (Fucales, Phaeophyceae) in the brackish – Planta 211: 555-562. Baltic Sea. – J. Phycol. 41: 1025-1038. Björck, S. 1995: A review of the history of the Bal- Bhattacharya, D. 1985: The demography of fronds tic Sea, 13.0-8.0 ka BP. – Quatern. Internat. 27: of Chondrus crispus Stackhouse. – J. Exp. Mar. 19-40. Biol. Ecol. 47: 217-231. Bolton, J. J. & Joska, M. A. P. 1993: Population Bierzychudek, P. 1985: Patterns of plant partheno- studies on a South African carrageenophyte: genesis. – Experimentia 41: 1255-1264. Iridaea capensis (Gigartinales, Rhodophyta). Billingham, M. R., Reusch, T., Alberto, F. & Serrão, – Hydrobiologia 261: 191-195. E. 2003: Is sexual reproduction more important Botstein, D., White, R. L., Skalnick, M. H. & Davis, at geographical limits? A genetic study of the R. W. 1980: Construction of a genetic linkage seagrass Zostera marina in the Ria Formosa, map in man using restriction length polymor- Portugal. – Mar. Ecol. Prog. Ser. 265: 77-83. phism. – Am. J. Hum. Genet. 32: 314-331. Bird, C. J., Greenwell, M. & McLachlan, J. 1983: Case, M. & Taper, T. J. 1986. On the coexistence Benthic marine algal fl ora of the north shore of and coevolution of asexual and sexual competi- Prince Edward Island (Gulf of St. Lawrence), tors. – Evolution 40: 366-387. Canada. – Aquat. Bot. 16: 315-335. Ceplitis, A. 2001: The importance of sexual and Bird, C. J., Saunders, G. W. & McLachlan, J. 1991: asexual reproduction in the recent evolution of Biology of Furcellaria lumbricalis (Hudson) Allium vineale. – Evolution 55: 1581-1591. Lamouroux (Rhodophyta: Gigartinales), a com- Chung, M. G. & Epperson, B. K. 1999: Spatial ge- mercial carrageenophyte. – J. Appl. Phycol. 3: netic structure of clonal and sexual reproduc- 61-82. tion in populations of Adenophora grandifl ora Bird, N. 1976: Studies on Gracilaria: ecology of an (Campanulaceae). – Evolution 53: 1068-1078. attached population of Gracilaria sp. at Barra- Coyer, J. A., Hoarau, G., Oudot-Le Secq, M.-P., chois Harbour, Colchester Co., N.S. – Proc. Inst. Stam, W. T. & Olsen, J. L. 2006: A mtDNA- Sci. 27: 144-158. based phylogeny of the brown algal genus Fu- Bird, N. L. 1977: Investigations on Furcellaria lum- cus (Heterokontophyta; Phaeophyta). – Mol. bricalis. Summary report, June 1975-August Phyl. Evol. 39: 209-222. 1977. – Intern. rept, Litex Industri, Glostrup, Coyer, J. A., Peters, A. F., Stam, W. T. & Olsen, J. L. Denmark. 36 pp. 2003: Post-ice age recolonization and differen- Bird, N. L., Chen, L. C.-M. & McLachlan, J. 1979: tiation of Fucus serratus L. (Phaeophyceae; Fu- Effects of temperature, light and salinity on caceae) populations in Northern Europe. – Mol. growth in culture of Chondrus crispus, Furcel- Ecol. 12: 1817-1829. laria lumbricalis, Gracilaria tikvahiae (Gigarti- Craigie, J. S. & Pringle, J. D. 1978: Spatial distri- nales, Rhodophyta) and Fucus serratus (Fucales, bution of tetrasporophytes and gametophytes in Phaeophyta). – Bot. Mar. 22: 521-527. four maritime populations of Chondrus crispus. Bird, N., McLachlan, J. & Grund, D. 1977: Stud- – Can. J. Bot. 47: 2910-2914. ies on Gracilaria. 5. In vitro life history of Dávila, J. A., Loarce, Y., Ramsay, L., Waugh, R. & Gracilaria sp. from the Maritime provinces. Ferrer, E. 1999: Comparison of RAMP and SSR – Can. J. Bot. 55: 1282-1290. markers for the study of wild barley genetic di- Bischof, K., Hanelt, D., Tüg, H., Karsten, U., Brou- versity. – Hereditas 131: 5-13. wer, P. E. M. & Wiencke, C. 1998: Acclimation Destombe, C., Godin, J., Nocher, M., Richerd, S. of brown algal photosynthesis to ultraviolet ra- & Valero M. 1993: Differences in response be- diation in Arctic coastal waters (Spitsbergen, tween haploid and diploid isomorphic phases Norway). – Polar Biol. 20: 388-395. of Gracilaria verrucosa (Rhodophyta: Gigarti- 28

