Disentangling Lecania

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 333

RIKKE REESE NÆSBORG

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 UPPSALA ISBN 978-91-554-6953-5 2007 urn:nbn:se:uu:diva-8183 Dissertation presented at Uppsala University to be publicly examined in Lindahlsalen, EBC, Norbyvägen 18A, Uppsala, Friday, September 28, 2007 at 09:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Abstract Reese Næsborg, R. 2007. Disentangling Lecania. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 333. 42 pp. Uppsala. ISBN 978-91-554-6953-5.

This thesis focuses on phylogenetic, taxonomic, ecological, and conservation aspects of the crustose lichen genus Lecania (Ramalinaceae, lichenized Ascomycota). Lecania has previously been defined on basis of relatively few morphological characters, and the genus had never been treated in molecular phylogenies. The molecular phylogeny of the genus is inferred from DNA sequences. Twenty-five species traditionally placed in Lecania are included in the study along with 21 species from closely related genera. Lecania is a polyphyletic genus. A well-supported monophyletic group containing 16 Lecania species, including the type species L. fuscella is discovered, i.e. Lecania s. str. Nine species formerly included in Lecania do not belong in the genus. A new species, L. belgica, is described. The relationships of a group of morphologically similar Lecania species, i.e. the L. cyrtella group are investigated using morphological and molecular methods. Haplotype network and phylogenetic analyses indicate that the included species, as conceived in the morphological examinations, all are monophyletic. Two new species, L. leprosa and L. madida,are described, L. proteiformis is resurrected from synonymy, and the known range of L. prasinoides is greatly expanded. The type species Lecania fuscella has become endangered in many countries. Twelve localities in Sweden where the species had been found historically are investigated, but L. fuscella is only recovered in one locality. The species composition in these 12 localities, 58 old and 5 new collections with L. fuscella is determined and analyzed. The vegetation community differs between the old and the new collections, and between the locality where the species is recovered and those where it is not. Lecania fuscella has not been able to adapt to environmental changes and now only appears in a specific type of vegetation community. The phylogenetic diversity of the species is calculated, but does not reflect the species’ evolutionary potential.

Keywords: Lecania, Ramalinaceae, Crustose lichens, Phylogeny, Taxonomy, Morphology, New species, Phylogenetic diversity, Rare species, ITS, mt-SSU, RPB2, IGS

Rikke Reese Næsborg, Department of Evolution, Genomics and Systematics, Systematic Botany, Norbyv. 18D, Uppsala University, SE-75236 Uppsala, Sweden

ISSN 1651-6214 ISBN 978-91-554-6953-5 urn:nbn:se:uu:diva-8183 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8183)

Distributor: Uppsala University Library, Box 510, SE-751 20 Uppsala www.uu.se, [email protected]

Following our will and wind we may just go where no one’s been. We’ll ride the spiral to the end and may just go where no one’s been. Spiral out. Keep going.

Maynard Keenan

Cover: Lecania fuscella, asci with spores and paraphyses. Illustration by the author.

Papers included in this thesis

This thesis is based on the following papers, which are referred to in the text by their Roman numerals: I Reese Næsborg, R., Ekman, S., & Tibell, L. 2007. Molecular phylogeny of the genus Lecania (Ramalinaceae, lichenized As- comycota). Mycological Research 111: 581–591. II Reese Næsborg, R. & van den Boom, P. P. G. Lecania belgica van den Boom & Reese Næsborg, a new saxicolous species from Western Europe. Submitted to Lichenologist. III Reese Næsborg, R. Taxonomic revision of the Lecania cyrtella group (Ramalinaceae, lichenized Ascomycota) based on molecular and morphological evidence. Submitted to Mycologia. IV Reese Næsborg, R. The phylogenetic diversity of Lecania fuscella does not reflect its evolutionary potential. Manuscript.

Paper I is reprinted with kind permission of the publisher.

All papers in this thesis were written by the first author, with comments and suggestions given by the co-authors. In paper I RRN is responsible for most of the laboratory work and two of the phylogenetic analyses.

Important note. In papers II and III the manuscripts contain descriptions of new species. These papers have been submitted for publication elsewhere, and in order to make it clear that the names of these new species are not validly published in this thesis the Latin diagnoses, which are necessary according to the International Code of Botanical Nomenclature, are omitted.

Contents

Introduction...... 9 Lichens ...... 10 Ramalinaceae ...... 10 Lecania...... 11 Aims ...... 15 Materials and methods ...... 16 Results and discussion ...... 17 Paper I and II...... 17 Paper III...... 19 Paper IV ...... 22 Dansk resumé...... 26 Laver ...... 26 Lavsystematik...... 27 Lecania og min forskning ...... 28 Svensk sammanfattning ...... 32 Lavar ...... 32 Lavsystematik...... 33 Lecania och min forskning...... 34 Acknowledgements...... 37 References...... 40

Abbreviations

bp. base pairs BP-ML Bootstrap Proportion Maximum Likelihood BP-MP Bootstrap Proportion Maximum Par- simony DD Data Deficient IGS InterGenic Spacer region ITS Internal Transcribed Spacer region LSU Large SubUnit mt-SSU mitochondrial Small SubUnit NMS Non-metric Multidimensional Scal- ing PD Phylogenetic Diversity PP Posterior Probability RPB2 RNA polymerase II second largest subunit s. lat. sensu lato (in a broad sense) s. str. sensu stricto (in a narrow sense) SSU Small SubUnit

