PhD-thesis © Lena Dahlman, 2003. Resource acquisition and allocation in Lichens Department of Ecology and Environmental Science Umeå University SE-901 87 Umeå

Front cover, designed by Lena Dahlman showing a section through the tripartite lichen Lobaria pulmonaria. The dark lump is a cephalodium, containing the cyanobacte- rial photobiont and the crown-shaped structure on the right is a fungal vegetative reproductive structure called the soralium with little ball-shaped soredia on top. The green layer is the colony of the photobiont Dictyochloropsis located in the medulla. This lichen’s nitrogen acquisition is studied in paper V, and the resource allocation patterns of tripartite lichens are more thoroughly investigated in papers I, II, and III

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ISBN 91-7305-496-8

2 PhD-thesis © Lena Dahlman Department of Ecology and Environmental Science Umeå University SE-901 87 Umeå ISBN 91-7305-496-8

Abstract Lichens are fascinating symbiotic systems, where a fungus and a unicellular alga, most often green (bipartite green algal lichens; 90% of all lichens), or a fi lamentous cyanobacterium (bipartite cyano- bacterial lichens; 10% of all lichens) form a new entity (a thallus) appearing as a new and integrated organism: in about 500 lichens the fungus is associated with both a cyanobacterium and an alga (tripartite lichens). In the thallus, the lichen bionts function both as individual organisms, and as a symbiont partner. Hence, in lichens, the participating partners must both be able to receive and acquire resources from the other partner(s) in a controlled way. Lichens are particularly successful in harsh terrestrial environments. In part this is related to their poikilohydric nature and subsequent ability to repeatedly become desiccated and hydrated.

Metabolic activity, i.e. photosynthesis, respiration, and for cyanobacterial lichens N2-fi xation, is limited to periods when the thallus is suffi ciently hydrated. Mineral nutrients are mainly acquired from dry or wet deposition directly on the thallus. Taken together it then appears that lichens are to a large extent passively controlled by their environment, making their control over resource allocation and acquisition particularly challenging. The aim of this thesis was to investigate resource acquisition and allocation processes in different lichens, and to see how these respond to changes in resource availability. This was done by following lichen growth in the fi eld during manipulation of water, light, and nutrient supply, and by assessing the responses of both the integrated thallus as well as the individual bionts. As a fi rst step, resource allocation and acquisition was investigated for a broad range of lichens aiming to determine the magnitude of metabolic variation across lichens. Seventy-fi ve lichen species were selected to cover as broad a spectrum as possible regarding taxonomy, morphology, habitat, and nitrogen require- ments. The lichens had invested their nitrogen resources so that photosynthetic capacity matched respiratory carbon demand around a similar equilibrium across the contrasting species. Regulation of lichen growth was investigated in another study, using the two tripartite species Nephroma arcti- cum and Peltigera aphthosa, emphasizing the contribution of both internal and external factors. The empirical growth models for the two lichens were similar, showing that weight gain is to a higher extent dependent on those external factors that regulate their photosynthesis, whilst area gain is more controlled by internal factors, such as their nitrogen metabolism. This might be inferred from another study of the same species, where nitrogen manipulations resulted in an undisturbed weight gain, a similar resource allocation pattern between the bionts, but a distorted area gain. Aiming to investigate lichen nitrogen relations even further, lichens’ capacities to assimilate combined nitrogen in the form of ammonium, nitrate and amino acids were assessed using 14 contrasting boreal species. All these had the capacity to assimilate all the three nitrogen forms, with ammonium absorption being more passive, and nitrate uptake being low in bipartite cyanobacterial lichens. Differences in uptake capacities between species were more correlated to photobiont than to morphology or substrate preferences. Finally, to investigate intra-specifi c plasticity in relation to altered nutrient supply, resource investments between photo- and mycobiont were investigated in the two bipartite green algal lichens Hypogymnia physodes aandnd Platismatia glauca iinn a llowow aandnd a hhighigh nnutrientutrient environ-environ- ment. In both species, more of the resources had been directed to the photobiont in the high nutrient environment also increasing their overall carbon status. Taken together, my studies indicate that in spite of the apparent passive environmental control on lichen metabolism, these symbiotic organisms are able to both optimize and control their resource acquisition and allocation processes.

3 List of papers

This thesis is based on the following publications, which will be referred to in the text by their respective Roman numerals.

I. Palmqvist K, Dahlman L, Valladares F, Tehler A, Sancho LG,

Mattsson J-E (2002) CO2 exchange and thallus nitrogen across 75 contrasting lichen associations from different climate zones. Oecologia. 133:295-306 II. Dahlman L, Palmqvist K (2003) Growth in two foliose tripartite lichens Nephroma arcticum and Peltigera aphthosa – empirical modelling of external versus internal factors. Functional Ecology (In Press) III. Dahlman L, Näsholm T, Palmqvist K (2002) Growth, nitrogen uptake, and resource allocation in the two tripartite lichens Nephroma arcticum and Peltigera aphthosa during nitrogennitrogen stress.stress. New Phytologist. 153: 307-315. IV. Dahlman L, Persson J, Näsholm T, Palmqvist K (2003) Carbon and nitrogen distribution in the green algal lichens Hypogymnia physodes and Platismatia glauca in relationrelation to nutrient supply.supply. Planta. 217:41-48. V. Dahlman L, Persson J, Palmqvist K, Näsholm T. (2003) Organic and inorganic nitrogen uptake in lichens. Submitted to Planta.

Papers I, II, III and IV are published with the kind permission of the publishers. Table of Contents

1. Symbiotic systems ______7

2. Why study lichens? ______7

3. General description of the lichen symbiosis ______8

4. Lichen morphology ______9

5. Lichen growth ______10

6. Resource acquisition in lichens ______11 6.1 Water ______11 6.2 Carbon______12 6.2.1 Photosynthesis______12 6.2.2 Respiration ______14 6.2.3 Limitations of carbon acquisition______14 6.3 Elements______15 6.2.1 Nitrogen______15 6.3.2 Limitations of nitrogen acquisition ______16

7. Resource allocation in lichen thalli ______17 7.1 Bipartite green algal lichens ______18 7.1.1 Carbon allocation ______18 7.1.2 Nitrogen allocation ______19 7.2 Bipartite cyanobacterial lichens ______20 7.2.1 Carbon allocation______21 7.2.2 Nitrogen allocation ______21 7.3 Tripartite lichens______22 7.3.1 Carbon allocation______22 7.3.2 Nitrogen allocation ______23

8. Aim of the thesis ______24

9. Methods and Markers for assessment of resource allocation _____24

10. Results ______29 10.1 General patterns of resource investments ______29 10.2 Growth of tripartite lichens______29 10.3 Implications of nitrogen manipulations for tripartite lichens ______30 10.4 Bipartite green algal lichens response to high nutrient availability ______31 10.5 Nitrogen uptake capacity in lichens ______32

11. Discussion ______33 11.1 How are resources distributed? ______33

5 11.1.1 General patterns of resource investments ______33 11.1.2 Comparing individual lichen species______34 11.1.3 Resource investment trends in different functional groups of lichens ______35 11.2 What resources do lichens assimilate?______36 11.2.1 Light and water assimilation in tripartite lichens ______36 11.2.2 Nitrogen assimilation ______37 11.3 How fast do lichens grow? ______38 11.3.1 Weight gain in tripartite lichens______38 11.3.2 Area gain in tripartite lichens______39 11.3.3 Extrapolating the model to all lichens ______39 11.4 How will enhanced nitrogen availability affect lichens? ______40 11.4.1 Effects on resource allocation patterns ______40 11.4.2 Effects on lichen growth ______41

12. Final conclusions ______42

13. Future prespectives______43

14. Acknowledgements______44

15. References ______45

6 1. Symbiotic systems

Symbiotic associations are often highly competitive and diverse. They are found in both marine and terrestrial environments all over the world. For example, over 90% of all plants are associated with fungi forming mycorrhizal connections. Thus, almost all plants are dependent on symbiotic interac- 1. Cetraria islandica (L.) Ach. – papers I; V tions in order to secure their growth and development. By forming a symbiotic interaction an organism can explore new niches and survive conditions in which they would otherwise be non-competitive or would not endure due to environmental constraints. On the other hand since the success and fi tness of a symbiotic organism is also dependent on the fi tness of its symbi- otic partner, a symbiotic organism must, apart from securing its own fi tness, ensure its symbiotic partner’s fi tness. These organisms are then forced both to allocate resources to and acquire resources from their symbiont partners in order to ensure their own survival.

2. Why study Lichens?

Lichens are fascinating symbiotic systems where two or three organisms form an entity, a thallus, appearing as a new and integrated organism (Smith et al., 1969). These symbiotic organisms are one of nature's most successful pioneers living in almost all terrestrial habitats and being particularly 1. Leptogium saturninum (Dickson) Nyl. – paper V important in the most barren and inhospitable parts of the world (Kappen, 1988), often thriving in habitats where few other organisms can survive. However, destruction of old growth forests and increased anthropogenic pollution has lead to the disappearance of these unique and important associations (Nitare, 2000). Despite of these threats, we lack basic knowledge on the function of lichens that can help us to understand their specifi c requirements and how different environmen- tal changes affect them. In part, this is because we do not know how the bionts function together within the lichen thallus. It is therefore of crucial importance that we investigate how the respective bionts function within the lichen thallus, both as a symbiotic partner as well as an individual organism.

7 3. General description of the lichen symbiosis

The lichen growth form can be regarded as a fungal nutritional strategy (Honegger, 1991), where the fungus lives as an ecologically obligate biotroph in symbiosis with algal and/or cyanobacterial photobionts that are extracel- 3. Nephroma arcticum (L.) lularly located endosymbionts of the lichen thallus. Highly stratifi ed Torss. – papers I; II: III; V heteromerous foliose and fruticose lichens are generally regarded as a mutualistic symbiosis and all associated bionts are assumed to increase their biological fi tness by the symbiotic state (Smith, 1992). The bio- logy of the crustose lichens, however, which is the major growth form of lichens, has not been adequately investigated. The lichen partners, a fungus (mycobiont) plus alga and/or cy- anobacterium (photobiont), form a thallus that appears as a new and integrated organism. This fungal nutritional strategy has evolved sev- eral times (Gargas et al., 1995) and lichens are taxonomically placed among other fungi with the lichen names referring to the fungal partner (Eriksson and Winka 1998). Around 13500 lichenized fungi have been described (Galun, 1988). (Although, the lichen symbiosis can contain up to three different organisms I will still for linguistic reasons refer to a lichen association as a species throughout the text). The majority of lichenized fungi (98%) are Ascomycetes, including all lichens investigated in this thesis. The vast majority (90%) of all lichens are associated with unicellular eukaryotic green algae (Chlorophyta). The genera Trebouxia aandnd Trentepohlia aarere mostmost ffrequent,requent, althoughalthough c. 40 algal genera have been recorded in lichens (Tschermak-Woess, 1988). In this thesis lichens associated with the genera Trentepohlia, Trebouxia, Coccomyxa, Dictyochloropsis, Pleurococcus, Prasiola andand Myrme- cia have been investigated. In 10-15% of all lichens cyanobacteria act either as the primary photobiont (bipartite cyanobacterial lichens) or as a secondary photobiont (tripartite lichens, around 500 species have

been identifi ed) fi xing atmospheric N2 (Tschermack-Woess, 1988). Cyanobacterial lichens investigated in this thesis are either associated with fi lamentous Nostoc sp.sp. oorr Stigonema sp.. The lichen symbiosis is poikilohydric and their water status is pas- sively controlled by the availability of water in their habitat. (Bewley, 1979; Blum, 1973). It is therefore generally assumed that the environ- ment passively controls their carbon and nitrogen budget with both photosynthetic activity and (for the c. 1300 lichens associated with

cyanobacterial photobionts (Tschermak-Woess, 1988) N2 fi xation being limited by water and light availability. Transport and distribution of the photosynthetic products from the photo- to the mycobiont may also

8 be environmentally controlled, being dependent on repeated drying and wetting of the thallus (Kershaw, 1985).