nales) exposed to artifi cial environmental condi- Eriksson, B. K. & Johansson, G. 2005: Effects of tions. – Hydrobiologia 260/261: 131-137. sedimentation on macroalgae: species-specifi c Destombe, C., Valero, M., Vernet, P. & Couvet, D. responses are related to reproductive traits. 1989: What controls the haploid-diploid ratio in – Oecologia 143: 438-448. the red alga, Gracilaria verrucosa? – J. Evol. Eriksson, O. 1986: Survivorship, reproduction and Biol. 2: 317-338. dynamics of ramets of Potentilla anserina on a DeWreede, R. E. & Green, L. G. 1990: Patterns of Baltic seashore meadow. – Vegetatio 67: 17-25. gametophyte dominance of Iridaea splendens Fierst, J., terHost, C., Kübler, J. E. & Dudegeon, S. (Rhodophyta) in Vancouver Harbour, Vancou- 2005: Fertilization success can drive patterns of ver, British Columbia, Canada. – J. Appl. Phy- phase dominance in complex life histories. – J. col. 47: 27-34. Phycol. 41: 238-249. Dixon, P. S. 1965: Perennation, vegetative propaga- Figueroa, F. L., Salles, S., Aguilera, J. Jiménez, C., tion and algal life histories, with special refer- Mercado, J., Viñegla, B., Flores-Moya, A. & Al- ence to Asparagopsis and other Rhodophyta. tamirano, M. 1997: Effetcs of solar radiation on – Bot. Gothob. 3: 67-74. photoinhibition and pigmentation in the red alga Dring, M. J. 1982: The Biology of Marine Plants. Porphyra leucosticta. – Mar. Ecol. Progr. Ser. – 199 pp. London, Edward Arnold. 151: 81-90. Düwel, L. 2001: Experimental Studies on Mac- Gabriel, W. & Bürger, R. 2000: Fixation of clonal roalgae Along the Salinity Gradient in the Baltic lineages under Muller’s ratchet. – Evolution 54: Sea. – Ph.D. Thesis, Bot. Inst. Univ. Copenha- 1116-1125. gen, Denmark. 77 pp. Gabrielsen, T. M., Brochmann, C. & Rueness, J. Dyck, L. J. & DeWreede, R. E. 1995: Patterns of sea- 2002: The Baltic Sea as a model system for sonal demographic change in the alternate isomor- studying post-glacial colonization and ecologi- phic stages of Mazzaella splendens (Gigartinales, cal differentiation, exemplifi ed by the red alga Rhodophyta). – Phycologia 47: 390-395. Ceramium tenuicorne. – Mol. Ecol. 11: 2083- Dyck, L. J., DeWreede, R. E. & Garbary, D. 1985: 2095. Life history phases in Iridaea cordata (Gigar- Gabrielson, P. W., Garbary, D. J., Sommerfeldt, M. tinaceae): Relative abundance and distribution R., Townsend, R. A. & Tyler, P. L. 1990: Phylum from British Columbia to California. – Jpn. J. Rhodophyta. – In: Margulis, L., Corliss, J. O., Phycol. 47: 225-232. Melkonian, M. & Chapman, D. J. (eds.), Hand- Ellstrand, N. C. & Roose, M. L. 1987: Patterns book of Protoctista: The Structure, Cultivation, of genotypic diversity in clonal plant species. Habitats and Life Histories of the Eucaryotic – Am. J. Bot. 74: 123-131. Microorganisms and Their Descendants Exclu- Engel, C., Åberg, P., Gaggiotti, O. E., Destombe, C. sive of Animals, Plants and Fungi. 914 pp. Jones & Valero, M. 2001: Population dynamics and and Bartlett Pub., Boston. stage structure in a haploid-diploid red seaweed, Gaggiotti, O. E. 1994: An ecological model for the Gracilaria gracilis. – J. Ecol. 89: 436-450. maintenance of sex and geographic partheno- Engel, C. R., Destombe, C. & Valero, M. 2004: genesis. – J. Theor. Biol. 167: 201-221. Mating system and gene fl ow in the red seaweed Gómez, A., Hughes, R. N., Wright, P. J., Carvalho, Gracilaria gracilis: effect of haploid-diploid G. R. & Lunt, D. H. 2007: Mitochondrial DNA life history and intertidal rocky shore landscape phylogeography and mating compatibility re- on fi ne-scale genetic structure. – Heredity 92: veal marked genetic structuring and speciation 289-298. in the NE Atlantic bryozoan Celleporella hyali- Erickson, D. L., Hamrick, J. L. & Kochert, G. D. na. – Mol. Ecol. 16: 2173-2188. 2004: Ecological determinants of genetic di- Gonzáles, J. & Meneses, I. 1996: Differences in the versity in an expanding population of the shrub early stages of development of gametophytes Myrica cerifera. – Mol. Ecol. 13: 1655-1664. and tetrasporophytes of Chondracanthus cham- 29