Introduction

Systematics is a field of science which addresses the diversity of life and the evolutionary relationships among living organisms. In order to understand these relationships several tools or disciplines are employed, i.e. identification, taxonomy, and phylogenetics. First, the organisms must be identified and described, since this is the foundation for deducing relationships. When organisms are properly identified and described, they can be named (taxonomy) and arranged in groups according to their evolutionary relatedness (phylogenetics). Sometimes, however, inference of a molecular phylogeny precedes description and naming of taxa. The evolutionary relatedness can be reconstructed using both morphological and molecular characters, the latter usually in the form of DNA sequences. Phylogenetic reconstructions are often elucidated by depicturing trees where monophyletic groups share the same common ancestor and, to some extent, the same derived characters. Systematics is therefore a fundamental discipline on which many other fields of biology rely. An area where systematics has both indirect and direct influence and im- pact is conservation biology. Indirectly, since biological diversity could not be assessed without proper identification, description, and naming of taxa. Recently, systematics has also offered more direct contributions to the field of conservation biology. Owing to the limited resources available for assign- ing protection of endangered species and/or habitats, it is often necessary to prioritize or rank the species/habitats requiring protection. Systematics offers objective means of setting these priorities by evaluating the biological distinctiveness of the species per se or of the area for one/more groups of organisms. The biological distinctiveness is reflected in genetically distinct lineages, and one method to estimate these genetically distinct lineages is the measure of phylogenetic diversity (PD, Faith 1992). This method uses the branch lengths of a phylogenetic tree. By summing branch lengths of all the branches corresponding to the minimum spanning path, the PD of a subset of taxa is obtained. Phylogenetic diversity thus assesses the conservation worth of those lineages in terms of the amount of evolution preserved. The inten- tion is to maximize protection of as much of the phylogeny of a set of taxa as possible. The gain in phylogenetic diversity that a specific species contrib- utes to a group of taxa (genus, family, etc.) can be calculated, and in this way the species can be evaluated when conservation priorities are made. When a group of taxa is considered for conservation it is desirable to be able to op-

9 timize the evolutionary potential, and thus the capacity to respond to future environmental changes (e.g. Soltis & Gitzendanner 1999, Forest et al. 2007). It has been proposed that PD represents the evolutionary potential (e.g. Soltis & Gitzendanner 1999, Forest et al. 2007), but PD does not necessarily reflect the capacity of specific lineages to respond to future environmental changes. If the genes used for calculation of PD does not code for the quantitative traits required for adaptation, PD may only reflect an arbitrary accumulation of genetic changes which has taken place in these particular genes during evolution. In order to use PD to assess the evolutionary potential it may be necessary to include selectively important genetic variation (Ouborg et al. 2006). Nevertheless, PD may currently be one of the best ways to estimate the evolutionary potential, since the initial response to an environmental change depends on the level of standing genetic variation. This standing genetic variation may in many cases be optimized in genetically distinct species.

Lichens Lichens are symbiotic systems which consist of a fungal partner, the mycobiont, and one or more algal/bacterial partners, the photobiont(s). The photobiont can be either a green algae or/and a cyanobacterium, and together the mycobiont and the photobiont form a tightly integrated structure. The photobiont produces carbohydrates which are utilized by the mycobiont, and the mycobiont provides the photobiont with a habitat in which it is able to sur- vive in otherwise inaccessible environments. Most mycobionts belong to the Ascomycetes which produce spores inside tiny sacs called asci (singular ascus). Dispersal of sexually produced spores in lichens is complicated because of the need for the fungal partner to find a suitable photobiont, many of which are not free-living. Other methods of dispersal in lichens include non-sexual propagation of vegetative structures such as e.g. isidia and soredia which contain both fungal hyphae and algae. Lichens are classified as fungi, i.e. a lichen’s name always refers to the fungal partner. Eleven of 61 orders in Ascomycota are exclusively lichenized, but several other orders have lichen representatives (Hibbett et al. 2007). Lichenization has evolved several times independently during the evolution of fungi, and lichenized and non-lichenized fungi are often found among each other in closely related groups.

Ramalinaceae In the early days of systematics morphological resemblance was the only tool available to classify organisms. This of course applied to lichen sys-

10 tematics as well, and features like growth form were deemed of utmost im- portance. Accordingly, families contained genera with one type of growth form, i.e. either foliose, fruticose, or crustose lichens, but rarely, if ever, a combination. Growth form, however, has developed in parallel in different systematic groups as adaptation to environmental conditions, and thus does not automatically reflect true relationships. As currently circumscribed, most families comprise genera with very different growth forms and appearances. Ramalinaceae was for a long time regarded as a family of fruticose lichens – the so-called cartilage lichens named after the genus Ramalina. A molecular phylogenetic study revealed that Bacidiaceae, which comprised a number of crustose genera including Lecania, is a younger synonym of Ramalinaceae (Ekman 2001), and thus should be included in Ramalinaceae. The family now contains 36 genera with fruticose, crustose and foliose taxa, e.g. Ba- cidia, Biatora, Cliostomum, Phyllopsora, Ramalina, Toninia, Vermilacinia, and Lecania (Eriksson 2006).

Lecania Lecania is a genus of inconspicuous crustose lichens which includes both corticolous, lignicolous, and saxicolous species (Mayrhofer 1988, James & Purvis 1992, Foucard 2001). It is a medium-sized genus comprising c. 40 species worldwide, mainly distributed in temperate areas (Kirk et al. 2001). The genus has largely been overlooked due to the inconspicuous nature of its members and difficulties in identification. For this reason the genus has only been treated in a few morphological revisions which just included the saxicolous species in specific geographical regions (Mayrhofer 1988, van den Boom 1992). Prior to my research the genus had only been included in a few molecular phylogenies (e.g. Ekman & Wedin 2000, Ekman 2001), and never with sufficient taxon sampling for achieving inference of its phylogenetic status. Circumscription of Lecania has traditionally been based on a few morphological characters. These are a crustose habit, lecanorine apothecia, septate spores, and more recently, the ascus apex structure. However, it is unlikely that one single or a few characters will reflect relationships cor- rectly, since it is difficult if not impossible to differentiate between homo- plastic and homologous characters. Instead, a combination of both morphological and molecular characters should be employed for inference of phylogenetic relationships. Morphologically the genus is characterized by the following features: the thallus is lichenized and crustose. In some species the thallus is covered with small propagules called blastidia, which can cover even the apothecial margin. The apothecia are lecanorine (with a thalline margin containing photobiont cells) to biatorine (without a true exciple, more or less convex, and

11 light-colored). The photobiont belongs to the green algae genus Trebouxia. The fungal hyphae in the excipulum are richly dichotomously branched but not anastomosed (Fig. 1), and they are often somewhat club-shaped toward the rim.