4. Lichen morphology

There are three major lichen morphologies; crustose, foliose, and fruticose. The major focus of this thesis is on foliose and fruticose lichens. Foliose lichens are dorsiventral with distinct upper and lower surfaces. These lichens are free from their substrates but usually attached to it by rhizines or similar 4. Platismatia glauca (L.) structures (Fig. 1a). The thallus develops into branching lobes. This W. Culb. - papers IV; V morphoform has a great range of thallus size and diversity (Hale, 1983). Fruticose lichens are more shrubby, hair-like or strap-shaped (Fig. 1b). The fruticose structure is often radial with a more or less hollow centre or a dense central cord and the thallus is often richly branched (Hale, 1983). The mycobiont comprises the majority of the biomass of a lichen, form- ing a morphologically complex three-dimensional structure above ground competing with plants for light, water and nutrients (Honegger, 1998).

Fig. 1 a, Peltigera malacea (Ach.) Funck; A schematic section through a typical foliose thalli. b, Cladina stellaris (Opiz) PPouzarouzar & Vezda;Vezda; A schematicschematic sectionsection throughthrough a typicaltypical ffruticoseruticose lichen.lichen.

9 Furthermore the fungus is obliged to secure adequate illumination, to facilitate the gas exchange of the photobiont cells as well as to provide suffi cient nutrient and water (Honegger, 1990). In the highly stratifi ed heteromerous lichens investigated in this thesis the mycobiont forms up to four distinct layers; an upper cortex, an alga containing layer, a medulla and, in most of the foliose species, a lower cortex (Fig. 1a,b; Büdel and Scheidegger, 1996; Jahns, 1988). The upper cortex serves as protection against high radiation and UV (Solhaug et al., 2003). The medullary layer makes up most of the internal thallus volume (Hale, 1983). These hyphae are usually long and loosely woven forming a cottony layer with a large internal air space that facilitates gas exchange (Honegger, 1993). In most cases the medullar hyphae are encrusted with crystalline secondary products making them hydrophobic, which further promotes gas exchange for the photobiont (Honegger, 1985). The algal layer is situated in the upper part of the medulla ensuring suffi cient illumination for photobiont photosynthesis, while providing protection against rapid water loss and intense solar radiation (Jahns, 1988; Rundel, 1988). The photobionts grow coordinately with the mycobiont and there are no unattached algae and/or bacteria in the lichen thallus (Honegger, 1991). The lower cortex often found in foliose lichens is an additional protective layer that can have a different structure from that of the upper cortex (Fig. 1a; Hale, 1983).

5. Lichen growth

Growth in lichens is the net result of resource acquisition and subsequent biosynthesis of cellular compounds (i.e. minus respiration and losses related to dispersal, fragmenta- tion, grazing or necrosis). Despite the environmental con- 5. Cladina stellaris (Opiz) trol of their metabolism, lichens, when wet and metabolically active, Pouzar & Vezda – paper V are able to convert incident light energy into new biomass as effi ciently as vascular plants (Palmqvist & Sundberg, 2000). Lichens are prima-

rily composed of carbohydrate ((CH2O)n) equivalents (Palmqvist and Sundberg, 2000). Factors that limit lichen photosynthesis, therefore, also limit their growth. Other nutrients, in particular nitrogen, are also needed for lichen growth. The formation of new lichen tissue requires the input of both carbon and mineral resources to the thallus, or alternatively, recycling of elements from aging thallus parts, as has been proposed for mat-forming lichens (Crittenden, 1991). Growth of the partners must further be coordinated so that neither becomes more favored than the other by environmental resource supplies (Smith et al., 1969).

10 Mature, fully differentiated thalli of foliose and fruticose lichens usu- ally have growth zones at the apical ends of the lobes (Hale, 1973). There is no meristematic cell division in lichens, as in multicellular plants. The apical growth is instead a result of the mycobiont growth pattern, in which the hyphae grow as separate tubular cells that extend apically (Gow, 1995). Non-lichenized fungi typically grow at their apices, ramifying into a mycelium (Wessel, 1993). The driving force for hyphal growth and extension is probably turgor pressure (Gow, 1995), with major structural wall components being manufactured directly on the extending apical plasma membrane (Gooday, 1995). Septum formation and nuclear division occur independently from the growth process at the very hyphal tip (Isaac, 1992). The same growth patterns are seen for lichenized fungi (Jans, 1988), with hyphal growth being most active in the marginal rim of the thallus (Honegger, 1993;1996), immediately behind the tips of the lobes (Armstrong and Smith, 1998). Adjacent to the hyphal apices, the photobiont cells divide at a rate somehow controlled by the mycobiont (Jahns, 1988; Armaleo, 1991; Hill, 1992). In older thallus parts, photobiont cell division is arrested (Hill, 1989; 1992).

6. Resource acquisition of lichens

Mineral elements are mainly acquired from dry or wet deposition directly on the thallus (Brown et al., 1994). The majority of the lichen thallus, including the surface, is composed of fungal hyphae and it is assumed that the mycobiont 6. Peltigera aphthosa (L.) absorbs most of the mineral nutrients and water. However, since the Willd. – papers I; II: III; V mycobiont lacks the capacity to fi x carbon it is entirely dependent on carbon derived from its photobionts in order to meet its metabolic carbon demand. The mycobiont is therefore obliged to secure its photo- bionts photosynthetic capacity and nutritional status in order to acquire a positive carbon balance.

6.1 Water Lichens are dependent on periods of rain, dew or high atmospheric hu- midity to achieve a satisfactory level of thallus hydration for metabolic activity (Kappen, 1988; Kershaw, 1985). Lichens, therefore, undergo frequent cycles of drying and wetting. The lichen mycobiont and its as- sociated photobiont/s must also be able to tolerate extended periods of desiccation (Kappen, 1988). The frequency and length of these cycles

11 affect both the carbon and nitrogen budget of lichens. Too short and infrequent periods of metabolic activity might lead to high rates of res- piration, incomplete recovery of photosynthesis and a massive leakage of carbon from the thallus due to re-hydration leakage (Dudley and Lechowicz, 1987). Water uptake is restricted to the fungal peripheral cortex, the out- ermost part of the lichen (cf. Honegger, 1993), and water moves pas- sively back and forth within the fungal derived apoplastic continuum (Honegger, 1991). The rates of water uptake and loss are dependent on the surrounding environment, the individual thallus maximum water holding capacity and water status. The two latter parameters are dependent of the thallus morphology, anatomy and coloration (Rundel, 1982). Due to their larger surface area to volume ratio fruticose lichens take up and loose water more rapidly compared to fl at foliose lichens. For the same reason, thick foliose lichens equilibrate more slowly with the surrounding air than thinner lichens (Gaussla and Solhaug, 1998). Moreover, the water holding capacity as well as the relative water content required for photosynthesis varies widely depending on lichen associa- tion and morphological constraints (Collins and Farrar, 1978; Lange et al., 1993; 1999). Fungal derived polyols might also be important in water homeostasis promoting passive water uptake and maintenance of osmotic potential (Smith et al., 1969; Farrar, 1988).

6.2 Carbon Although lichens can assimilate exogenously added polyols (Armstrong and Smith, 1996), it must still be assumed that the majority of the acquired carbon originates from the photobiont’s photosynthesis.

6.2.1 Photosynthesis Photosynthesis uses the energy of light to generate stored chemical en- ergy in the form of complex organic compounds. In addition to reduc-

tion of CO2 and assimilation of carbohydrates, the photosynthetic light reaction provides energy for other reactions, including the assimilation of nitrogen and biosynthesis of fatty acids (Björkman, 1981). Although the basic principles of photosynthesis are the same in both cyanobacte- rial and green algal lichens, there are differences that signifi cantly affect carbon acquisition (cf. Palmqvist, 2000; Kershaw, 1985; Lange et al., 1999). In lichens with green algal photobionts (Trebouxia), photosyn- thesis can occur if the thallus is allowed to equilibrate with air with a water potential above approximately minus 10 MPa (Lange et al.,

12 1986), however, only 20-30% of full photosynthesis can be achieved without liquid water (Scheidegger et al., 1995). When liquid water is added, green algal lichens with Trebouxia photobiontsphotobionts cancan induceinduce 75-80% of maximal photosynthesis within 10 min, with a complete induction after 30 min (Palmqvist, 2000). This indicates that these photobionts are well protected from desiccation, and that activation and/or repair processes are rapid. The rapid onset of photosynthesis in response to hydration makes Trebouxia sp.sp. a ssuitableuitable pphotobionthotobiont iinn green algal lichens that are thin and quickly equilibrate with the water level of their surrounding habitat.

The Nostoc pphotobionthotobiont cancan onlyonly performperform pphotosynthesishotosynthesis aandnd N2 fi xation after wetting with liquid water (Lange et al., 1986). This is because desiccation of Nostoc disruptsdisrupts thethe energyenergy transfertransfer fromfrom thethe phycobiline pigments to photosystem II (Bilger et al., 1989), and this disruption can only be restored when rehydration occurs with liquid water. Moreover both cyanobacterial photosynthesis and N2 fi xation require up to an hour for full induction (Palmqvist, 2000), thus indi- cating that these bipartite cyanobacterial lichens need a prolonged wet period to bring about a positive carbon gain. The Nostoc andand Trebouxia photobiontsphotobionts hhaveave a carboncarbon concentratingconcentrating mechanism (CCM) that enhances photosynthesis under CO2 limiting conditions that often occur in water saturated thalli (cf. Lange et al., 1993; Palmqvist et al., 1994; 1998; Badger et al., 1998; Palmqvist, 2000). The Coccomyxa pphotobiont,hotobiont, however,however, lackslacks a CCM,CCM, andand hashas a higher Rubisco content and other Rubisco characteristics relative to algae with a CCM (Palmqvist et al., 1995; 1998). The tripartite lichens studied in this thesis (Peltigera aphthosa aandnd Nephroma arcticum) are associated with a Coccomyxa greengreen algalalgal photobiont.photobiont. UnlikeUnlike lichenslichens with a Trebouxian photobiont that quickly induces photosynthesis, lichens with Coccomyxa aalgaelgae requirerequire uupp ttoo oonene hhourour fforor ccompleteomplete induction of photosynthesis (Palmqvist, 2000). The slow induction of photosynthesis could be dependent on the higher Rubisco content, which may require a more prolonged activation (Palmqvist et al., 1994). Coccomyxa algaealgae cancan alsoalso induceinduce photosynthesisphotosynthesis withwith waterwater vaporvapor (Lange(Lange et al., 1986). Thus, the tripartite lichens investigated in more detail in this thesis can assimilate photosynthetic carbon activated by water vapor, when not fully hydrated to induce N2 fi xation. The difference in time-span required for full induction of photosynthesis in different photobionts probably affects the growth pattern and carbon balance of lichen species. Lichen photosynthesis is also limited by available light. Within a habitat irradiances varies both seasonally and daily as well as spatially.

13 Both algae and cyanobacteria can to a certain extent adjust their photo- synthetic apparatus relative to the available irradiance. Lichen photo- synthesis is generally saturated at low irradiances (100-400) µmol quanta m-2 s-1 (Gauslaa and Solhaug, 1996), whilst a few lichens are adapted to high light (Kershaw, 1985, Lange et al., 1999). Shade adapted lichens, especially cyanobacterial lichens, seem particularly vulnerable to photoinhibition (Demmig-Adams et al., 1990a; b), and when exposed to high irradiances they become depleted of Chl a and irreversibly photoinhibited (Gauslaa and Solhaug, 1996; 1999). All lichens, however, seem to be more tolerant of high irradiances in desiccated conditions (Gauslaa and Solhaug, 1996).

6.2.2 Respiration Respiration serves to energize growth, transport, and assimilation proc- esses, and to convert assimilated carbon into compounds that can be used for growth and maintenance (Lambers et al., 1998). In contrast, respiration is detectable within 2-4 minutes after rehydration of lichens (Smith and Molesworth, 1973). Unlike photosynthesis, respiration in all lichens can be induced by water vapor. Therefore, lichens can respire although not wet enough for induction of photosynthesis (Cowan et al., 1979a; b). Respiration also increases signifi cantly with tempera- ture, with the potential to acclimate to prevailing temperatures in the particular habitat (Kershaw, 1985; Lambers et al., 1998). Respiration increases with increasing nitrogen content due to the increased energy demands related to protein turnover (Lambers, 1985). Lichens with high nitrogen contents therefore often have a higher respiratory load relative to low nitrogen lichens (Sundberg et al., 1999). In lichens this relation is less tight, compared to plants, since tripartite and bipartite cyanobacterial lichen often invest a large part of their nitrogen into non-respiring chitin in their cell walls (Sundberg et al., 1999). Since, the mycobiont represents the major biomass in lichens it might also be assumed that the mycobiont requires the majority of respiration, but the respiration of individual bionts of the lichen symbiosis has not been fully studied.