issoi (C. Ag.) Kützing from Puerto Aldea, north- depth distribution. – Mar. Ecol. Progr. Ser. 149: ern Chile. – Aquaculture 47: 91-107. 255-266. Graham, L. D. & Wilcox, L. 2000: Algae. – 640 pp. Hannach, G. & Santelices, B. 1985: Ecological differ- Prentice-Hall, Inc., NJ. ences between the isomorphic reproductive phas- Grant, W. S. & Bowen, B. W. 1998: Shallow popu- es of two species of Iridaea (Rhodophyta: Gigar- lation histories in deep evolutionary lineages of tinales). – Mar. Ecol. Prog. Ser. 47: 291-303. marine fi shes: insights from sardines and ancho- Hansen, J.E. & Doyle, W.T. 1976: Ecology and vies and lessons for conservation. – J. Hered. 89: natural history of Iridaea cordata (Rhodophyta: 415-426. Gigartinaceae): Population structure. – J. Phy- Guillemin, M.-L., Destombe, C., Faugeron, S., Cor- col. 47: 273-278. rea, J.A. & Valero, M. 2005: Development of Hansson, B. & Westerberg, L. 2002: On the correla- microsatellite DNA markers in the cultivated tion between heterozygosity and fi tness in natu- seaweed, Gracilaria chilensis (Gracilariales, ral populations. – Mol. Ecol. 11: 2467-2474. Rhodophyta). – Mol. Ecol. Notes 5: 155-157. Hawkes, M. W. 1990: Reproductive strategies. – In: Gustafsson, B. G. 2001: Quantifi cation of water, Cole, K. M. & Sheath, R. G. (eds.), Biology of salt, oxygen and nutrient exchange of the Bal- the red algae, pp. 305-347. Cambridge Univer- tic Sea from observations in the Arkona Basin. sity Press, Cambridge. – Cont. Shelf. Res. 21: 1485-1500. Hay, M. E., Duffy, J. E., Pfi ster, C. A. & Fenical, W. Gysels, E. S., Hellemans, B., Pampoulie, P. & 1987: Chemical defense against different marine Volckaert, F. A. M. 2004: Phylogeography of the herbivores: are amphipods insect equivalents? common goby, Potomaschistus microps, with – Ecology 68: 1567-1580. particular emphasis on the recolonization of the Hay, M. E., Pawlik, J. R., Duffy, J. E. & Fenical, W. Mediterranean and the North Sea. – Mol. Ecol. 1989: Seaweed-herbivore-predator interactions: 13: 403-417. host plant specialization reduces predation on Hällfors, G., Niemi, Å., Ackefors, H., Lassig, J. & small herbivores. – Oecologia 81: 418-427. Leppäkoski, E. 1981: Biological oceanography. Herbert, P. D. N. 1987: Genotypic characteristics of – In: Voipio, A. (ed.), The Baltic Sea, pp. 219- cyclic parthenogens and their obligately asexual 274. Amsterdam. derivatives. – In: Stearns, S. C. (ed.), The Evolu- Hämmerli, A. & Reusch, T. B. H. 2002: Local ad- tion of Sex and its Consequences, pp. 175-195. aptation and transplant dominance in genets of Birkhäuser, Basel. the marine clonal plant Zostera marina. – Mar. Hemmi, A. & Jormalainen, V. 2004: Genetic and en- Ecol. Prog. Ser. 242: 111-118. vironmental variation in performance of a ma- Hämmerli, A. & Reusch, T. B. H. 2003: Inbreeding rine isopod: effects of eutrophication. – Oecolo- depression infl uences genet size distribution in a gia 140: 302-311. marine angiosperm. – Mol. Ecol. 12: 619-629. Hoarau, G., Coyer, J. A., Veldsink, J. H., Stam, W. T. Hammock, E. A. & Young, L. J. 2004: Functional & Olsen, J. L. 2007: Glacial refugia and recolo- microsatellite polymorphism associated with di- nization pathways in the brown seaweed Fucus vergent social structure in vole species. – Mol. serratus. – Mol. Ecol. 16: 3606-3616. Biol. Evol. 21: 1057-1063. Holmsgaard, J. E., Greenwell, M. & McLachlan, Hancock, J. M. 1999: Microsatellites and other sim- J. 1981: Biomass and vertical distribution of ple sequences: genomic context and mutational Furcellaria lumbricalis and associated algae. mechanisms. – In: Goldstein, D. B. & Schlöt- – Proc. Int. Seaweed Symp. 10: 309-314. terer, C. (eds.), Microsatellites. Evolution and Hommersand, M. H. & Fredericq, S. 1990: Sexual Applications, pp. 1-9. Oxford Univ. Press. reproduction and cystocarp development. – In: Hanelt, D., Melchersmann, B., Wiencke, C. & Nult- Cole, K. M. & Sheath, R. G. (eds.), Biology of sch, W. 1997: Effects of high light stress on pho- the red algae, pp. 305-347. Cambridge Univer- tosynthesis of polar macroalgae in relation to sity Press, Cambridge. 30