Figure 1. Dichotomously branching excipular hyphae in Lecania (example from L. cyrtella). A few algal cells from the genus Trebouxia are interwoven between the hyphae. The scale bar is 10 m. Illustration by the author.

The apothecia usually have a brownish pigmentation, which is distinctly unevenly distributed and gives the apothecia a characteristic dotted appearance, especially when the apothecia are moist. The paraphyses are fairly easily separated in potassium hydroxide, and they often thicken gradually toward the apex.

Fig. 2. Examples of paraphyses in Lecania. At the left paraphyses with only slightly swollen terminal cells in L. erysibe, and right paraphyses with strongly swollen terminal cells in L. proteiformis. The latter are surrounded by a dark pigment cap which gives the apothecia a pronounced dotted appearance. The scale bar is 10 m. Illustration by the author.

12 The terminal cell can be up to 6–7 m wide, and the apex is sometimes surrounded by a dark pigment ‘cap’ (Fig. 2). The ascospores are hyaline, ellipsoid, fusiform, or oblong, and usually 0–3-septate (Fig. 3). The spores are often somewhat curved, but they never have ornamentation or a perispore.

Figure 3. Examples of spores in Lecania. At the left two non-septate fusiform spores from L. cyrtellina, in the middle two 1-septate ellipsoid spores from L. cyrtella, and at the right two 3-septate oblong spores from L. fuscella. The scale bar is 5 m. Illustration by the author.

When the asci are treated with first potassium hydroxide and then iodine, the ascus apex or tholus is stained enabling determination of the apex structure. The tholus has a low, blunt, conical ocular chamber, and a high, conical axial body with or without a surrounding zone that is darker than the rest of the stained apex (Fig. 4).

Figure 4. The two types of ascus apices found in Lecania. At the left the conical axial body is surrounded by a darker zone, this type of apex is sometimes referred to as a Biatora-type. At the right the apex surrounding the conical axial body is of uniform color, sometimes referred to as a Bacidia-type apex. The scale bar is 10 m. Illustration by the author.

13 Conidia in Lecania are of two types: 0–1-septate microconidia which are strongly curved, sometimes sigmoid and 0.5–1 m wide, and 0–1-septate macroconidia which are more or less curved and 1–2 m wide. Most members of Lecania do not produce secondary substances. An exception is L. aipospila where triterpenoids have been detected (van den Boom & Brand 2005). Since both corticolous, lignicolous and saxicolous species are found in the genus it is not possible to make generalizations of the ecological preferences. However, it appears that several of the corticolous species are early coloniz- ers. They are dispersed by spores, regenerate easily, and can potentially colonize widespread areas. As a consequence of the early colonizer strategy they usually do not withstand competition very well, and thus continuously require suitable substrates for colonization. For this reason many corticolous species do not cover large areas, but are more commonly scattered over the substrate. The saxicolous species are in general more tolerant to competition and can sometimes cover large surfaces (e.g. L. aipospila and L. erysibe), but some saxicolous species appear to be more sensitive to competition e.g. L. rabenhorstii and L. atrynoides. Several species in Lecania are uncommon or rare in Sweden, but only two species appear on the Swedish Red List, i.e. L. fuscella and L. koerberiana (Gärdenfors 2005). Both are listed in the data deficient category (DD) illus- trating the very limited knowledge of the distribution and ecological preferences of many of the species in the genus. Many lichens have very narrow ecological requirements, e.g. they may require presence of old trees of a specific species, and such lichens are often used as indicators of biodiversity and biotopes worthy of protection (e.g. Nitare 2000). No Lecania species are included in these lists despite the rareness of some of the species. One important qualification for an indicator species is that it should be relatively easy to find and identify in the field; qualifications that none of the rare Le- cania species satisfy. The very small size of Lecania apothecia (usually less than 1 mm), their inconspicuous appearance, and the scattering of the apothecia renders them very hard to find, and identification in the field is at best very uncertain and usually impossible. Apart from difficulties in finding and identifying the different species, the knowledge of the preferences and ecological demands of most of them is much too limited to infer anything about the habitat where they are found.

Aims This thesis concentrates on different aspects of the genus Lecania, and includes phylogenetic, taxonomic, ecological, and conservation approaches. Paper I deals with the overall phylogenetic relationships of the genus, and aims at testing the monophyly of a representation of the species traditionally included in Lecania. In Paper II, the aim was to formally describe one of the new species included in the phylogeny in Paper I. In Paper III, I wanted to investigate the relationships of a group of morphologically similar species in Lecania s. str., i.e. the Lecania cyrtella group. This was obtained by employing both morphological and molecular methods. In Paper IV, the reduction in distribution and the phylogenetic diversity of the type species of the genus, Lecania fuscella, was investigated. Possible reasons for the disappearance and implications concerning conservation of the species are discussed.