6.2.3 Limitation of carbon acquisition In summary, carbon gain in lichens is limited by: 1, The lichen thallus water content, since rates of photosynthesis and respiration are depend- ent on the water status of the thallus (Lange et al., 1986; Scheidegger et al., 1995). 2, The length and frequency of the active period, with too

14 short and infrequent periods of activation leading to a negative carbon balance. 3, The photobiont’s photosynthetic capacity, where different photobionts with different nutritional status and CCM have different rates and characteristics of photosynthesis (Palmqvist et al., 1998). 4, The lichen photosynthesis, when wet and active, is also dependent on the amount of irradiance that reaches the thallus. There is a direct re- lationship between photosynthetic rate and available irradiance (Nash, 1996). 5, The carbon demand of the lichen symbiosis. Photosynthesis in plants is regulated by the carbon demand (the sink) and is up regulated in response increased carbon demands (Field and Mooney, 1986). It can therefore be hypothesized that an increased nutritional status of a lichen would lead to an increased carbon sink, resulting in enhanced photosynthesis co-mediated by enhanced nutrient investments in the photobiont.

6.3 Elements The essential elements required by plants are hydrogen, carbon, oxygen, nitrogen, potassium, calcium, magnesium, phosphorus, sulfur, chlorine, boron, iron, manganese, zinc, copper and molybdenum (Epstein, 1972). These elements are also required in lichens and acquisition of these is believed to be by a direct dry or wet deposition on the thallus surface (Nash, 1996). However, studies have suggested phosphate and nitrogen might also be translocated from older dying parts of the thallus towards the growing apices (Crittenden, 1991; Hyvärinen and Crittenden, 1998; 2000; Ellis et al., 2003). This thesis has focused on the acquisition and allocation of nitrogen.

6.3.1 Nitrogen Nitrogen uptake by lichens is the sum of the nitrogen uptake by its individual bionts, although the individual or relative uptake rates of the associated bionts have never been quantifi ed. Since the mycobiont represents the majority of lichen biomass, controls water uptake and encloses the photobionts in a fungally derived hydrophobic sheath, it must be assumed that the mycobiont also conducts the majority of nitrogen absorption from external nitrogen resources. Nitrogen uptake by lichenized fungi has not been fully investigated but must be assumed to be comparable to other ascomycete fungi such as those forming ecto-mycorrhiza. With uptake of nitrate, ammonium and amino acids in plants, algae, cyanobacteria and fungi being medi- ated by a range of different plasma membrane transporters exhibiting

15 varying affi nities, transport rates, and regulatory mechanisms (cf. Quoreshi et al., 1995; Grossman and Takahasi, 2001; Williams and Miller, 2001; Bhattacharya et al., 2002; Wipf et al., 2002; Javelle and Morel, 2003). It can be assumed that similar assimilation mechanisms are active in the lichen symbiosis. Nitrogen assimilation in plants, fungi, cyanobacteria and algae is also under strict control by several factors, such as cellular nitrogen- and carbon status (i.e. Cho et al., 1981; Marzluf, 1981; Grace, 1997; Grossman and Takahasi, 2001; Williams and Miller, 2001). Assuming that lichen nitrogen uptake is controlled in a similar way, the nitrogen uptake rates will not be constant for a particular lichen species. In 10% of lichens the mycobiont is associated with cyanobacteria.

These lichens have the intrinsic capacity to fi x N2 from the atmosphere (Friedl and Büdel, 1996). The cyanobacterial photobiont in these lichens provides both photosynthate and fi xed ammonium to their symbiotic partners (Rai, 1988). This leads to a signifi cantly higher nitrogen status of cyanobacterial lichens relative to the green algal lichens that are solely dependent on nitrogen depositions directly on their thallus surfaces to meet their nitrogen demands (Smith, 1992). The enzyme complex

responsible for N2 fi xation is extremely oxygen sensitive (Potts, 1996; Fay, 1992), which makes it diffi cult to combine with oxygenic photo- synthesis. Filamentous Nostoc, ssuchuch aass tthathat ffoundound iinn tthehe cyanobacterialcyanobacterial lichens investigated in this thesis, has solved this dilemma by letting the

N2 fi xation occur in structurally and physiologically differentiated cells where the oxygen tension is kept low. These cells, called heterocysts, are protected from oxygen produced in the neighboring vegetative cells where oxygenic photosynthesis occurs (Potts, 1996).

6.3.2 Limitation of nitrogen acquisition It can thus be hypothesised that nitrogen uptake in lichens will be restricted by: 1, The nitrogen available for absorption in the lichen habitat. 2, Endogenous carbon status of the lichen (assimilation of nitrogen, apart from being an energy dependent process, also leads to additional respiratory costs (Lambers, 1985), therefore its nitrogen uptake requires a high endogenous carbon to nitrogen ratio. 3, The nutritional demands of the lichen and endogenous nitrogen concentra- tions of the lichen symbionts, as most organisms down-regulate their nitrogen uptake in response to high endogenous nitrogen concentra- tions (Williams and Miller, 2001).

16 7. Resource allocation in lichen thalli

In plants, there is a trade-off between nitrogen investment into carbon acquiring shoots on the one hand, and carbon expending roots on the other (Chapin et al., 1987). We might then hy- pothesize that there is a similar trade off in lichens between carbon acquiring photobionts and the carbon demanding mycobiont. In addition, the dominant non-photosynthetic fungus has not only basic nitrogen requirements for proteins, nucleic acids etc., but also an additional nitrogen requirement for fungal cell wall (chitin) synthesis. These expenditures are inevitable, since mycobiont growth is indeed required for increasing thallus area, increasing both potential light interception and mineral acquisition. 7. A cross-section of Translocation of metabolites from photo- to mycobiont and vice Peltigera aphthosa (L.) versa mmayay bbee eenvironmentallynvironmentally ccontrolled,ontrolled, bbeingeing ddependentependent oonn repeatedrepeated Willd. – papers I; II: III; V drying and wetting of the thallus (Kershaw, 1985; Richardson, 1993). (courtesy of Kjell Olofsson) Despite this strong environmental infl uence on their metabolism, lichens must be able to maintain a positive energy balance in order to grow and survive. The lichen photobiont affects the nutritional status (Rai, 1988), pho- tosynthetic characteristics (Palmqvist, 2000) as well as the exchange of metabolites (Richardson et al., 1967; Rai, 1988). The carbon products excreted from the various photobionts also differ in form, quantity and transfer rates (Hill, 1972; Richardson et al., 1967; Sturgeon, 1982; Fahselt, 1994). Moreover, the fungal to photobiont interface differs depending on photobiont associations, suggesting various mechanisms for resource translocation depending on the partners involved (Honegger, 1984; 1991). When studying functional responses as well as resource acquisition and allocation in different lichens it is therefore reasonable to classify them according to photobiont association. In my studies I have therefore divided the lichens into three functional groups: 1, bipartite green algal lichens – a symbiosis between a mycobiont and a green algal photobiont. 2, bipartite cyanobacterial lichens – a symbiosis between a mycobiont and a cyanobacterium. 3, Tripartite lichens – a symbiosis between a mycobiont, a green algal photobiont, and a nitrogen fi xing cyanobacterium. The direction of the major carbon and nitrogen fl uxes for the different functional groups are summarized in Fig. 2.

17 Fig. 2 A schematic picture of the major routes of carbon and nitrogen fl uxes in bipartite cyanobacterial lichens; tripartite lichens; bipartite green algal lichens.

7.1 Bipartite green algal lichens Since the mycobiont represents the majority of lichen biomass it may be assumed that the mycobiont accounts for the majority of nitrogen and phosphorus acquisition and translocation within a thallus. The lichen mycobiont associated with green algae that has been studied in more detail belongs to the Parmeliacean taxa. These fungal hyphae invade the Trebouxian photobionts by means of intrapariental haus- toria indicating a highly coordinated developmental process in both bionts (Honegger, 1991). The mycobiont also encloses the photobiont in a hydrophobic proteinaceous fungal-derived layer (cf. Honegger, 1991; Fahselt, 1994). The enclosing of the algal cells results in a mycobiont-photobiont interface where nutrients as well as water are exchanged between the bionts (cf. Honegger, 1991) thereby allowing the mycobiont to fully control both infl ux and effl ux to the photobiont (Richardson, 1999; Honegger, 1998). By the enclosing of the algae the mycobiont might be speculated to induce a nitrogen (Honegger, 1993) or phosphate (Feige and Jensen, 1992) limitation on the photobiont, leading to a reduced algal cell division and induced algal production of carbohydrate storage polyols such as ribitol (cf. Honegger, 1993).

7.1.1 Carbon allocation

Photosynthetically assimilated CO2 in Trebouxia-lichens is rapidly metabolized into ribitol (Lines et al., 1989) a storage polyol that is

18 only synthesized to a small extent in free-living algae (Sturgeon, 1985). The mechanism behind the symbiotically induced shift in carbon metabolism or the extensive excretion of the fi xed carbon of the green algal photobiont has not been fully investigated. The different control mechanisms that have been suggested are: 1, Fungal control over algal nitrogen status, by depriving the photobiont of nitrogen the fungus can then induce production of storage polyols (Feige and Jensen, 1992). 2, The mycobiont inhibits phosphate uptake of the photobiont, re- tarding protein synthesis leading to increased storage and retarded cell division (Feige and Jensen, 1992). 3, Fungally derived secondary phenolic substances that affect the algal metabolism (Honegger, 1993). 4, Mycobiont derived products, for instance phenolic secondary com- pounds that might specifi cally inhibit cell division (Hill, 1989). The major carbohydrate fl ux from the green algal photobiont seems to occur during the repeated rehydration events. Outside of the photo- biont ribitol is rapidly metabolized by the mycobiont into arabitol, and further to arabinose, ribinose, fructose, and fi nally mannitol (Lines et al., 1989; Jensen et al., 1991). The assimilated carbon is thereby made unavailable to the photobiont (Feige and Jensen, 1992), which must however still be able to retain enough carbon to meet its own energetic requirements. The fl ux of ribitol might be induced by damage to the algal membranes caused by desiccation (Honegger, 1991). The fl ux of polyols is massive during the fi rst 15 min of desiccation followed by a negligible leakage within an hour (Dudley and Lechowitz, 1987). Sundberg (1999) suggests that a water-related inhibition might decrease carbon transfer between photo- and mycobiont during prolonged water saturation, again indicating the need for repeated drying and wetting cycles for Trebouxiod lichens. The outfl ow of polyols is well correlated to the polyol content of the thallus (Dudley and Lechowitz, 1987), and the carbon translocation seems to be a passive process over a concentra- tion gradient. It can be hypothesized that the fungus enhances ribitol outfl ow by rapidly converting it into mannitol and thus hindering reabsorption by the photobiont.

7.1.2 Nitrogen allocation Little is known about the nitrogen metabolism of bipartite green algal lichens. No studies of nitrogen translocation between the bionts of these lichens have been preformed, but it might be speculated that carbon and nitrogen translocation are restricted to desiccation and rehydration events. In Ascomycete mycorrhiza, nitrogen transfer be- tween the fungus and plant is thought to occur predominately in the

19 form of amino acids (Martin and Botton, 1993). Transfer of nitrogen from the mycobiont to its green algal photobiont in lichens is yet to be demonstrated, it might however be speculated that if there is an active nitrogen transfer it would, as in mycorrhiza, be predominantly in the form of amino acids. These are, however, theoretical speculations and nitrogen translocation within green algal lichens is a fi eld that certainly merits further studies. It should be noted, that studies have shown a relatively high nitrate reductase activity in the green algal photobionts indicating that a large assimilation of nitrate could have been conducted by the photobiont (Avalos and Vicente, 1984; Crittenden, 1996). This indicates that even though nitrate had been taken up in the fungal apoplast it had not been absorbed by the mycobiont. It can therefore be speculated that the bionts compete for the nitrogen dissolved in the fungal apoplast. It can further be speculated that in the case of nitrate the photobiont might be better at utilizing this nitrogen source relative to the mycobiont. In support of this notion, fungi generally have relatively low capacity to utilize nitrate (cf. Hawkins et al., 2000), whilst green algae readily assimilate this compound (Grossman and Takahasi, 2001). Lichens living under extremely nutrient limited conditions might also reallocate soluble nitrogen from older dying parts as a nitrogen resource (Crittenden, 1991; Hyvärinnen and Crittenden, 1998; Ellis et al., 2003).