Hughes, J. S. & Otto, S. P. 1999: Ecology and the The ecology and evolution of clonal plants, pp. evolution of biphasic life cycles. – Am. Nat. 1-29. Backhuys Publishers, Leiden. 154: 306-320. Kornfeldt, R.-A. 1979: Makroalgers biomassa längs Johannesson, K. & André, C. 2006: Life on the Hallandskusten. (Biomass of macroalgae along margin: genetic isolation and diversity loss in the coast of Halland, SW Sweden.) – Svensk. a peripheral marine ecosystem, the Baltic Sea. Bot. Tidskr. 73: 131-138. – Mol. Ecol. 15: 2013-2029. Korpelainen, H., Kostamo, K., Karttunen, H. & Vir- Johansson, G. 2002: Factors affecting the distribu- tanen, V. 2007: Isolation and characterization tion of rocky-shore macroalgae on the Swedish of microsatellite loci for twenty common bryo- coast. Morphological, physiological, reproduc- phyte species. – Submitted manuscript. tive and genetic aspects. – Acta Universitas Up- Kostamo, K., Blomster, J., Korpelainen, H. & saliensis, Uppsala. 34 pp. Maggs, C. A. 2007: The development of new Kain, J. M. 1984: Plocamium cartilagineum in the microsatellite markers of Ulva intestinalis and Isle of Man: why are there so many tetrasporo- the transferability of microsatellite markers phytes? – Br. Phycol. J. 19: 195. across species of Ulvales. – Manuscript. Kangas, P., Autio, H., Hällfors, G., Luther, H., Nie- Kristiansen, A. A., Pedersen, P. M. & Moseholm, mi, Å. & Salemaa H. 1982: A general model of L. 1994: Salinity-temperature effects on growth the decline of Fucus vesiculosus at Tvärminne, and reproduction of Scytosiphon lomentaria south coast of Finland in 1977-81. – Acta Bot. (Fucophyceae) along the salinity gradient in Fennica 118: 1-27. Danish waters. – Phycologia 33: 444-454. Karentz, D. & Lutze, L. H. 1990: Evaluation of bio- Lazo, M. L., Greenwell, M. & MaLachlan, J. 1989: logically harmful ultraviolet radiation in Ant- Population structure of Chondrus crispus Stack- arctica with a biological dosimeter designed for house (Gigartinaceae, Rhodophyta) along the aquatic environment. – Limnol. Oceanogr. 35: coast of Prince Edward Island, Canada: Distri- 549-561. bution of gametophytic and sporophytic fronds. Karlsson, J., Kuylenstierna, M. & Åberg, P. 1992: – J. Exp. Mar. Biol. Ecol. 47: 45-58. Contribution to the seaweed ﬂ ora of Sweden: Levring, T. 1940: Studier über die Algenvegetation new or otherwise interesting records from the von Blekinge, Südschweden. – 179 pp. PhD dis- west coast. – Acta Phytogeogr. Suec. 78: 49-63. sertation. University of Lund, Lund. Kautsky, L. & Kautsky, H. 1989: Algal species di- Lewis, W. M. 1985: Nutrient scarcity as an evolu- versity and dominance along gradients of stress tionary cause of haploidy. – Am. Nat. 125: 692- and disturbance in marine environments. – Veg- 701. etatio 83: 259-267. Lindgren, A. & Åberg, P. 1996: Proportion of life Kirk, J. T. O. 1983: Light and photosynthesis in cycle stages of Chondrus crispus and its popula- aquatic ecosystems. – 401 pp. Cambridge Uni- tion structure: a comparison between a marine versity Press, Cambridge. and an estuarine environment. – Bot. Mar. 47: Kirk, J. T. O. 1994: Optics of UV-B radiation in nat- 263-268. ural waters. – Arch. Hydrobiol. B., 43: 1-16. Lobban, C. S. & Harrison, P. J. 1994: Seaweed Ecol- Kirst, G. O. 1990: Salinity tolerance of eukaryotic ogy and Physiology. 