15 Materials and methods

In Paper I (and also Paper II and IV) three gene regions were used, two from the nuclear genome; the internal transcribed spacer region (ITS) and the first part of the RNA polymerase II second largest subunit (RPB2); and one from the mitochondrial genome; the mitochondrial small subunit (mt- SSU). The ITS region is located between the small subunit (SSU) and the large subunit (LSU) in the ribosomal protein factory with the 5.8S inserted between ITS-1 and ITS-2. The ribosomal DNA exists in numerous copies (Baldwin et al. 1995). In Lecania and related genera the region is c. 500 base pairs (bp.) long. RPB2 encodes the second largest subunit of RNA polymerase II, which is the holoenzyme responsible for transcription of mRNA. It is a low-copy gene (Liu et al. 1999) and the part used here is c. 980 bp. long. Mitochondria are cell organelles with their own DNA coding for some structural proteins and enzymes involved in respiration. The structure of the ribosomal DNA in the mitochondria resembles that in the nucleus, but usually exists in only one copy (Bruns & Szaro 1992). The mt-SSU region in Lecania and related genera is c. 760 bp. long. The ITS and mt-SSU have frequently been used in lichens (e.g. Mattsson & Wedin 1998, LaGreca 1999, Ekman 2001, Andersen & Ekman 2005, Crewe et al. 2006), whereas RPB2 has only been employed recently (e.g. Reeb et al. 2004, Midlikowska et al. 2006). In Paper III two gene regions were used. The ITS region and the last part of the intergenic spacer region (IGS), also from the nuclear genome. The IGS is located between LSU and SSU in the ribosomal DNA where it is present in numerous copies (Sugita et al. 2002). The part used in Paper III is c. 350 bp. long. The use of IGS in lichens is also fairly recent (e.g. Printzen & Ekman 2002, Lindblom & Ekman 2005). To infer the phylogenetic relationships several different analyses methods were employed. In Paper I (and also Paper II and IV) the main tool was Bayesian inference and additional measures of branch support were obtained by performing maximum parsimony and maximum likelihood bootstrap analyses. In Paper III haplotype network analyses were complemented with maximum parsimony and Bayesian inference analyses. For more details see Paper I and III. In Paper IV non-metric multidimensional scaling (NMS) was employed to infer similarities in species composition between different localities. Fur- thermore, phylogenetic diversity measures (PD) were used to ascertain biological distinctiveness. For more details see Paper IV.

16 Results and discussion

Paper I and II Paper I is the first molecular study of the phylogeny of Lecania. As men- tioned above, Lecania has often been circumscribed on basis of a few morphological characters, and I suspected that the genus would display an exten- sive amount of polyphyly. In order to elucidate the phylogenetic relationships in the group 25 species traditionally placed in Lecania and 21 species from closely related genera were included in the analyses. It was revealed that Lecania indeed is a polyphyletic genus (Fig. 5). However, a monophyletic group comprising 16 Lecania species, including the type species L. fuscella, was recovered in the analyses. As a consequence, recognition of Lecania s. str. is proposed. Nine species formerly assigned to the genus do not belong in Lecania s. str., and some of these are only very distantly related to Lecania s. str. The species which should be excluded from Lecania are: L. baeomma, L. glauca, L. gerlachei, L. brialmontii, L. racovitzae, L. hyalina, L. chlorotiza, L. naegelii, and L. furfuracea. Unfortunately, the cor- rect affiliations for most of these taxa are not revealed in this study owing to too sparse taxon sampling. However, L. hyalina has already been included in Biatora as B. globulosa (Printzen 2004), and affiliations for L. gerlachei, L. brialmontii and L. racovitzae are suggested in Paper I (see below). Lecania s. str. is divided into two major groups with high branch support: a group of saxicolous species, which I refer to as the L. inundata group (L. aipospila, L. inundata, L. turicensis, L. atrynoides, and L. sp. 1) and a group consisting of both saxicolous and corticolous species, which I call the L. cyrtella group (L. sylvetris, L. dubitans, L. sambucina, L. hutchinsiae, L. cyrtella, L. erysibe, and L. sp. 2). The type species, L. fuscella, is not included in any of these groups (Fig. 5). According to the phylogenetic analyses some morphologically similar species in Lecania s. str. are not closely related. This applies to L. rabenhorstii, which is only distantly related to the L. inundata group despite close morphological resemblance with L. inundata. Another example is L. sambucina, which has often been regarded a synonym of L. cyrtella. These species are separated mainly on basis of the number of spores in the asci. Le- cania cyrtella has 8 spores per ascus, whereas L. sambucina has 10–16. Nevertheless, my analyses clearly demonstrate that L. sambucina is a separate species.

Figure 5. Bayesian majority rule consensus tree based on ITS, mt-SSU and RPB2 sequences with average branch lengths. Branch support, Bayesian posterior probabilities (PP) > 0.5, maximum likelihood bootstrap proportions (BP-ML) and maximum parsimony bootstrap proportions (BP-MP) > 50 %, are displayed at the nodes.

For more detailed descriptions of the morphological similarities and differences between these two species see Paper III. Lecania cyrtellina has also been confused with L. cyrtella, but is only distantly related to this species. Close examinations reveal that these species are not too hard to separate morphologically. Lecania cyrtellina has non-septate, very narrow spores (Fig. 3) and a much lower hymenium than L. cyrtella (see Paper III for more detailed descriptions of both species). Finally, L. hutchinsiae and L. sylves-

18 tris are shown not to be closely related despite morphological similarities. These species are mainly separated morphologically by differences in the thallus, and the color and translucency of the apothecium. Lecania hutchinsiae is found on non-calcareous rocks, while L. sylvestris prefers calcareous rocks (more details in Paper III). Apart from the recovery of a monophyletic Lecania s. str., the analyses produced a couple of interesting results involving other genera. First, it can be noted that the included representatives from the genus Bilimbia (B. mi- crocarpa, B. lobulata, and B. sabuletorum) form a well-supported group (Fig. 5, Bilimbia s. str.), thus the recent exclusion of these species from My- cobilimibia (Hafellner & Türk 2001, Hafellner & Coppins 2004) are supported by molecular data. Second, the three Antarctic ‘Lecania’ species, L. gerlachei, L. brialmontii, and L. racovitzae, form a well-supported group (Fig. 5, Thamnolecania). These three species are all Antarctic endemics. The first two of these three species are sometimes referred to as Thamnolecania and recognition of this genus is a possibility. An alternative to recognition of these two genera could be a new, wider circumscription of Bilimbia (Bilimiba s. lat., Fig. 5). This clade comprises the Bilimbia group, the morphologically similar Bacidia fuscoviridis, the ‘Thamnolecania’ group plus Lecidea sphaerella and Catillaria croatica. An advantage of recognizing a wide Bilimbia would be placement of some ‘hard-to-place’ species (B. fuscoviridis, L. sphaerella, and C. croatica). These three species obviously have no affiliation to the genera in which they are currently placed. Two undescribed species were included in the phylogenetic study. One of these, Lecania sp. 1 = L. belgica, (Fig. 5) is formally described as a new species in Paper II. It is a saxicolous species which is only known from the type locality in Belgium. The other undescribed species, L. sp. 2, was treated together with the rest of the L. cyrtella group in Paper III (see below).