7.2 Bipartite cyanobacterial lichens The Nostoc inin cyanobacterialcyanobacterial llichensichens areare eembeddedmbedded inin a gelatinousgelatinous sheath. This gelatinous sheath is composed of polysaccharides, mainly glucans (Honegger, 1991). In bipartite heteromerous Peltigera lichenslichens with a Nostoc pphotobiont,hotobiont, thin-walledthin-walled intragelatinousintragelatinous fungalfungal protru-protru- sions invade the cyanobacterial gelatinous sheath (Honegger 1991). These protrusions are found close to the cyanobacterial cells but unlike the green algal lichens the mycobiont never penetrates or adheres to the bacterial cell walls (Honegger, 1991). These thin-walled intrage- latinous protrusions are lateral outgrowths of the aerial hyphae of the outermost part of the medullary layer and they lack the hydrophobic cell wall surface layer (Honegger, 1991). Water and dissolved nutrients can therefore be taken up by or be released by these fungal protrusions. As, for the bipartite green algal lichens described above, the mycobi- ont forms an apoplastic continuum in which water and nutrients are translocated (Honegger, 1991).

20 7.2.1 Carbon allocation Glucose is the major carbohydrate transfer product in all Nostoc ccon-on- taining lichens (Richardson and Smith, 1966; 1968). Once taken up by the mycobiont the glucose is, as in the bipartite green algal lichen, quickly and irreversibly metabolized into mannitol, via the pentose phosphate pathway (Lines et al., 1989). The exact mechanism behind the induction of carbohydrate export and mass transfer from the photo- to mycobiont has not been identifi ed. The movement of the photosynthetic glucose from the bacterial photobiont into the fungal apoplast is faster compared to the green algal photobiont photosynthate transfer (Richardson et al., 1968). This could however, be due to higher turnover rates in the relatively smaller carbon pool of Nostoc comparedcompared to Trebouixa sp. and Coccomyxa sp.sp. (Sundberg,(Sundberg, 11999).999). TheThe glucoseglucose transfer is insensitive to various inhibitors, indicating that glucose re- lease is a passive diffusion (Feige and Jensen, 1992). How this diffusion is controlled, however, has not been fully elucidated, although it has been suggested that the passive extraction is controlled by a glucan/ glucose equilibrium (Feige and Jensen, 1992). High exogenous glucose concentrations inhibits glucose excretion, suggesting that the myco- biont induces glucose export by maintaining a concentration gradient over the cyanobacterial membranes by rapidly metabolizing excreted glucose into mannitol. Therefore, even though there is an initially high carbohydrate transfer upon rehydration, the photosynthate transfer in cyanobacterial lichens is not as dependent on reoccurring drying and wetting cycles, as in green algal lichens. This is also seen in the different activity patterns of the two lichen groups where cyanobacterial lichens can remain active when wet for up to several days after rehydration, whilst green algal lichens dry out within a couple of hours (Palmqvist and Sundberg, 2000; Green et al., 1994).

7.2.2 Nitrogen allocation As described in section 6.3.1, cyanobacterial Nostoc hashas thethe intrinsicintrinsic capacity to fi x N2. In the bipartite cyanobacterial lichen P. canina tthehe assimilated N2 is exported into the fungal apoplast in the form of am- monium (Rai, 1988). Inhibition experiments have shown that 90% of the released fi xed N2 was in the form of ammonium, however, only 50% of this nitrogen was exported from the cyanobacteria (Rai et al., 1983), thus indicating that a large part of the fi xed nitrogen was retained for cyanobacterial metabolism. The mechanism of ammonium excretion of lichenized cyanobacteria has not been fully elucidated, however, as glutamine synthetase activity in lichenized cyanobacteria

21 is lower relative to free-living Nostoc, mmostost ooff tthehe fi x exedd a mammoniummonium probably cannot be utilized for bacterial growth and is therefore ex- creted. Furthermore, it has been suggested that the mycobiont decreases the membrane potential across the cyanobaterial plasma membrane increasing the excretion (Rai et al., 1984). The excreted ammonium is translocated into the apoplast of the mycobiont (Feige and Jensen, 1992). In the fungal intragelatinous protrusions it has been suggested that the major translocation pathway for the absorbed ammonium is via a highly active glutamate dehydrogenase into glutamate and via transaminases into alanine or aspartate that is further translocated via the fugal hyphae into the main thallus (Rai, 1988). In cyanobacterial lichens alanine has been proposed as the main form of fungal nitrogen transfer within the hyphae (Rai, 1988).

7.3 Tripartite lichens The approximately 500 known tripartite lichen species are sophisticated symbiotic systems where the mycobiont is associated with both a carbon

assimilating green alga and an N2 fi xing cyanobacterium (Tschermak- Woess, 1988; Honegger, 1991 ). The fungal cyanobacterial interface is the same as in bipartite cyanobacterial lichen (see above). In tripartite lichens the cyanobacteria is always located in a specialized structure thus completely separated from the algae (Hyvärinen et al., 2002). Several different green algal photobionts are found in tripartite lichens. The two tripartite lichens investigated in more detail in this thesis are associated with Coccomyxa ssp..p.. IInn ccontrastontrast ttoo TTrebouxioidrebouxioid aalgallgal lichenlichen nnoo hhausto-austo- rial complex is formed between the mycobiont and the Coccomyxa ccellsells (Honegger, 1991). Coccomyxa cellscells havehave a thinthin trilaminartrilaminar outermostoutermost wallwall layer of membrane-like appearance composed of sporopollenin (Brunner and Honegger, 1985). The sporopollenin-containing outermost wall might play a key role in the symbiotic relationship between the Coccomyxa photobionts and their mycobiont counterpart. No haustorial complex is formed between Coccomyxa andand itsits mycobiontmycobiont andand onlyonly a simplesimple wall-wall- to-wall apposition with a tight adhesion of the hydrophobic cell wall surfaces of the mycobiont are formed. As in the other functional lichen groups, the tripartite lichen system forms a fungal derived apoplastic continuum (Honegger, 1991).

7.3.1 Carbon allocation The associated Coccomyxa sp. photobiont excretes ribitol into the fungal apoplast. It is, however, unknown how the carbohydrates are trans-

22 located from the Coccomyxa ccellsells ttoo tthehe ttightlyightly aadheringdhering mmycobiontycobiont hyphae. The sporopollenin containing outermost layer is permeable to relatively small molecules but not to compounds with higher molecu- lar weights (Brunner and Honegger, 1985). As described in section 6.2.1 Coccomyxa, relative to Trebouxia, needs a longer induction of its photosynthesis upon rehydration. Moreover, a 13C-NMR study of carbon fl uxes showed a decrease of carbohydrate transfer in bipartite Platismatia glauca iinn rresponseesponse ttoo pprolongedrolonged wwaterater ssaturation.aturation. NNoo ssuchuch pattern was obvious for the tripartite lichen Peltigera aphthosa iindicatingndicating that the carbohydrate excretion of Coccomyxa iiss nnotot aass ddependentependent oonn repeated desiccations as Trebouxia (Sundberg,(Sundberg, 11999).999). TThehe nnitrogenaseitrogenase activity of the cyanobacteria in the tripartite cephalodia is suggested to be self-reliant and not dependent on the fi xed carbon from the Coccomyxan photobiont (Rai, 1988). Nostoc associatedassociated withwith tripartitetripartite lichens excrete glucose to a lower extent than free-living and bipartite Nostoc, mmostost probablyprobably duedue toto theirtheir higherhigher heterocystheterocyst frequencyfrequency andand thusthus lower photosynthetic activity and a larger endogenous carbon demand for their own metabolism (Rai, 1988).

7.3.2 Nitrogen allocation

The cyanobacterially derived N2 in tripartite lichens is exported to the fungal apoplast in the form of ammonium as in bipartite lichens (Fig. 2; Rai, 1988). The heterocyst frequency in Nostoc isis muchmuch higherhigher inin the cephalodia of tripartite lichens relative to free-living and lichenized Nostoc inin thethe bipartitebipartite lichenlichen symbiosissymbiosis (Rai,(Rai, 1988).1988). A relativelyrelatively largerlarger part of the fi xed N2 is released into the fungal apoplast, 95%, com- pared to the 50% that is excreted from bipartite cyanobacterial Nostoc (Rai et al., 1980; 1983). This suggests that the Nostoc ccontainedontained inin thethe cephalodia structure has a lower metabolic nitrogen demand relative to Nostoc associatedassociated inin a bipartitebipartite cyanobacterialcyanobacterial lichen.lichen. Furthermore,Furthermore, due to a 10-fold lower cyanobacterial biomass in tripartite compared to bipartite lichens (Rai et al., 1980; 1983), the N2 assimilation is lower in the tripartite lichens leading to lower apoplast concentrations of ammonium and a lower nitrogen content of the whole thallus (Rai, 1988). The Nostoc ssymbiontymbiont pprovidesrovides fi x exedd n initrogentrogen nonott o nonlyly t oto t hthee mycobiont but it provides nearly all the fi xed nitrogen requirements of the green algal photobiont as well (Rai, 1988). In tripartite lichens ammonium derived from the cyanobacteria contained in the cepha- lodia is the same as the above described for bipartite cyanobacterial lichens (Rai, 1988). There are thus two possible allocation paths for the assimilated N2 to reach the Coccomyxan photobiont. The assimi-

23 lated ammonium may be directly excreted into the fungal apoplast with possible passive further allocation by the water fl ow towards the photobiont, giving a situation where the two non-fi xing symbionts compete for the released ammonium. Another possible route would be that there is an active fungally controlled nitrogen allocation within the hyphae, where alanine is actively or passively transferred from the mycobiont into the photobiont.

8. Aim of this thesis

The aim of this thesis was to map resource acquisition and allocation patterns in lichens, and to see how these processes responded to changes in resource availability. This was done by following lichen growth in the fi eld 8. Cladina Stellaris under varying conditions - manipulating light, water and nutrient (Opiz) Pouzar & Vezda availability. During the work, markers for assessing the metabolic – paper V status of the whole lichen thallus as well as its individual bionts were developed. By comparing growth and resource allocation patterns in lichens growing under contrasting conditions we could follow lichens response to environmental changes.

9. Methods and markers for assessment of resource allocation

When studying resource acquisition and allocation it is important to have markers and methods to measure growth, carbon acquisition and expenditures rates, as well as changes in major metabolic pools. When I started my thesis research we had methods to study lichen

carbon economy (CO2 gas exchange and fl uorescence measurements – see paper I) and to follow growth (transplantation technique more thoroughly described in papers II, III and Palmqvist and Sundberg, 2000). We could also study when and for how long lichens became hydrated, which temperatures they were exposed to in their habitat as well as the amount of irradiance reaching the thalli (described in Palmqvist and Sundberg, 2000 and paper II). There was, however, a need to extend existing techniques for measuring functional markers, which could assess the metabolic status of the lichen, both as a whole and of the individual bionts. A large part of my thesis has therefore

24 been focused on further developing and modifying existing methods and to apply these methods to lichens under different environmental constraints. Methods developed and modifi ed for this thesis include; quantitation of chlorophyll, ergosterol, chitin, protein, amino acids, and soluble carbohydrates and the assessment of nitrogen uptake via 15N isotopes. We have also worked with actual fi eld data on lichen growth to generate an empirical model for growth in tripartite lichens. Bold methodsmethods aarere discusseddiscussed inin moremore detaildetail below.below.