1st ed. – 362 pp. Cambridge marine algae. – Ann. Rev. Plant Physiol. Plant Univ. Press. Mol. Biol. 41: 21-53. Luo, H., Mörchen, M., Engel, C. R., Destombe,C., Kirst, O. & Wiencke, C. 1995: Ecophysiology of Epplen, J. T., Saumitou-Laprade, P. & Valero, M. polar algae. – J. Phycol. 31: 181-199. 1999: Characterization of microsatellite markers Klimeš, L., Klimešová, J., Hendriks, R. & van in the red alga Gracilaria gracilis. – Mol. Ecol. Groenendael, J. 1997: Clonal plant architecture: 8: 700-702. a comparative analysis of form and function. Luttikhuizen, P. C., Drent, J. & Baker, A. J. 2003: – In: de Kroon, H & van Groenendael, J. (eds.), Disjunct distribution of highly diverged mito- 31

chondrial lineage clade and population subdivi- and genetic variation in clonal plants. – In: de sion in a marine bivalve with pelagic larval dis- Kroon, H. & van Groenendael, J. (eds.), The persal. – Mol. Ecol. 12: 15-29. ecology and evolution of clonal plants, pp. 185- Lüning, K. 1981: Light. – In: Lobban C. S. and 210. Backhuys Publishers, Leiden. Wynne, M. J. (eds.), The biology of seaweeds, Morgante, M., Hanafey, M. & Powell, W. 2002: Mi- pp. 326-355. Blackwell Scientifi c Publishers, crosatellites are preferentially associated with Oxford. nonrepetetive DNA in plant genomes. – Nature Mabel, B. K. & Otto, S. P. 1998: The evolution Gen. 30: 194-200. of life cycles with haploid and diploid phases. Nei, M. 1978: Estimation of average heterozygos- – Bioessays 20: 453-462. ity and genetic distance from a small number of Maegewa, M., Kunieda, M. & Kida, W. 1993: The individuals. – Genetics 89: 583-590. infl uence of ultraviolet radiation on the photo- Niemi, Å. 1975: Ecology of phytoplankton in the synthetic activity of several red algae from dif- Tvärminne area, SW coast of Finland. II. Pri- ferent depths. – Jpn. J. Phycol. 41: 207-214. mary production and environmental conditions Maggs, C. A. 1988: Intraspecifi c life history vari- in the archipelago and the sea zone. – Acta Bot. ability in the Florideophycidae (Rhodophyta). Fennica 105: 1-73. – Bot. Mar. 31: 465-490. Nikula, R. & Väinölä, R. 2003: Phylogeography of Maggs, C. A. 1998: Life history variation in Dasya Cerastoderma glaucum (Bivalvia: Cardiidae) ocellata (Dasyaceae, Rhodophyta). – Phycolo- across Europe: a major break in the Eastern gia 37: 100-105. Mediterranean. – Mar. Biol. 143: 339-350. Maggs, C. A. & Cheney, D. P. 1990: Competition Nikula, R., Strelkov, P. & Väinölä, R. 2007: Diver- studies of marine macroalgae in laboratory cul- sity and trans-Arctic invasion history of mito- ture. – J. Phycol. 26: 18-24. chondrial lineages in the North Atlantic Ma- Mäkinen, A., Kääriä, J. & Rajasilta, M. 1988. Fac- coma balthica complex (Bivalvia: Tellinidae). tors controlling the ocurrence of Furcellaria – Evolution 61: 928-941. lumbricalis (Huds.) Lamour. and Phyllophora Oliveira, W. J., Pádua, J. G., Zucchi, M. I., Camar- truncata (Pallas) Zinova in the upper littoral of go, L. E. A., Fungaro, M. H. P. & Vieira, M. L. the archipelago of SW Finland. – Kieler Meeres- C. 2005: Development and characterization of forsch., Sonderh. 6: 1404-1446. microsatellite markers from the yellow passion Mathieson, A. C. 1982: Reproductive phenology fruit (Passifl ora edulis f. fl avicarpa). – Mol. and sporeling ecology of Chondrus crispus Ecol. Notes 5: 331-333. Stackhouse. – In: Tsuda, R. T. & Chiang, Y.