Paper III

Most of the saxicolous Lecania species included in the phylogenetic study in Paper I have already been treated in various morphological revisions (Mayrhofer 1988, van den Boom 1992). I thus chose to concentrate on the relationships in the Lecania cyrtella group, and I extended the group to include L. cyrtellina since this species frequently has been confused morphologically with L. cyrtella. The relationships were studied utilizing both molecular and morphological methods. Eleven species were included in the study: L. cyrtella, L. cyrtellina, L. dubitans, L. erysibe, L. hutchinsiae, L. sambucina, and L. sylvestris which all were included in the phylogeny study in Paper I, and furthermore, L. leprosa, L. madida, L. prasinoides, and L. proteiformis which morphologically appeared to belong in this group. Le-

19 cania leprosa (= L. sp. 2 from Paper I) and L. madida are new to science, and are formally described in Paper III. Lecania leprosa is a saxicolous species from Eastern Europe. It is the sister to L. erysibe (Figs. 5 & 7), which it closely resembles morphologically, but the new species contains a pigment, Hertelii-green (Meyer & Printzen 2000), never detected in L. erysibe. Fur- thermore, some anatomical and morphological differences separate the species. Lecania madida is a corticolous species from the Pacific Northwest of North America. The species resembles L. cyrtella morphologically, but is probably more common or might even replace L. cyrtella in the area. Le- cania madida has a preference for very moist habitats as opposed to the dry, nutrient-rich habitats of L. cyrtella. Additionally, a number of morphological and anatomical differences separate the species. Lecania prasinoides and L. proteiformis have been described earlier, but have not been fully recognized. Lecania prasinoides has previously only been reported from Russia and Eastern Europe, but in this paper the distribution range is extended to include the Baltic countries, the Nordic countries, and Western Canada. The species resembles L. cyrtella and L. cyrtellina, but can be separated from these by a number of anatomical and morphological differences as well as a preference for very humid habitats. Lecania proteiformis has during the last century been treated as a variety of L. erysibe which it resembles somewhat morphologically, but both morphological and anatomical characters and molecular evidence indicate that it is a species of its own (Figs. 6 & 7). A key to all the species and species descriptions are provided in Paper III.

Figure 6. The 95 % probability haplotype network based on IGS and ITS sequences. One line represent one mutational step and open circles represent haplotypes not present in the analysis. The size of the ovals is proportional to the number of haplotypes sharing the same sequence.

Figure 7. One of 4700 most parsimonious trees based on IGS and ITS sequences. Branch support is shown at the nodes and includes bootstrap support > 50 % above the branches and posterior probabilities > 0.5 below.

Lectotypes are designated for three species, i.e. L. cyrtella, L. erysibe, and L. sambucina. The morphological distinctions between the species in this group are very well supported by molecular evidence. In the haplotype network estimation (Fig. 6) only specimens from the same taxa cluster in the same networks. Furthermore, the phylogenetic reconstructions (Fig. 7) indicate well- supported monophyletic groups for all the included species, pointing to a high degree of autapomorphies within the species. However, the interspecific relationships are not resolved in this investigation, most likely owing to a high degree of homoplasy among the closely related taxa or inability of the chosen gene regions to resolve the interspecific relationships. Most likely,

21 addition of more gene regions is needed to clarify the relationships within the genus.

Paper IV In order to establish the molecular phylogeny of Lecania in Paper I it was necessary to request fresh material of most of the included species, since obtaining intact DNA from old material is often very difficult. A soundly based phylogeny had to include the type species, L. fuscella, otherwise no conclusions about the relationships of the genus could be drawn. However, getting hold of fresh material of this species proved to be very difficult even though lichenologists all over the world were contacted; apparently it is a very rare species everywhere. This assumption is confirmed by information on distribution and frequency. Apart from being listed as endangered in Sweden (Gärdenfors 2005), the species appears on Red Lists in at least two additional countries, and it is considered extremely rare or extinct in a number of other countries. Since herbarium material indicates that the species was more frequent earlier, the reduction seems to have occurred rather recently. I therefore decided to investigate localities in Sweden, where the species had previously been found. This was done in order to recover L. fuscella, add to the limited knowledge of its ecological preferences, and try to explain the reduction in occurrence. Lecania fuscella was only recovered in one of 12 reinvestigated localities and no new findings in other localities were registered.

Figure 8. Distribution of Lecania fuscella in Sweden. At the left the distribution between 1859 and 1968. represents 51 old collections on the mainland and 5 old collections from Gotland, all with presence of L. fuscella. At the right distribution after 1980 is shown. represent the only locality where L. fuscella was recovered, the 11 reinvestigated localities where the species was absent, and the 5 new collections from Gotland where L. fuscella is present.

22 The past and present distribution of L. fuscella in Sweden is illustrated in Fig. 8. The total species composition was determined in 58 old herbarium collections and in the 12 revisited localities, as it was hypothesized that changes in associate species composition might provide some clue as to why the species have diminished so greatly in number. Most of the species encountered in the investigation were common and only nine of the 44 species found in the old collections were not encountered in the reinvestigation. However, the NMS analysis comparing species composition in 58 old collections with five new collections from Gotland (Sweden) show small differences in the type of vegetation community where L. fuscella is encountered. The new collections cluster together in the outskirts of the plot indicating some differences in species composition between the old and the new collections (, Fig. 9).