Chlorophylls are the pigments primarily responsible for harvesting the light energy used in photosynthesis. Chlorophyll a (Chl(Chl a) is well correlated with photosynthetic capacity both within and across lichen associations (Tretiach and Pecchiari, 1995; Valladares et al., 1996; paper I). Chl a cancan alsoalso bebe usedused toto assessassess photobiontphotobiont biomassbiomass (paper(paper I).I). The nitrogen costs of the chloroplast proteins are 40-fold greater than for Chl a ((ChapinChapin eett aal.,l., 11987).987). ThusThus oonene ccanan ttheoreticallyheoretically ccal-al- culate the amount of nitrogen invested in the photobiont, as was done in paper IV. One should bear in mind, however, that the Rubisco and Chlorophyll b ccontentontent mmightight vvaryary dependingdepending onon llightight andand nnitrogenitrogen conditions.

Chlorophyll is extracted in MgCO3 saturated dimethyl sulphoxide (60°C for 40 min). The method for extracting and quantitating chloro- phyll is described in detail in Palmqvist and Sundberg (2001).

Ergosterol is the principal sterol of fungal plasma membranes (Griffi n, 1994; Smith and Read, 1997; Ekblad et al., 1998). Ergosterol correlates with the amount of metabolically active fungal tissue (paper I; Sundberg et al., 1999). This marker was also suggested to be correlated to basal fungal respiration rates of lichens (Sundberg et al., 1999). Ergosterol is sensitive to high irradiances and quickly deteriorates in light. It is there- fore also a potential marker for fungal damage due to high irradiances, as described previously (Trigos and Ortega-Regules, 2002). Ergosterol is extracted in 99.5% ethanol, disrupting all membranes, and thereafter quantitated on HPLC. The method for extracting and quantitating ergosterol is described in detail in Dahlman et al. (2001)

Chitin is a nitrogen containing cell wall component found in most fungi (Griffi n, 1994). Chitin content is correlated to total fungal biomass, metabolically active or not. Chitin has also been used as a marker for total fungal biomass in ectomycorrhizal systems (Ekblad and Näsholm, 1996). The extent to which chitin occurs in cell walls seems to be related to the type of photobiont associated with the lichen

25 (Schlarmann et al., 1990). The relative chitin content of a lichen is, on an inter-specifi c scale, well correlated with nitrogen status, where high nitrogen lichens have a higher chitin content relative to other cell wall components, as described by Boissierè (1987). This is also seen in Paper I where the chitin content is well correlated to nitrogen status, with the chitin content varying from 0.2 to 97 mg g-1 dw and nitrogen content varying from 1 to 50 mg g-1 dw (the N content of chitin is 6.3%). The differences in cell walls between green algal and cyanobacterial lichens may be related to the relative abundance of available carbon and nitrogen, with the wall acting as a carbon sink when the C:N ratio is high and as a nitrogen sink when the ratio is low (Jennings, 1989). Although chitin is a reliable marker for total fungal biomass, it is a somewhat crude and non-plastic marker on an intra-specifi c scale. It is therefore my belief that this marker per dw is best used as a complement to ergosterol as has been done for mycorrhizal roots, where the chitin to ergosterol ratio has been used as a relative measure of metabolically inactive to active fungal biomass (Ekblad et al., 1998). Chitin is extracted by acid hydrolysis into glucosamine residues after the cell walls have been treated with NaOH to remove proteins and amino acids that could interfere with the HPLC analysis. The method for extracting and quantitating chitin is described in detail in Dahlman et al. (2001).

Amino acids are nitrogen containing organic acids. They are the units from which protein molecules are built. Being building blocks for various proteins and cell wall components, the pool size of soluble amino acids measured here is a direct measurement of the nitrogen pool available for the lichen’s metabolism. Glutamine, glutamate, alanine and aspar- agine are the dominant amino acids in the lichen symbiosis (Jäger and Weigel, 1978; Rai, 1988). Some lichens also have large arginine pools (Planelles and Legaz, 1987), which can be degraded by sequential actions of arginase and urease to provide both nitrogen and carbon for synthetic activities, indicating that this amino acid is a potential storage form for lichens (Fahselt, 1994). It has also been suggested that arginine derived urea could be mobilized under conditions of deprivation (Blanco et al., 1984). The accumulation of arginine could thus be a way of avoiding the toxic effects of surplus ammonium assimilation, as shown for the lichen, Xanthoria parietina, (Silberstein et al., 1996) and also as seen in paper IV. The location of the amino acid pools in lichens is assumed to be in the mycobiont (Rai, 1988), since the mycobiont can store excess amino acids in hyphal vacuoles (Jäger and Weigel, 1978; Davis, 1986), while the photobiont lacks such storage compartments.

26 Amino acids were extracted in 10 mM HCl and analyzed as their 9-fl urorenylmethylchloroformate (FMOC) derivatives on HPLC. The method is described in detail in paper IV and developed from Näsholm et al. (1987).

Total protein content Proteins make up the largest nitrogen pool in lichens (Rai, 1988), it is a costly pool with a high respiratory demand (Lambers, 1985). No quantitations of individual proteins were performed for this thesis, since in paper IV the main focus was to simply quantitate the total N content in the protein pool. We therefore chose a crude way of measuring proteins, by simply denaturing them into amino acids and then measuring the total amount of amino acids contained in proteins. With this simple method the ability to differentiate whether the proteins are located in the mycobiont or photobiont is lost. The method is however a simple and straight forward way of quantitating nitrogen protein pools in lichens. The amino acids were quantitated as above and the method is described in more detail in paper IV, modifi ed from Nordin and Näsholm, 1997 and Steinlein et al., 1993.

Soluble carbohydrates Various roles have been proposed for poly- ols in lichen metabolism (Corina and Munday, 1971; Farrar, 1973; Richardson, 1985; Farrar, 1988), but a clear consensus has not been reached. Sugar alcohols may be important for the water status of a fungus and may promote passive water uptake (Smith et al., 1969, Farrar 1988). Hydroxyl groups of polyols may also help to maintain membrane integrity during periods of dessication (Farrar, 1976). Polyols may also constitute a reserve of carbon and reducing power (Hult and Gatenbeck, 1978; Hult et al., 1980; Farrar, 1988), with mannitol in particular considered as a form of low molecular weight storage (Sturgeon, 1985). Polyols seem to vary signifi cantly in response to different environmental conditions and can constitute up to 10% of the thallus dry weight (Palmqvist, 2000).

Mannitol isis thethe mostmost abundantabundant polyolpolyol inin AscomycetesAscomycetes (Pfyffer(Pfyffer et al., 1990) and is one of the many polyols produced in lichens (Vicentet and Legaz, 1988). Mannitol is produced entirely by the mycobiont (Feige, 1978). It is considered a form of low mo- lecular weight storage (Sturgeon, 1985). Thus mannitol can be used as a fungal specifi c marker for the lichen carbon storage pool.

27 Ribitol iiss pproducedroduced ssolelyolely bbyy tthehe ggreenreen aalgallgal pphotobionthotobiont aandnd iiss ssubse-ubse- quently exported into the mycobiont where it is rapidly metabolized

(Feige and Jensen, 1992). Assimilated CO2 is rapidly synthesized into ribitol (Lines et al., 1989), a storage polyol (Sturgeon, 1985) that is then excreted into the lichen symbiosis (Richardson et al., 1967). Ribitol is therefore a marker of both the green algal photobiont carbon pool as

well as CO2 assimilation in the lichen.

Glucose isis thethe majormajor translocationtranslocation productproduct ofof photosyntheticallyphotosynthetically fi xxeded carbon from cyanobacteria in lichens (Smith et al., 1969). Glucose can be located in both the photo- (both green algal and cyanobacterial) and the mycobiont, being a direct product of photosynthesis as well as a substrate for respiratory metabolism in both partners (cf. Fahselt, 1994),

Arabitol isis foundfound predominantelypredominantely inin thethe mycobiontmycobiont andand isis regardedregarded asas a more readily respired substrate than mannitol (Lewis and Smith, 1967; Armstrong and Smith, 1993), showing a more pronounced variation on a day-to-day basis. Arabitol is therefore a more readily mobiliseable reserve than mannitol (Armstrong and Smith, 1993), as has also been suggested for ectomyccorrizhal fungi (Jennings and Lysek, 1996). Thus the amount of soluble arabitol could be viewed as a marker for carbon directly available for fungal metabolism.

The specifi c compartmentalization of the various soluble carbohydrates thus gives a method to assess specifi c biont carbon availability as done in paper IV. Comparing storage and respiratory carbohydrates also gives a crude method of estimating surplus carbon within the thallus. When measuring these compounds, however, great care must be taken when collecting lichens. For instance, collecting a thallus at the end of a prolonged active period with a newly activated lichen, the variation in the soluble carbohydrate pools could be more dependent of the transfer mechanism than functional differences in metabolic pools. The soluble carbohydrates were extracted in water and derivatized to trimethylsilyl (TMS)-derivatives and quantitated through gas chro- matography-mass spectrometry analysis. The method is described in detail in paper IV, modifi ed from Katona et al., (1999).

28 10. Results

10.1 General patterns of resource investments. With an aim to understand lichen resource allocation patterns and nutrient acquisition and how these processes respond to different environmental conditions, we collected a large background data set in order to determine the magnitude of the metabolic variation across lichens. The 75 lichen species were selected to cover as broad a spectrum as possible regarding taxonomy, morphology, habitat, and nitrogen requirements. The lichens had various morphologies, and represented seven photobiont and 41 mycobiont genera. The lichens were divided into previously described functional groups i) bipartite green algal lichens, ii) bipartite cyanobacterial lichens, and iii) tripartite lichens. Photosynthetic capacity, steady-state respiration, thallus nitrogen concentration, and concentrations of chlorophyll, chitin and ergosterol, were measured. We examined whether lichens might, like plants (Brouwer, 1962), fi t into a generalized C:N equilibrium model. Chl a, chitin and ergosterol were used as indirect markers for photobiont activity, fungal biomass and fungal active fungal tissue, respectively.

• Across lichens, thallus nitrogen concentrations ranged from 1 to 50 mg g-1 dw, net photosynthesis varied 50-fold, and respiration 10-fold. On average, green algal lichens had the lowest, cyanobacterial Nostoc lichens the highest and tripartite lichens intermediate nitrogen concentrations. • Chl a, ergosterol, and chitin increased with thallus nitrogen concentration, and lichens with high Chl a and nitrogennitrogen concentrations also had high rates of both photosynthesis and respiration. • The Chl a-to-ergosterol ratio was similar across all species, indicating that the ratio of photobiont cells to metabolically active fungal tissue was similar irrespective of nitrogen status, morphology and habitat. • From these data it can be suggested that lichens invest their nitrogen resources so that their photosynthetic capacity matches their respiratory carbon demand around a similar (positive) equilibrium across species.

10.2 Growth of tripartite lichens In paper II we examined on an intra-specifi c level how internal and external factors contribute to lichen growth. Light, water and nutrient

29 supplies were manipulated during three months in the fi eld for the tripartite lichens Nephroma arcticum (L.) Torss. and Peltigera aphthosa (L.) Willd. Concomitant measurements of weight gain, area gain, and microclimatic conditions were made. Parallel investigations of nutrient investments in photo- versus mycobiont tissue, using the markers Chl a, ergosterol, chitin and total nitrogen content were also performed.

• The growth models were similar for both lichens. Peltigera aphthosa grew faster than Nephroma arcticum when exposed to the same environmental conditions. • Around 80% of the variation in weight was explained by variation in light received during wet and metabolically active periods, Chl a concentration and areaarea gain. All threethree parameters had a positive effect on weight gain. • Around 80% of the variation in area gain was explained by variation in weight gain, initial thallus specifi c weight, ergosterol and chitin. The area gain was correlated positively to weight gain and thallus specifi c weight, and correlated negatively to ergosterol and chitin. • The higher capacity for growth of P. aphthosa comparcompareded to N. arcticum could be explained by the lichen’s higher water holding capacity, prolonging its active time in the light, and higher Chl a to ergosterolergosterol ratio - i.e. its relativelyrelatively higher investments in photobiont versus mycobiontmycobiont tissue.

10.3 Implications of nitrogen manipulations for tripartite lichens In order to study the tripartite lichens Nephroma arcticum and Peltigera aphthosa responseresponse toto enhancedenhanced nitrogennitrogen availability,availability, a mildmild nitrogennitrogen stress was introduced by weekly irrigations with different nitrogen forms for three months in the fi eld. The total added nitrogen dosage was 500 mg m-2 (representing a nitrogen deposition of 20 kg ha-1 and year–1), 15N was used to assess nitrogen uptake in the different treatments. In a parallel experiment I removed all visible cephalodia in order to prevent

N2 fi xation, thereby reducing nitrogen availability. The lichen species responses to the manipulations of nitrogen availability were measured by concomitant measures of weight gain, area gain, and microclimatic conditions. The response to the nitrogen manipulation by individual bionts was measured using the markers Chl a, ergosterol, chitin and total nitrogen content.