-M. Olsen, J. L., Stam, W. T., Coyer, J. A., Reusch, T. (eds.), Proceedings of the Republic of China- B. H., Billingham, M., Boström, C., Calvert, E., United States Cooperative Science Seminar on Christie, H., Granger, S., La Lumiere, R., Mil- Cultivation and Utilization of Economic Algae. chakova, N., Oudot-Le Secq, M. P., Pocaccini, Univ. Guam Mar. Lab., Mangilao, pp. 33-40. G., Sanjabi, B., Serrao, E., Veldsink, J., Widdi- Mathieson, A. C. & Burns, R. L. 1975: Ecological combe, S. & Wyllie-Echeverria, S. 2004: North studies of economic red algae. V. Growth and re- Atlantic phylogeography and large-scale popu- production of natural and harvested populations lation differentiation of the seagrass Zostera ma- of Chondrus crispus Stackhouse in New Hamp- rina L. – Mol. Ecol. 13: 1923-1941. shire. – J. Exp. Mar. Biol. Ecol. 47: 137-156. Pacheco-Ruíz, I. & Zertuche-González, J. A. 1999: May, G. 1986: Life history variations in a predomi- Population structure and reproduction of the car- nantly gametophytic population of Iridaea cor- rageenophyte Chondracanthus pectinatus in the data (Gigartinaceae, Rhodophyta). – J. Phycol. Gulf of California. – Hydrobiologia 398/399: 47: 448-455. 159-165. McLellan, A. J., Prati, D., Kaltz, O. & Schmid, Palmieri, D. A., Bechara, M. D., Curi, R. A., B. 1997: Structure and analysis of phenotypic Gimenes, M. A. & Lopes, C. R. 2005: Novel 32

polymorphic microsatellite markers in section Rietema, H. 1993: Ecotypic differences between Caulorrhizae (Arachis, Fabacae). – Mol. Ecol. Baltic and North Sea populations of Delesseria Notes 5: 77-79. sanguinea and Membranoptera alata. – Bot. Phillips, B. 1994: Ecological differences between the Mar. 36: 15-21. isomorphic phases of Mazzaella lilacina (Rho- Rietema, H. 1995: Ecoclinal variation in Rhodomela dophyta, Gigartinaceae): (1) spore production, confervoides along a salinity gradient in the North (2) recruitment specialization, (3) resistance to Sea and Baltic Sea. – Bot. Mar. 38: 475-479. removal by wave action. – 113 pp. MSc Thesis, Röhner, M. R., Bastorp, R. & Jürss, K. 1997: Genet- University of British Columbia, Vancouver. ic differentiation in Hediste diversicolor (Poly- Piquot, Y., Petit, D., Valero, M., Cuguen, J., de La- chaetea: Neredididae) for the North Sea and the guerie, P. & Vernet, P. 1998: Variation in sexual Baltic Sea. – Mar. Biol. 130: 171-180. and asexual reproduction among young and old Rönnberg, O., Ådjers, K., Ruokolahti, K. & Bonde- populations of the perennial macrophyte Spar- stam, M. 1992: Effects of ﬁ sh farming on ganium erectum. – Oikos 82: 139-148. growth, epiphytes and nutrient content of Fucus Piriz, K. L. 1996: Phenology of a Gigartina skotts- vesiculosus L. in the Åland archipelago, north- bergii Setchell et Gardner population in Chubut ern Baltic Sea. – Aquat. Bot. 42: 109-120. province (Argentina). – Bot. Mar. 39: 311-316. Rosenvinge, L. K. 1917: The marine algae of Den- Provan, J., Wattier, R. A. & Maggs, C. A. 2005: mark. Contributions to their natural history II. Phylogeographic analysis of the red seaweed Rhodophyceae II (Cryptonemiales). – K. Dan- Palmaria palmata reveals a Pleistocene marine ske. Vidensk. Selsk. Skr. 7 Række, Naturvi- glacial refugium in the English Channel. – Mol. densk. Math. Afd. VII, 2: 155-238. Ecol. 14: 709-803. Sagert, S., Forster, R. M., Feuerpfeil, P. & Schu- Ramus, J. 1983: A physiological test of the theory bert, H. 1997: Daily course of photosynthesis of complementary chromatic adaptation. II. and photoinhibition in Chondrus crispus (Rho- Brown, green and red seaweeds. – J. Phycol. 19: dophyta) from different shore levels. – Eur. J. 173-179. Phycol. 32: 363-371. Raven, J. A. 1999: Constraints on marine algal in- Salisbury, E. J. 1942: The reproductive capacity of vasion of low-salinity environments: Sex in the plants. – G. Bell & Sons, London. Baltic. – J. Phycol. 35: 210-212. Santelices, B., Aedo, D., Hormazábal, M. & Flores, Richards, C. M., Brownson, M., Mitchell, S. E., V. 2003: Field testing of inter- and intraspeciﬁ c Kresovich, S. & Panella, L. 2004: Polymorphic coalescence among mid-intertidal red algae. microsatellite markers for inferring diversity – Mar. Ecol. Progr. Ser. 250: 91-103. in wild and domesticated sugar beet (Beta vul- Santelices, B., Correa, J. A., Aedo, D., Flores, V., garis). – Mol. Ecol. Notes. 