Figure 9. Similarities in species composition between collections shown on a NMS graph based on presence/absence data of 50 species in 63 collections. represents 5 new collections from Gotland, represents 5 old collections from Gotland, and represents 53 old collections from 12 provinces in Sweden, all with presence of Lecania fuscella. represents 2 new collections from Gotland and Uppland in Swe- den, and 2 old collections from Gotland and Uppland, all with presence of Dip- lotomma alboatrum.

Five old collections from Gotland (, Fig. 9) cluster in the vicinity of four of the new Gotland collections suggesting that the differences in species composition partly are a result of differentiations between different geographical regions. However, other factors may also influence the result. An interesting associated lichen is Diplotomma alboatrum which prefers dry, sun-exposed, nutrient-rich bark. This species was encountered in all five new Gotland collections with presence of L. fuscella. It was also found in the only reinvestigated locality where L. fuscella was recovered, and in nine of the 58 old collections. It was not found in any of the reinvestigated localities without presence of L. fuscella. To get an indication of similarities in species

23 composition between the ecological niches of L. fuscella and D. alboatrum, four D. alboatrum collections were included in the statistical analysis (, Fig. 9). None of these collections held any L. fuscella. The Diplotomma collections group together close to the five new Gotland collections (Fig. 9). This indicates that the vegetation community where L. fuscella is now encountered is becoming more similar to that of D. alboatrum. The slight shift in vegetation community is probably the consequence of a deterioration of most of the habitats the species used to inhabit. This has resulted in a reduction of the number of habitats that satisfies the demands of L. fuscella, and since the species has not been able to adapt to these environmental changes it has decreased greatly in number. The NMS analysis of the 12 reinvestigated localities (Fig. 10) also shows differences in species composition between the locality with and those without L. fuscella. The locality where L. fuscella was recovered has a different vegetation community than the 11 localities where the species was not found. The small sample sizes in both analyses calls for caution in the interpretation of the results.

Figure 10. Similarities in species composition between localities shown on a NMS graph based on presence/absence of 98 species in 12 localities. represents the locality where Lecania fuscella was encountered and represents the localities where the species was absent.

A possible explanation for the considerable reduction of Lecania fuscella could be that acidification and especially acid rain has impaired the living conditions of the species by increasing substrate pH and possibly even by direct toxic effects on the lichen. Even though ambient acidification has decreased during the last couple of decades, recovery of the species has most likely been hampered by 1) no specimens were left to disperse spores and/or 2) inability of the lichen to compete with the nitrogen-tolerant/-loving species that tend to dominate species composition in many habitats nowadays.

24 In any case, it appears that L. fuscella has not been able to respond to the environmental changes it has experienced over the last half century. The species is becoming increasingly isolated in the few localities where is lin- gers. Small populations are more prone to genetic drift and loss of genetic variation, thus the risk of extinction due to rapid environmental changes will increase. In addition to the attempt to recover Lecania fuscella and explain the reduction of the species, the biological distinctiveness was determined by calculation of the species’ phylogenetic diversity (PD) using branch lengths in the phylogenetic tree obtained in Paper I. It was uncovered that L. fuscella is a genetically distinct lineage in the genus, thus preservation of the species would protect a considerable amount (13 %) of the biological distinctiveness in the genus. However, this investigation questions the proposed connection between genetically distinct lineages and their evolutionary potential (e.g. Soltis & Gitzendanner 1999, Forest et al. 2007). A taxon with a high PD has accumulated feature diversity and theoretically has a higher possibility of possessing a feature that can respond to future environmental changes. Still, a high PD does not ensure variation in the quantitative traits responsible for adaptation to environmental changes, and this seems to be the case in this investigation. Despite a high PD L. fuscella has not been able to respond to any of the repeated changes it has been exposed to. It may therefore be necessary to include genes coding for selectively important variation in the phylogenetic analyses of PD if the objective is that PD should represent the evolutionary potential.

One final remark, after having conducted field work in the Pacific Northwest in USA I am now in a position to demonstrate that there are no Lecania in Paradise – at least not in Paradise, Montana!

25 Dansk resumé

Laver Laver er symbiotiske systemer som består af en svamp (mykobionten) og enten en grønalge eller en cyanobakterie (fotobionten). Man kan derfor ikke kalde en lav for en organisme for hvad taler man så om, svampen eller fotobionten? Normalt er en symbiose et forhold, hvor begge parter har fordele af at leve sammen, men man har fundet ud af, at svampen ofte parasiterer på fotobionten i begrænset omfang, og at fotobionten forhindres i at forplante sig sexuelt, når den lever inde i svampen. Hvorfor lever disse to partnere så overhovedet sammen? Både svamp og fotobiont har trods alt fordele af sameksistensen. Via fotosyntese producerer fotobionten sukker og andre karbohydrater, som udnyttes af svampen. Til gengæld beskytter svampen fotobionten mod stærkt lys og forsyner den med vand. Svampens vigtigste symbiotiske funktion er dog nok, at den giver fotobionten et levested, som gør, at fotobionten kan trives på levesteder (habitater) hvor den aldrig ville overleve fritlevende, f. eks. på næringsfattige sten, tør bark eller i ørkener. En lav består af løv (thallus) og frugtlegemer. Løvet indeslutter fotobionten og er oftest lagdelt med overbark, et fotobiontlag, et marvlag og hos nogle laver underbark. Løvet kan have mange forskellige udformninger, og af praktiske grunde opdeler man ofte laverne efter deres udseende: bladformede laver, som har bredt, fladt bladlignende løv, buskformede laver, som har rundt, grenet busklignende løv og skorpelaver, som har fladt løv der er helt fastvokset til underlaget. Udformningen af frugtlegemerne er også mangfoldig; de kan være åbne og skiveformede (apothecium) eller næsten lukkede som en lille kugle (perithecium), og der findes mange variationer af disse to grundformer. Uanset udformningen produceres sporerne dog i små sække (asci – ental ascus). Nogle begreber som anvendes i lav-terminologien er illustreret i Figur 11. Når en lav skal formere sig sexuelt, dvs. når den producerer sporer, er det kun svampepartneren, som spredes. Derfor må svampen finde fotobiontceller af den rigtige slags for at kunne udvikles til en lav; ikke en helt let opgave for mange fotobionter findes ikke fritlevende. Svampen løser ofte dette problem ved at bruge de alger, den kan få fat i som en midlertidig løsning, og så senere stjæle den rigtige type fotobiontceller fra andre laver. Mange laver formerer sig også vegetativt ved at producere isidier (små barkklædte udvækster på løvet som let kan brække af. De består