30 • Estimated ammonium uptake was 4 times higher than the nitrate uptake. • The greatest recovery of the added 15N was found in the thallus margins, indicating an active translocation of assimilated nitrogen into the growing margins or that the assimilation of the added nitrogen was higher at the margins. • Both the nitrogen addition and deprivation affected thallus expansion rates in both species, whilst weight gain and key component concentrations were largely unaffected. • The nitrogen manipulations resulted in a distorted area gain. The decreased area gain might make them inferior competitors for light relative to the surrounding mosses, leading to a secondary shading effect. The distorted area gain might explain why these lichens disappear from areas with high nitrogen loads.

10.4 Bipartite green algal lichens response to high nutrient availability In order to explore the intra-specifi c plasticity in nutrient allocation pat- terns we measured the nutrient balance of the two green algal (Trebouxia) lichens, Hypogymnia physodes (L.)(L.) Nyl.Nyl. andand Platismatia glauca (L.)(L.) W.W. Culb. in a high and a low nutrient environment. These associations were the only two epiphytic lichens that had survived in a 14 year old forest fertilization experiment with nitrogen loads up to 100 kg ha-1 year-1. Intensively fertilized thalli were compared with untreated control thalli, concomitant measurements of Chl a, ergosterol, and chitin con- centrations were preformed. To assess the lichen’s nitrogen status, total nitrogen, proteins and amino acids were also measured. For carbon status we measured glucose, the photobiont (Trebouxia) export product ribitol, and the mycobiont specifi c carbohydrates arabitol and mannitol.

• In both lichen species the fertilized thalli had 2-3 fold higher protein and nitrogen concentrations, 5-10 fold higher Chl a concentrations, while ergosterolergosterol and chitin werewere merelymerely doubled. • The fertilized thalli had invested relatively more nitrogen in their photobiont tissue relative to their mycobiont, leading to 4-5 fold higher ribitol concentrations, while the fungal carbohydrates did not increase to the same extent.

31 • The amino acid arginine had increased 60-fold. Arginine accumulation possibly avoided toxic effects of accumulated ammonium, albeit binding a signifi cant fraction of assi milated carbon. • The two lichens had responded to the high nitrogen load by rerouting their resource partitioning in favor of the photobiont, increasing their carbon gain capacity.

10.5 Nitrogen uptake capacity in lichens. In order to examine short-term nitrogen uptake capacities from dif- ferent nitrogen sources, 14 lichen species associated with different photobionts (green algal, cyanobacterial or both), representing vari- ous morphologies (fruticose or foliose), and micro-habitat preferences (epiphytic or terricolous) were investigated. Nitrogen was supplied un- der non-limiting conditions as an amino acid mixture, ammonium, or nitrate, using 15N to quantitate uptake. Thallus nitrogen, amino acid and soluble polyol concentrations, and the biont specifi c markers chlorophyll (Chl) a (photobiont)(photobiont) andand ergosterolergosterol (mycobiont)(mycobiont) wwereere alsoalso measured, in order to characterize the lichen associations and to test whether any of these metabolites or markers were correlated to nitrogen uptake capacity. Measurements of passive versus aactivective uuptakeptake ooff tthehe various nitrogen sources were performed, using the Carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibitor.

• In all lichens investigated the ammonium uptake capacity was the highest. • The CCCP treatments indicate that ammonium uptake was to a larger extent passive relative to amino acid and nitrate uptake. • Nitrate uptake varied depending on the associated photobiont, being particularly low in bipartite cyanobacterial lichens. • Our data shows that all investigated lichens have the capacity to utilize amino acids indicating that amino acid uptake seems to be a general capacity amongst lichens. • The relative uptake of ammonium, nitrate, and amino acids was primarily dependent on the associated photobiont, while morphology and habitat were of less importance.

32 11. Discussion

11.1 How are resources distributed?

11.1.1 General patterns of resource investments Paper I shows that the associated photobiont signifi cantly affects the nitrogen status of lichens, where bipartite cyanobacterial lichens gener- ally have a higher nitrogen content relative to tripartite and bipartite green algal lichens (Fig. 3). Furthermore, there is a general pattern where the Chl a-to-ergosterol ratio is similar across all investigated lichens, indicating that the ratio of photobiont cells to metabolically active fungal tissue is balanced indifferent of nitrogen status (Fig. 3). These data suggest that lichens invest their nitrogen resources so that its photosynthetic capacity matches its respiratory carbon demand around a similar (positive) equilibrium across species. There were also lichen species, however, that signifi cantly differed from the general trend, with a higher Chl a toto ergosterolergosterol ratioratio e.g.e.g. asas forfor thethe twotwo lichenlichen speciesspecies investigated in paper IV. The data set from paper I, however, shows that lichens with high Chl a andand nitrogennitrogen concentrationsconcentrations alsoalso havehave highhigh ratesrates of both net photosynthesis and respiration (Fig. 4). This supports the general model which suggests that carbon expenditures in lichens are well balanced to photosynthetic capacity. This correlation emphasizes that mycobionts must ensure suffi cient nitrogen investments in the photobiont population to achieve effi cient photosynthesis to meet its concomitant fungal carbohydrate demands.

Fig.3 The relationship between Chl a : Ergoste- rol ratio and total thallus nitrogen (mg g-1 dw) across 75 lichen species. Regression line given with ±95% confi dence interval. Color represents photobiont group: white-green bipartite, grey-tripartite, dark grey-Nostoc bipartite. SymbolSymbol represents morphology: square-crustose, triangle- fruticose, circle-foliose, diamond-homoio- merous. Exception: Dermatocarpon spp. (circle with cross).

33 Fig. 4.

Light saturated net CO2 fi xation (NP) and

dark CO2 effl ux (R) rates as a function of thallus Chl a concentration of the boreal and temperate/subtropical lichens measured at o ambient CO2 and 15 C. All samples with the exception of N-fertilised Hypogymnia physodes (white diamond with cross) and Ephebe lanata (black(black upside-downupside-down triangle with cross) were pooled in the regression analysis (straight line) yielding the following linear equations; NP = 10.1±2.1[Chl a] + 5.9±2.8 with adj r2=0.20 and p=0.0000, R = 3.3±0.5[Chl a] + 3.0±0.7 with adj r2=0.30 and p=0.0000. Regression line given with ±95% confi dence interval. Colour represents photo- biont group: white-green bipartite, grey-tripartite, dark grey-Nostoc bipartite. Symbol represents morphology: square- crustose, triangle-fruticose, circle-foliose, diamond-homoiomerous. Exception: Derma- tocarpon spp. (circle with cross).

11.1.2 Comparing individual lichen species Two lichen species that exhibited a signifi cantly different pattern of resource allocation, compared to the general trend, displaying an ex- tremely high Chl a toto ergosterolergosterol ratioratio werewere thethe bipartitebipartite cyanobacterialcyanobacterial lichen Ephebe lanata whichwhich containscontains Stigonema sp.sp. andand thethe antarcticantarctic bipartite green alga lichen Mastodia tesselata whichwhich containscontains Prasiola crispa. There are two possible explanations of this pattern. One, these photobionts’ photosynthetic capacities and effi ciencies as lichen pho- tobionts have not been thoroughly investigated. These two photobiont genera might then be speculated to have less effi cient photosynthesis,

although this was not seen in CO2 exchange measurements (paper I). A second possibility is that these photobionts are less lichenized, meaning that the mycobiont is less effi cient in acquiring and translocating carbon from the photobiont, indicating that a larger photobiont population is needed in order to obtain a positive carbon balance. These speculations require further investigations. If we interpret the data set of paper I as indicating that there is an optimization of nitrogen investments within a lichen thallus, we can hypothesize that there should be a balanced Chl a toto ergosterolergosterol ratioratio

34 indifferent of nutrient status. One could test this hypothesis by expos- ing an individual lichen species to a nutrient gradient. In paper I, two bipartite green algal lichens were collected from habitats with extremely high nitrogen availability, the Antarctic Usnea aurantico-atra aandnd tthehe boreal Hypogymnia physodes. Both lichens had dramatically increased their resource investments in favor of the photobiont in these environments. In paper IV this phenomenon was further investigated and the green algal lichen Platismatia glauca sshowedhowed tthehe ssameame sshifthift iinn rresourceesource iinvest-nvest- ments between the bionts in response to increased nitrogen availability. However, there are also lichen species (Evernia prunastri, Nephroma arcticum, Peltigera aphthosa and Xanthoria parietina) that follow the general trend and do not alter their resource investment patterns in response to high nitrogen loads (Gaio-Oliveira et al., 2003a; b, paper III). (This phenomenon will be further discussed in section 11.4.1).

11.1.3 Resource investment trends in different functional groups of lichens When processing the data set from paper I in more detail, investigating the patterns within the different functional groups (Bipartite green algal lichens, tripartite lichens, and bipartite cyanobacterial lichens), there is a trend of increased Chl a : ergosterol ratio with increasing nitrogen concentration within the bipartite green algal group (Fig. 5). Where a large fraction of the lichens of this group with high nitrogen contents had a higher nitrogen investment in their photosynthetic capacity, refl ected in their higher Chl a : ergosterol ratio, others followed the general trend of a constant ratio(Fig. 5). This trend was not displayed in tripartite and bipartite cyanobacterial lichens (Fig. 3).

Fig. 5 The relationship between Chl a : Ergosterol ratio and total thallus nitrogen (mg g-1 dw) within bipartite green algal lichens. Symbol repre- sents morphology: square-crustose, triangle-fruticose, circle-foliose, diamond-homoiomerous. Exception: Dermatocarpon spp. (circle with cross).

35 11.2 What resources do lichens assimilate?

11.2.1 Light and water assimilation in tripartite lichens. As described in section 6.1-6.2, assimilation of carbon (photosynthesis) and water are two well-studied processes (cf. Larson, 1981; Coxson et al., 1983; Kershaw, 1985; Lange et al., 1986; Honegger, 1991; Cowan et al., 1992; Richardson, 1993; Green et al., 1994; Gauslaa and Solhaug, 1999). In paper II the two tripartite lichens Nephroma arcticum and Peltigera aphthosa werewere placedplaced inin threethree differentdifferent irradianceirradiance levels;levels; low,low, intermediate, and high. The intermediate light level corresponded to these lichens’ natural light conditions. In the higher light these lichens could not increase their photosynthetic capacity to the same magnitude as irradiances were increased. It can be speculated that these two lichen species were, not limited by available irradiance in the high light treat- ment; they were rather limited by their photobionts’ capacities to assimi-

late photons and to fi x CO2. In contrast, in the low light treatment P. aphthosa hadhad increasedincreased inin areaarea ratherrather thenthen weightweight relativerelative toto thethe mediummedium light treatment, indicating that full photosynthesis was not induced in the low light resulting in a low carbon gain that was not satisfactory to retain normal growth. This phenomenon was also seen in a reciprocal transplantation of Lobaria pulmonaria bbetweenetween SwedenSweden andand PortugalPortugal (Gaio-Oliveira et al., 2003c). The Swedish lichens could not acclimate to the higher irradiances in Portugal and the Portuguese lichens could not obtain a positive carbon balance in the lower irradiances received in Sweden (Gaio-Oliveira et al., 2003c). The limitation in light optima could have several plausible explana- tions, one being that a mycobiont of the same fungal species could be associated with different algal strains with differing light optima, where some strains are capable of inducing full photosynthesis at low irradi- ances while other strains are acclimated to higher irradiances. Moreover when moving lichens used to high irradiances to lower irradiance levels their photosynthetic capacity and Rubisco content might be excessive preventing a full induction of photosynthesis. When moving lichens to higher irradiances the photosynthetic apparatus might be insuffi cient, and before suffi cient investments in the photosynthetic apparatus have been made the lichen cannot fully access available resources. A second possible, negative side effect when increasing the available irradiances is that this might lead to an accelerated evaporation of water from the thallus, reducing the active time and further reducing the amount of carbon and nitrogen acquired into the symbiosis. Dramatically increased irradiances might also lead to photoinhibition of the photobiont (Gauslaa and Solhaug, 1996; Demmig-Adams, 1999a; b).