4: 143-145. Hormazábal, M. & Sanchez, P. 1999: Conver- Richerd, S., Couvet, D. & Valero, M. 1993: Evo- gent biological processes in coalescing Rhodo- lution of the alteration of haploid and diploid phyta. – J. Phycol. 35: 1127-1149. phases in life cycles. II. Maintenance of the hap- Santelices, B., Correa, J. A., Meneses, I., Aedo, D. lo-diplontic cycle. – J. Evol. Biol. 6: 263-280. & Varela, D. 1996: Sporeling coalescence and Richerd, S., Perrot, V., Couvet, D., Valero, M. & intraclonal variation in Gracilaria chilensis Kondrashov, A. S. 1994: Deleterious mutations (Gracilariales, Rhodophyta). – J. Phycol. 32: can account for the maintenance of the haplo- 313-322. diplont cycle. – In: Beaumont, A. R. (ed.), Ge- Saunders, G. W. & Hommersand, M. H. 2004: As- netics and evolution of aquatic organisms, pp. sessing red algal supraordinal diversity and tax- 263-280. Chapman and Hall, New York. onomy in the context of contemporary system- Rietema, H. 1991: Evidence for ecotypic divergence atic data. – Am. J. Bot. 91: 1494-1507. between Phycodrys rubens populations from the Schwenke, H. 1971: Water movements 5.2. Plants. Baltic Sea and North Sea. – Bot. Mar. 34: 375- – In: Kinne, O. (ed.), Marine Ecology I. Envi- 381. 33

ronmental factors (2), pp. 1091-1121. Wiley- Suomalainen, E., Saura, A. & Lokki, J. 1987: Cytol- Interscience, London. ogy and Evolution in Parthenogenesis. – 216 pp. Scrosati, R. 1998: Population structure and dy- CRC Press, Boca Raton. namics of the clonal alga Mazzaella cornuco- Svedelius, N. 1901: Studier öfver Östersjöns hafsalg- piae (Rhodophyta, Gigartinceae) from Barkley ﬂ ora. – 140 pp. PhD dissertation. University of Sound, Paciﬁ c coast of Canada. – Bot. Mar. 47: Uppsala, Uppsala. 483-493. Tatarenkov, A., Bergström, L., Jönsson, R. B., Ser- Scrosati, R., Garbary, D. J. & McLachlan, J. 1994: rão, E. A., Kautsky, L. & Johannesson, K. 2005: Reproductive ecology of Chondrus crispus Intriguing asexual life in marginal populations (Rhodophyta, Gigartinales) from Nova Scotia, of the brown seaweed Fucus vesiculosus. – Mol. Canada. – Bot. Mar. 47: 293-300. Ecol. 14: 647-651. Serrão, E. A., Brawley, S. H., Hedman, J., Kautsky, Taylor, A. R. A. 1975: The Chondrus crispus – Fur- L. & Samuelsson, G. 1999: Reproductive suc- cellaria fastigiata community at Campbell’s cess in Fucus vesiculosus (Phaeophyceae) in the Cove, Prince Edward Island. – 72 pp. Tech. Baltic Sea. – J. Phycol. 35: 1-2. Rept. Ser., 88, Industr. Dev. Br., Fish. Mar. Serv. Serrão, E. A., Kautsky, L. & Brawley, S. H. 1996: Environment Canada, Ottawa. Distributional success of the marine seaweed Tokida, J. & Yamamoto, H. 1965: Syntagmatic ger- Fucus vesiculosus L. in the brackish Baltic Sea mination of tetraspores in Pachymeniopsis yen- correlates with osmotic capabilities of Baltic doi. – Phycologia 5: 15-20. gametes. – Oecologia 107: 1-12. Väinölä, R. 1992: Evolutionary genetics of marine Smith, R. I. 1964: On the early development of Mysis spp. (Crustacea: Mysidacea). – Mar. Biol. Nereis diversicolor in different salinities. – J. 114: 539-550. Morph. 114: 437-464. Väinölä, R. 2003: Repeated trans-Arctic invasions Smith, R. I. 1977: Physiological and reproductive in littoral bivalves: molecular zoogeography of adaptations of Nereis diversicolor to life in the the Macoma baltica complex. – Mar. Biol. 143: Baltic Sea and adjacent waters. – In: Reish, D. J. 935-946. & Fauchauld, K. (eds.), Essays on polychaetous Väinölä, R. & Hvilsom, M. M. 1991: Genetic di- annelids, pp. 449-476. In memory of Dr. Olga vergence and a hybrid zone between Baltic and Hartman, Allan Hancock Foundation, Univ. North Sea Mytilus populations (Mytilidae, Mol- South. Cal., Los Angeles. lusca). – Biol. J. Linn. Soc. 43: 127-148. Squirrel, J., Hollingsworth, P. M., Woodhead, M., Valatka, S., Mäkinen, A. & Yli-Mattila, T. 2000: Russell, J., Lowe, A. J., Gibby, M. & Powell, W. Analysis of genetic diversity of Furcellaria lum- 2003: How much effort is required to isolate nu- bricalis (Gigartinales, Rhodophyta) in the Baltic clear microsatellites from plants? – Mol. Ecol. Sea by RAPD-PCR technique. – Phycologia 39: 12:1339-1348. 