26 af svampehyfer og alger) eller soredier (et mellignende pulver som består af små klumper af alger, som er omgivet af svampehyfer). På den måde slipper laven for at skulle finde en fotobiont, eftersom både svamp og fotobiont er med i samme pakke.

Lavsystematik Svamperiget, som er mere i slægt med dyr end med planter, inddeles i syv rækker. De fire største rækker er piskesvampe (Chytridiomycota), koblingssvampe (Zygomycota), sæksvampe (Ascomycota) og basidiesvampe (Basidiomycota), og disse er igen inddelt i klasser, ordener, familier, slægter og arter. Denne inddeling er opbygget hierarkisk således at f. eks. en orden kan bestå af flere familier, som igen kan bestå af flere slægter. Det højeste niveau i hierarkiet kaldes en art. Inddelingen af svamperiget (og alle andre riger) baseres på systematiske grupper, dvs. alle arter i en gruppe stammer fra samme forfader. Når man har en sådan gruppe, hvor alle arter stammer fra samme nærmeste forfader, kalder man gruppen monofyletisk. Hvis man derimod har en gruppe af arter, hvor nogle arter har én stamfader og andre en anden, kaldes gruppen polyfyletisk. Den videnskablige disciplin, hvor man inddeler arter efter deres slægtskabsforhold, kaldes systematik. Systematik inddeles ofte i flere underdiscipliner: identifikation, hvor arten identificeres og beskrives, taxonomi, hvor de beskrevne arter gives et navn, og fylogeni, hvor de navngivne arter deles ind i systematiske grupper alt efter slægtskabsforhold. Slægtskabsforholdene kan man udrede vha. morfologiske (udseende) og/eller molekylære karakterer, de sidstnævnte oftest med hjælp af DNA sekvenser. For at gøre slægtskabsforholdene overskuelige, konstruerer man stamtræer, som beregnes på baggrund af ligheder og forskelligheder mellem de inkluderede arter. Når man beskriver, navngiver og klarlægger slægtskabsforholdene hos laver er alting baseret på svampepartneren. De fleste laver tilhører rækken af sæksvampe, men der findes også nogle lavrepræsentanter i basidiesvampene. Sæksvampene inddeles i 61 ordener, og 11 af disse er helt licheniserede, dvs. alle medlemmer er laver. Oftest finder dog man licheniserede og ikke- licheniserede svampe i samme gruppe som hinandens nærmeste slægtninge. Eftersom licheniserede svampe findes i så mange forskellige grupper, inklusive i basidiesvampene, kan de umuligt have samme nærmeste forfader alle sammen. Man kan derfor konkludere, at dette samspil mellem svamp og fotobiont er opstået mange gange parallelt i forskellige grupper under evolutionen. Da man begyndte at inddele arter i systematiske grupper, havde man ikke andre redskaber end de morfologiske karakterer. Derfor regnede man med, at f. eks. en lavs vækstform var vigtig, og systematiske grupper indeholdt enten

27 bladformede laver, buskformede laver eller skorpelaver, men ikke en blanding. Senere har man dog fundet ud af, at vækstformen er udviklet som reaktion på miljømæssige påvirkninger, og ikke altid fordi de er nært beslægtede. Muligheden for at undersøge det genetiske slægtskab med hjælp af DNA analyser har vist, at der er flere familier, som indeholder både busk-, blad- og skorpelaver. Et eksempel kommer fra familien Ramalinaceae hvor den lavslægt jeg har arbejdet med, Lecania, hører hjemme. Ramalinaceae bestod tidligere udelukkende af buskformede laver – familien er opkaldt efter slægten Ramalina som på dansk kaldes grenlaver – men en molekylær undersøgelse af slægtskabsforholdene mellem denne familie og Bacidiaceae, en familie som indeholdt skorpelaver, viste at de var så nært beslægtede at man ikke kunne adskille de to forskellige familier. Eftersom Ramalinaceae er det ældste navn, er det ifølge systematiske regler det som gælder, og familien omfatter nu derfor både busk-, blad- og skorpelaver.

Lecania og min forskning Lecania er en slægt af små, uanselige skorpelaver. Thallus er oftest tyndt og/eller gråligt, apothecierne er brune–brunsorte og for det meste mindre end 1 mm. Der findes ca. 40 arter spredt over jorden, mest i de tempererede egne. De forskellige Lecania-arter vokser på sten, bark og ved. Slægten er i stor udstrækning blevet overset af systematikere, nok fordi at den er så lidet iøjenfaldende og desuden ikke let at identificere. Derfor er hele slægten aldrig blevet behandlet sammen i moderne tid. I stedet har man defineret slægten ud fra relativt få morfologiske karakterer såsom, at arterne er skorpeformede, har lecanorin egenrand (se Fig. 11 for forklaring), to–fire- cellede sporer, og at ascustoppen har en bestemt udformning som ses, når man behandler den med først kaliumhydroxid og så jod. Problemet med at basere slægtskab på en eller få karakterer er, at man aldrig kan være sikker på, at de træk som ser ens ud virkelig er udviklet på samme måde, dvs. at de kommer fra samme forfader. Det er derfor meget bedre at basere sine slægtskabsrevisioner på mange karakterer og gerne både morfologiske og molekylære karakterer. På den måde kommer man ikke så let til at lægge for stor vægt på ligheder som i virkeligheden ikke kan sammenlignes. Inden jeg begyndte min forskning fandtes der som sagt ikke så meget information om slægten. Et par morfologiske revisioner af de stenlevende arter i slægten fra specifikke geografiske områder, og et par molekylære undersøgelser af familien hvor Lecania kun var repræsenteret med en eller to arter. Derfor kunne man ikke vide noget om slægtskabet indenfor gruppen, og der var altså al mulig grund til at mistænke, at den gruppe af arter som man traditionelt har inkluderet i Lecania ikke var monofyletisk. I artikel I undersøgte jeg det molekylære slægtskab imellem 25 arter fra Lecania og 21 arter fra andre slægter som jeg vidste fra en tidligere