36 Fig. 6 Nitrogen uptake (µmol g-1 dw h-1) of the added nitrogen sources. White bars represents + total amino acid uptake of a mixture of 11 amino acids (n=4), gray bars represent NH4 - uptake rates (n=3), and black bars NO3 uptake rates (n=3). Bars indicate mean uptake rates (µmol g-1 dw h-1), while error bars display SE.

11.2.2 Nitrogen assimilation The lichen bionts although in a lichenized state are individual enti- ties that still functions independently, meaning that the uptake rates measured in this thesis are the sum of individual uptake rates of the lichen bionts. All investigated lichen species had the capacity to take up amino acids, although with widely varying rates (Fig. 6). In aver- age tripartite lichens had signifi cantly lower uptake rate compared to bipartite green algal and cyanobacterial lichens. With the exception of Nephroma bellum, ammonium uptake rates were generally signifi cantly higher than the amino acid or nitrate uptake rates (Fig. 6). In contrast to the amino acid uptake, the tripartite lichens had signifi cantly higher rates of ammonium uptake as compared to bipartite green algal and cyanobacterial lichens. Nitrate uptake rates were low in bipartite cyano- bacterial lichens, intermediate in the tripartite lichens, and highest in the bipartite green algal lichens (Fig. 6). The reason for the discrepan- cies in ammonium and amino acid uptake rates between bipartite and tripartite lichen can only be speculated upon and further studies are needed to answer this question. The signifi cant correlation between nitrate uptake rates and associated photobiont supports the idea with a relatively high nitrate absorption by the green algal photobiont (cf. section 7.1.2; Crittenden, 1996; Avalos and Vicente, 1984).

37 The inhibitor Carbonyl cyanide m-chlorophenylhydrazone (CCCP) specifi cally inhibits ATP-dependent transporters across cellular plasma membranes (Martin et al., 1991). CCCP signifi cantly decreased the nitrogen uptake in lichens indicating that nitrogen uptake and trans- port are active, ATP and proton gradient dependent processes. CCCP inhibits nitrate uptake and amino acid uptake to a similar extent, while ammonium uptake is less sensitive (paper V). This suggests that ammonium absorption is to a higher extent passive than amino acid and nitrate absorption. This has been suggested in earlier ammonium uptake studies (e.g. Brown et al., 1994). The passive uptake of am- monium could be a possible explanation to the discrepancy in relative nitrogen uptake rates in the fi eld (paper III) and in non-limiting lab- conditions (paper V), where there is a relatively larger nitrate uptake in the short-term experiment than in the fi eld. These contrasting results may arise because the fi eld nitrate is easily washed off while ammonium, as a cation is adsorbed to the negatively charged groups present on hyphal cell walls remaining attached to cell walls for later assimilation (Brown et al., 1994). Even though we now know that lichens have the capacity to assimi- late exogenously added nitrogen, the most limiting factor for nitrogen absorption must still be nitrogen available for uptake in a lichen’s habitat. The limitation of available nitrogen can be speculated to be particularly important for green algal lichens while, for cyanobacterial

lichens that have the intrinsic capacity of N2 fi xation nitrogen avail- ability in the habitat might be of lesser importance. There is therefore a great need for further studies to characterize the nitrogen availability of different lichen habitats before we can evaluate the importance of different nitrogen sources.

11.3 How fast do lichens grow?

11.3.1 Weight gain in tripartite lichens As described previously, lichen metabolism and hence growth is re- stricted to when these poikilohydric organisms are wet. It is therefore imperative for lichen growth that they remain wet and in the light for a long enough period to acquire a positive net carbon gain (Palmqvist, 2000). In lichens there is a strong correlation between intercepted light

during wet active periods (Iwet) and weight gain demonstrating that lichens are primarily limited by water and thereafter by available light, limiting their photosynthetic activity (Palmqvist, 2000). Moreover,

since cephalodial N2–fi xation is also limited both by water availability

38 and photosynthetically transduced energy (Rai, 1988); Iwet may subse- quently control the accumulation of weight in the form of combined nitrogen as well. This suggests that in tripartite lichens photosynthetic activity, and possibly also the N2 fi xation activity, might increase with the Chl a cconcentrationoncentration ofof thethe thallus.thallus. ThisThis ideaidea isis supportedsupported byby thethe strong correlation between photosynthetic capacity and Chl a concen-concen- tration shown in paper I (Fig. 4). In addition to Iwet and Chl a, area gain also affects the lichen’s weight gain, since light is absorbed on an area basis. Therefore, it might be more advantageous for a lichen to invest new resources into new area rather than in thickening the thallus, i.e. a thallus that continues to invest gained weight (resources) in new area will stimulate further resource acquisition.

11.3.2 Area gain in tripartite lichens Contrary to weight gain, area gain appears to be more strongly regulated by internal rather than external factors. In the identical model for both Nephroma arcticum and Peltigera aphthosa, weight change and initial thallus specifi c weight (g -2m , accumulated energy and carbon from photosynthesis) had a positive effect on area gain, while the two fungal specifi c markers chitin (g -2m , total fungal biomass), and ergosterol (g m-2, active fungal tissue) had a negative effect on area gain, indicating a trade-off where investments in non-photosynthetic tissue (mycobiont tissue) are needed to increase the area for intercepting light.

11.3.3 Extrapolating the model to all lichens Even though the growth model for tripartite lichens cannot be directly extrapolated to all lichens, the model has a functional basis and it is therefore possible to hypothetically extrapolate it to all lichens. An increased nitrogen investment in photosynthetic capacity should, based on our results, lead to increased area and weight gain in lichens. This is also seen in the literature where nitrogen additions to bipartite green algal lichens in most cases increase thallus growth (cf. Crittenden et al., 1994). This phenomenon, with an increased growth in response to enhanced nitrogen availability, could be speculated to be dependent on their nitrogen-limited conditions, at least when exposed to non- limiting irradiances (see section 11.2.1). Unlike bipartite green algal lichens, enhancing nitrogen avail- ability for bipartite cyanobacterial lichens does not enhance lichen growth, as seen for tripartite lichens in papers III and Sundberg et al. (2001), additional exogenous nitrogen might inhibit cyanobacterial

39 lichens area gain. The distorted area gain in N2 acquiring lichens is

most probably due to a down-regulated N2 fi xation activity of the cephalodia following the external nitrogen additions (Hällbom and Bergman, 1983). This down regulation might be speculated to lead

to a decreased the nitrogen availability in the thallus. Due to the N2 fi xing nature of cyanobacterial lichens, increasing active time in light,

and thereby increasing the internal N2 fi xation rather than increasing nitrogen depositions, would probably lead to increased nitrogen status of a lichen, and thus an increased growth.

11.4 How will enhanced nitrogen availability affect lichens?

11.4.1 Effects on resource allocation patterns In paper IV the two green algal lichens Platismatia glauca and Hypogymnia physodes exhibited large metabolic plasticity, being able to cope with both nutrient poor and nutrient rich conditions. The lichens increased their thallus nitrogen concentration 2-3 fold and shifted their nitrogen investments in favour of the photobiont, resulting in an increased net carbon assimilation capacity and increased soluble carbon-pools in both the photo- and mycobiont (Fig. 7). Moreover, the lichens had accumulated a large pool of arginine in response to the excessive nitrogen supply, thereby possibly avoiding the toxic effects of accumulated ammonium. The two lichens apparently coped with the increased nitrogen loads by rerouting their resources. Measurements of net photosynthesis that were conducted on thalli of Hypogymnia physodes (in(in paperpaper I)I) showedshowed a highhigh vitalityvitality andand photosyntheticphotosynthetic capac-capac- ity in the fertilized lichens. Moreover in H. physodes, respiration only increased 3-fold in the fertilized thalli relative to the 8-fold increase in photosynthesis also indicating an increased carbon gain in the lichens. This indicates that green algal lichens apparently have the capacity to deal with surplus nitrogen. In a parallel study Xanthoria paretina aandnd Evernia prunastri diddid notnot re-routere-route theirtheir resourceresource investmentinvestment inin responseresponse to an enhanced nitrogen availability, maintaining a balanced Chl a toto ergosterol ratio (Gaio-Oliveira et al., 2003a; b). The same pattern was seen in paper III, where the two tripartite lichens Nephroma arcticum and Peltigera aphthosa ddidid notnot reroutereroute theirtheir resourceresource aallocationllocation patternpattern in response to a enhanced nitrogen availability, indicating that no gen- eral response in lichen resource allocation patterns towards enhanced nitrogen availability can be predicted.

40 Fig. 7 Relative carbon investments in soluble carbohydrates and arginine. Circle area is directly proportional to total carbon in the fi ve compounds, in av- erage being 8.7 mg g-1 dw in Hypogymnia physodes CCtrtr - tthalli;halli; 116.76.7 mgmg g-1 dw in H. physodes F - thalli; 9.1 mg g-1 dw in Platismatia glauca CCtrtr - tthalli;halli; 18.9 mg g-1 dw in P. glauca F - thalli. arab = arabitol; arg = arginine; glu = glucose; man = mannitol; rib = ribitol.

11.4.2 Effects on lichen growth As suggested in section 11.3.4 nitrogen additions to tripartite and bi- partite cyanobacterial lichens could impair their growth. This was also seen in paper III where the tripartite lichens Nephroma arcticum and Peltigera aphthosa wwherehere exposedexposed ttoo mmildild nnitrogenitrogen sstresstress thatthat distorteddistorted their growth pattern reducing their area growth. This distorted area gain might be a possible explanation of the disappearance of these lichens in response to increased nitrogen availability. In section 11.3.3 the growth model predicted that moderate nitrogen additions to green algal lichens might enhance their growth. In paper IV, where a dramatic shift in nitrogen investments occurred, growth was unfortunately not followed. Since, we did not measure any growth rates in paper IV or

41 Gaio-Oliveira et al., 2003a; b we cannot speculate on how additional nitrogen affects area gain in green algal lichens. Since lichens are found in harsh and nutrient limited environments, their competitive strategy is probably to occupy niches where other plants or bryophytes cannot grow. Bipartite green algal lichens are often slow growing (Rogers, 1990), due to their low nitrogen and Chl a contentcontent asas also predicted by our growth model. For terricolous lichens a distorted area gain in response to increase nitrogen availability (as in paper III) is particularly unfortunate since the major limiting factor of lichen growth is light received when wet (paper II). In response to the increased nitro- gen availability the surrounding bryophytes and plants that might be more effi cient in utilizing and allocating the surplus nitrogen for further growth, can shade terricolous lichens. This phenomenon was apparent in the fi eld site of papers II and III where the underlying and neighbouring bryophytes increased their growth dramatically in response to the added nutrients (unpublished observations). The added nitrogen thus gives a competitive advantage to plants that can utilize the added nitrogen more effi ciently and thereby out-compete the lichens for the above ground space. This suggested pattern of a changed species composition in response to increased nutrient availability has been described amongst plants, where species better at utilizing the additional nitrogen out competes slow growing plants changing the habitat structure (Bobbink et al., 1998). Additional biotic effect of increased nitrogen avalibility includes, as described for plants an higher frequency of parasitic fungi infections (Strengbom, 2002), this is also a probable scenario for lichen communities.