109-117. Stam, W. T., Olsen, J. L. & Coyer, J. A. 2000: Post- Valle, G. 1993: TA-repeat microsatellites are close- glacial recolonization and biogeographic pat- ly associated with ARS consensus sequences in terns in the North Atlantic. – Phycologia 40s, yeast chromosome III. – Yeast 9: 753-759. 46. van Oppen, M. J. H., Draisma, S. G. A., Olsen, J. Stöcklin, J. 1999: Differences in life history traits L. & Stam, W. T. 1995a: Multiple trans-Arctic of related Epilobium species: clonality, seed size passages in the red alga Phycodrys rubens: evi- and seed number. – Folia Geobot. 34: 7-18. dence from nuclear rDNA ITS sequence. – Mar. Stöcklin, J. & Winkler, E. 2004: Optimum reproduc- Biol. 123: 179-188. tion and dispersal strategies of a clonal plant in van Oppen, M. J. H., Olsen, J. L. & Stam, W. T. a metapopulation: a simulation study with Hier- 1995b: Genetic variation within and among acium pilosella. – Evol. Ecol. 18: 563-584. North Atlantic and Baltic populations of the 34

benthic alga Phycodrys rubens (Rhodophyta). Wilder, J. & Hollocher, H. 2001: Mobile elements – Eur. J. Bot. 30: 251-260. and the genesis of microsatellites in dipterans. Wærn, M. 1952: Rocky-shore algae in the Öre- – Mol. Biol. Evol. 18: 384-392. grund Archipelago. – Acta Phytogeogr. Suec. Winkler, E. & Fischer, M. 2001: The role of vegeta- 30: 1-298. tive and seed dispersal within habitats for opti- Wallentinus, I. 1984: Partitioning of nutrient up- mal life histories of clonal plants: a simulation take between annual and perennial seaweeds study. – Evol. Ecol. 15: 281-301. in the Baltic archipelago area. – Hydrobiologia Wright, P. J. & Reed, R. H. 1990: Effects of osmotic 116/117: 363-370. stress on gametic size, rhizoid initiation and Wallentinus, I. 1991: The Baltic Sea gradient. – In: germling growth in fucoid algae. – Br. Phycol. Mathieson, A. C. & Nienhuis, P. H. (eds.), Inter- J. 25: 149-155. tidal and Littoral Ecosystems. Ecosystems of the Yoon, H. S., Müller, K. M., Sheath, R. G., Otto, F. World 24: 83-108. Elsevier, London. D. & Bhattacharya, D. 2006: Defi ning the major Watts, P. C. & Thorpe, J. P. 2006: Infl uence of con- lineages of red algae (Rhodophyta). – J. Phycol. trasting larval developmental types upon the 42: 482-492. population-genetic structure of cheilostome bry- Zamorano, J. & Westermeier, R. 1996: Phenology ozoans. – Mar. Biol. 149: 1093-1101. of Gigartina skottsbergii (Gigartinaceae, Rho- West, J. A. 1970: The conspecifi city of Heterosipho- dophyta) in Ancud Bay, southern Chile. – Hyd- nia asymmetria and H. densiculata and their life robiologia 47/327: 253-258. histories in culture. – Madroño 20: 313-319. Zane, L., Bargelloni, L. & Patarnello, T. 2002: West, J. A., Zuccarello, G. C. & Kamiya, M. 2001: Strategies for microsatellite isolation: a review. Reproductive patterns of Caloglossa species – Mol. Ecol. 11: 1-16. (Delesseriaceae, Rhodophyta) from Australia Zenkevitch, L. 1963: Biology of the seas of the and New Zealand: multiple origins of asexuality U.S.S.R. – 955 pp. Interscience Publishers, New in C. leprieurii. Literature review on apomixis, York. mixed-phase, bisexuality and sexual compatibil- Zuccarello, G. C., Burger, G., West, J. A. & King, ity. – Phycol. Res. 49: 183-200. R. J. 1999: A mitochondrial marker for red algal intraspecifi c relationships. – Mol. Ecol. 8: 1443- 1447.