12. Final conclusions

Being poikilohydric, lichen’s activity, resource acquisition, and alloca- tion, are passively controlled by the water availability in their habitats. In spite of this, paper I indicates that there seems to be an optimiza- tion of resource allocation between the bionts of the lichen symbiosis. Lichens invest their nitrogen resources so that their maximal carbon input capacity match their respiratory carbon demand around a similar (positive) equilibrium across species. The results thus demonstrate an optimization of resource partitioning in response to available resources. From empirical data we obtained an growth model for lichens, as performed in paper II, predicting terricolous tripartite lichen growth in boreal habitats. The growth model reveals that these are highly regulated symbiotic interactions where enhanced carbon harvesting

42 potential, co-mediated by a larger photobiont population and a higher water holding capacity, increases growth. There is also a discrepancy in nitrogen uptake dependent on the associated photobiont, where high nitrogen containing lichens associated with the Nostoc photobi-photobi- ont absorb nitrate to a lower extent than lichens associated with green algae. Paper V shows that lichens can absorb both intact amino acids as well as ammonium. There was also a discrepancy in uptake rates for ammonium and amino acids in-between bipartite and tripartite lichen systems. Enhancing nitrogen availability to lichens leads to several differ- ent responses: Tripartite lichens display a distorted area gain while maintaining a balanced Chl a toto ergosterolergosterol ratio.ratio. A similarsimilar responseresponse with a maintained Chl a to ergosterol ratio was also seen in the two lichen species Evernia prunastri aandnd Xanthoria paretina ((Gaio-Gaio- Oliveira et al., 2003a; b). Hypogymnia physodes aandnd Platismatia glauca on the other hand, (paper V) displayed a signifi cant shift in Chl a to ergosterol ratio co-mediated by a higher soluble carbohydrate pool and a dramatic increase in arginine synthesis indicating a re- allocation of resources in response to the enhanced nitrogen status.

13. Future prespectives

Shifts in allocation patterns under changing environmental conditions have been experimentally proven to maximize plant growth (Robinson and Rorison, 1988). It is also assumed that environmental limitations, or excesses that reduce resource use will also reduce resource uptake (Chapin, 1991). As described above, unlike plants, all lichens are pas- sively controlled, with both their carbon and nitrogen budgets being dependent on the water availability in their habitat. In spite of these constraints the lichen symbiosis seems to be optimized with a balanced carbon and nitrogen budget in response to their available resources, being able to adjust their resource allocation patterns in relation to both nitrogen stress and other habitat constraints. Several important questions remain to be answered for the lichen symbiosis amongst which are; How fast and to what extent can the lichen symbiosis adjust to changed environmental constraints and what factors determine different lichen species plasticity towards change? - Why do some lichens re-route their nutrient investments while others retain a balanced Chl a toto ergosterolergosterol rratio?atio? DoDo thethe ad-ad- justments to changed environmental constraints occur via increasing

43 specifi c photobiont or mycobiont activity? - Which one of the symbionts dominates the lichen symbiosis, which biont acts as the sink and which one is the source? On which scale lays the specifi city of the lichen symbiosis? - Which are the crucial steps of recognition and does the mycobiont choose a photobiont after its metabolic needs or vice versa?

14. Acknowledgments

Thanks to Heidi Döring, Henrik Hedenås, Gisela Oliveira, Torgny Näsholm and Kristin Palmqvist for providing comments and thoughts about earlier version of this thesis. I would also like to thank Amanda Cockshutt and Anna Crewe for correcting my English. I am also grate- ful to Kjell Olofsson for providing the picture of the cross section of Peltigera aphthosa. The work in this thesis has been funded by grants from The Centre for Environmental Research (CMF) (993194), The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) (24.0795/97); (23.0345/99), and by the Gunnar and Ruth Björkman foundation.

44 15. References

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46 Cowan DA, Green TGA, Wilson AT (1979b) Lichen metabolism 2. Aspects of light and dark physiology. New Phytologist 883:3: 761-769.761-769. Cowan IR, Lange OL, Green TGA (1992) Carbon-dioxide exchange in lichens: determination of transport and carboxylation characteristics. Planta 1187(2):87(2): 282-294282-294 Coxson DS, Brown D, Kershaw KA (1983) The interaction between

CO2 diffusion and the degree of thallus hydration in lichens: some further comments. New Phytologist 93:93: 247-260.247-260. Crittenden (1991) Ecological signifi cance of necromass production in mat-forming lichens. Lichenologist 23:23: 3323-33123-331 Crittenden PD, Kalucka I, Oliver E (1994) Does nitrogen limit the growth of Lichens? Cryptogamic Botany 4:4: 142-155142-155 Crittenden PD (1996) The effect of Oxegen depravation on inorganic nitrogen uptake in an Antarctic macrolichen. Lichenologist 28(4): 347-354 Crittenden PD (1998) Nutrient exchange in an Antarctic macrolichens during summer snowfall-snow melt events. New Phytologist 139: 697-707 Dahlman L, Zetherström M, Sundberg B, Näsholm T, Palmqvist K (2001) Measuring ergosterol and chitin in Lichens. In Protocols in lichenology – culturing, biochemistry, physiology and use in biomonitoring. (eds. Kranner I., Beckett R., Varma A.), Chapter 21: 348-362 Springer, Berlin. Davis RH (1986) Compartmental and regulatory mechanisms in the arginine pathways of Neurospora crassa andand Saccharomyces cerevisiae. Microbiological Reviews pp.pp. 280-313280-313 Demmig-Adams B, Adams WW III, Czygan F-C, Schreiber U, Lange OL (1990a) Differences in the capacity for radiation-less energy dissipation in the photochemical apparatus of green and blue-green algal lichens associated with differences in cartenoid composition. Planta 180:180: 582-589582-589 Demmig-Adams B, Marguas C, Adams WW III, Meyer A, Kilian E, Lange OL (1990b) Effects of high light on the effi ciency of photochemical energy conversion in a variety of lichen species with green and blue-green phycobionts. Planta 180:180: 400-409400-409 Dudley SA, Lechowicz MJ (1987) Losses of polyols through leaching in subarctic lichens. Plant physiology 83:83: 813-815813-815 Ekblad A, Näsholm T (1996) Determination of chitin in fungi and

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57 Tack talet till lavar

Ofta har jag fått frågan varför lavar? Hur kan en människa som ofta uttalet en så stark skepsis till naturen och som aldrig riktigt har förstått storheten med naturutfl ykter välja att dedikerad så mycket tid till lavar? Hade någon för tio år sedan förslagit att jag skulle dispu- tera på lavar vid Umeå Universitet, hade jag nog skrattat gott - kallat människan galen och stilla undrat var Umeå låg (i Svergie?). Bara tanken att läsa biologi hade tett sig skrattretande och lavar visste jag knappt att det existerade. Så nu står jag här och skall försvara min avhandling på lavar, varför? Ja, den största och klaraste orsaken till varför är nog en prof. Leif Tibell. Han hävdade att lavar styrdes helt och hållet av svampen och att vilken alg som var associerad till laven var helt ovidkommande för artbegreppet. Feminist som jag är, iden- tifi erade jag algen som kvinnan i detta ”äktenskap” och förkastade detta uttalande som orimligt, sedan dess har jag jobbat för algens synliggörande i laven. Någon gång längs vägen har dock feministen i mig erkänt att man inte kan männskligfi era lavar och bara låtit sig fascineras av denna spännande symbios.

Kikki, jag är än idag imponerad att du vågade satsa på mig en ob- stinat liten liten 23 åring. Att vara din doktorand har varit det job- bigaste men samtidigt också roligaste och mest utvecklande jag har genomlevt. Du har krävt mycket av mig men samtidigt har du gett lika mycket tillbaka. Du har alltid haft tid för att prata om allt ifrån lavar till oväsentligheter, du har gett mig mer än vad jag kan förklara i ord. Torgny, du var visst menad att ”bara” vara biträdande handle- dare i mitt projekt, men jag har nog aldrig riktigt förstått konceptet biträdande. Jag har skamlöst använt ditt labb och ditt kunnande fullt ut - trots att du hävdar att du tycker att lavar är konstiga har du tillfört oändligt mycket till projektet och till mig. Tack för att du har tagit dig an mig, speciellt under Kikkis frånvaro – det betydde otroligt mycket! Margareta, min ”tredje” handledare – du har varit en klippa att ha med sig i labbet. Trots att jag har kleggat ihop ventiler, skruvat sönder HPLC apparater och desperat ringt in dig till labbet under helger på grund av små konstiga misstag har du aldrig klagat. Med en stoisk tålmodighet har du handlett mig igenom laboratorie-

58 världens labyrint. Du är dessutom en mycket trevlig arbetspartner i och utanför labbet – jag uppskattar alla diskussioner och meningsut- byten om allt ifrån arbetsmiljö frågor till dryftande om all världens ”talibaner”. Gisela, without you the work of this thesis would have been half as fun to do. I am very grateful for all fun and interesting discussions about everything. But also, you have been very important in shaping my scientifi c question and knowledge about lichens. A big chocolate pastry awaits you at Wayne’s. Jörgen, nog var jag allt lite rädd för dig i början (du var ju så lång) men du har varit en makalös sparringpartner under min avhandling. – Du har utmanat mig, gjort mig både tokförbannad och tokglad. Utan dig hade min avhandling inte varit var den var idag. Oskar, Jonas Ö, Åsa, mina rookies;) utan kvävegruppen ingenting – utanför och i labbet, till skogs, på en klippvägg, vid en piazza i Rom, snorklandes i Medelhavet, fi skandes eller bara över en middag. Jag vill även tacka Jonas G för all GC-MS hjälp och för alla livsfunderingar. Alla ekofysiologer, nya & gamla medlemmar av N-gruppen – som alltid kul att forska med er. Tack för all hjälp under åren! På EG har jag även varit lyckligt lottad att få ha arbetat ibland många begåvad lavforskare – ni har varit till stor hjälp och nöje – Tack! Jan-Eric – för att du fi ck mig att bli forskare. Till slut vill jag tacka Kalle som har tillbringat sommaren med att ”vattna” mina lavar – inte riktigt teknisk fysik, men bra nära...

Åsa – du ska bara veta vad jag gillar dig, alla dessa timmar i KBC, vid tygerna, över en fl aska vin (eller 3) eller var som helst – du har varit där och förgyllt det mesta. Tina, Tuss, Anna, Maria – Utan er hade denna tid varit urbota trist, tack för alla sekunder! Henrik, Mattias, Jocke, Nic – det är alltid lika trevligt att snacka fåglar!EG, nya som gamla inhabitanter, trots att vi är så många och trots att man kanske inte alltid kan alla namn så känner man sig ändå hemma. Så många trevliga att nämna så lite tid – känn er alla träff ade och omtyckta! (speciellt vårt vackra gäng på våning 3). Skoglig genetik och växtfysiologi, mitt andra ”hem” befolkat av en drös av trevliga doktorander, TA-personal och forskare alltid på gång, alltid under- bara. Ni är alldeless för många för att nämna vid namn men inte för att tycka om. Tack för att jag fi ck stanna förbi ett tag.

59 Alla sekreterare och TA personal både på EG och SLU. Ni har un- derlättat mitt arbete och min vardag oändligt! Alltid redo att hjälpa när jag har stormat in med mina panikutryckningar – Toktack!

Britta Norberg – jag tror att både du och jag har tappat räkningen på antalet säkertskort du har fått lov att utställa pga av mitt eviga slarv, jag börjar få slut på ursäkter – men tro mig jag är tacksam förvarenda ett!

Min makalös hög av vänner! Även om ni inte alltid förstår vad jag håller på med, ler ni överseende och får mig att tänka på annat ett tag. Borlänge, vi hänger fortfarande ihop hela högen och det är mi- rakulöst, midsommar, juldagen ströhelger.Pernilla, Johanna, Hanna, Andreas, Anders, Daniel, Jonas, Mats, Pudding, Per, Magnus och alla andra. Norrland, hade aldrig anat att jag skulle ha så trevligt här – tack alla! Jonas – det fi nns få jag hellre hänger med. Ellinor & Patrik – middagsvänner, squashpartners, sambos, hyresvärdar, gäster. Lars, Olle, Katarina, Ankin, Roland, Mattias, Linn, Louise, Maria & många många många fl er…Alla ni vänner i förskingringen jag vet att jag borde höra av mig oftare – Martin, Axel, Annika, Ulrika, Anders, Johan och en massa fl er – känn er inte bortglömda bara omtyckta!

Mitt genetiska ursprung. Tack för att ni har gjort mig till den jag är - utan er ingen Lena och en massa, massa färre historier. Mamma, Pappa, Lisa, Mormor, faster, moster, farbror, Markus & Lillemor. Pär – Ingen har nog haft en bätttre storebror - fast vår resa lär nog bli ett intressant experiment.

Jag vill slutgiltigen tacka bolaget Grafi t– som räddade denna avhan- dling i sitt slutskedet med ett välbehövligt sladdbyte – & Mattias (Print & Media, Umeå universitet) som har tålmodigt hjälpt mig med layouten på min avhandling – Tack!

Ingenting utan vänskap, Tack!

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