28 undersøgelse var nærbeslægtede med Lecania. Undersøgelsen viste at Lecania var polyfyletisk og at ni af de arter som traditionelt har tilhørt slægten ifølge DNA sekvenserne ikke hører til denne slægt (se Fig. 5). En monofyletisk gruppe på 16 Lecania-arter, inklusive typearten Lecania fuscella, som beskrivelsen af slægten er baseret på, blev dog også fundet i analysen. Jeg foreslår derfor at Lecania bare skal omfatte disse 16 arter plus eventuelt andre arter som ikke var inkluderet i vores undersøgelse. Jeg viste også, at Lecania sambucina ikke er et synonym for Lecania cyrtella som man tidligere har troet, men en egen art. I analysen indgik der to nye, ubeskrevne Lecania-arter (sp. 1 og sp. 2, Fig. 5). Den ene af disse (sp. 1) bliver formelt beskrevet og navngivet (Lecania belgica) i artikel II. Eftersom de stenlevende arter i Lecania allerede er blevet revideret og beskrevet morfologisk et par gange, valgte jeg at koncentrere min næste undersøgelse (artikel III) om en gruppe af både trælevende og stenlevende arter – Lecania cyrtella-gruppen. Arterne i denne gruppe ligner hinanden morfologisk, og flere af dem er ofte blevet forvekslet med hinanden. Jeg beskrev derfor arterne indgående morfologisk og lavede desuden en nøgle, så man kan adskille arterne. For at underbygge mine morfologiske resultater analyserede jeg DNA sekvenser fra et antal individer af hver art og resultatet understøttede alle de arter jeg havde udskilt morfologisk (Fig. 6 & 7). To nye, ubeskrevne arter (den ene sp. 2 fra artikel I, Lecania leprosa, og den anden en ny art fra Nordvestre USA, Lecania madida) bliver beskrevet formelt og navngivet i denne artikel. To andre arter i undersøgelsen, Lecania prasinoides og Lecania proteiformis, blev beskrevet i slutningen af 1800- tallet/begyndelsen af 1900-tallet, men af den ene eller anden grund har de ikke været rigtigt anerkendt som arter. I denne undersøgelse kan det dog bekræftes, at begge er arter som bør anerkendes i Lecania. Den ene (Lecania prasionides) er tidligere kun fundet i Rusland og Østeuropa, men jeg kunne påvise at den også findes i de Baltiske lande, Norden og Vestre Canada. Som sagt tidligere var det vigtigt at have slægtens typeart, Lecania fuscella, med i den fylogentiske undersøgelse. Hvis den ikke havde været med, ville jeg ikke have kunnet afgøre, om den monofyletiske gruppe af Lecania arter jeg fandt virkelig kunne kaldes Lecania, eftersom slægtsnavnet altid følger typearten, og man ikke uden videre kan gå ud fra at den ville havne i samme gruppe. Når man skal ekstrahere DNA fra en lav er det for det meste vigtigt, at man har ganske frisk materiale – ikke ældre end 3–5 år. DNA nedbrydes nemlig ret hurtigt i mange laver, og det bliver meget svært at få et resultat. Jeg var derfor nødt til at have fat i frisk materiale, men det var lettere sagt end gjort. Selv om jeg kontaktede lichenologer overalt i verden var der kun ganske få som kunne hjælpe mig med Lecania fuscella. Det virkede altså, som om Lecania fuscella er sjælden i de fleste områder, og dette bekræftes når man ser på oplysninger om udbredelse og forekomst. Arten er rødlistet i Danmark, Sverige og Tyskland og meget sjælden eller forsvundet i mange andre lande. Jeg besluttede derfor at opsøge lokaliteter i

29 Sverige, hvor arten tidligere er fundet for at se om arten stadig fandtes der og for at øge informationen om dens økologi – dvs. dens valg af levested og miljø. Tolv gamle lokaliteter blev besøgt men Lecania fuscella blev bare genfundet et sted (artikel IV). Artssammensætningen af øvrige laver blev også undersøgt, både i de gamle indsamlinger og i de lokaliteter som jeg besøgte. Det viste sig, at de fleste andre laver i de gamle indsamlinger var temmelig eller meget almindelige; af de 44 arter jeg idenficerede i de gamle indsamlinger, var der kun ni (ud over Lecania fuscella som ikke blev fundet på 11 lokaliteter) som ikke blev fundet igen. Hvorfor er Lecania fuscella så forsvundet, hvis de fleste andre arter stadig findes? Min teori er, at syreregn har påvirket arten i negativ retning, enten ved at forsure barken på de træer hvor laven vokser eller mere direkte gennem giftige effekter. I de sidste årtier er forsurningen og syreregnen mindsket meget, men det virker ikke som om arten kan komme sig. Dette kan have flere årsager. Det kan være fordi laven simpelthen allerede er forsvundet og således ikke kan sprede sine sporer eller måske fordi den ikke kan modstå konkurrencen fra den gruppe af laver, hvis levevilkår forbedres af de mange næringsstoffer, som i dag spredes fra f. eks. landbrug og udstødning fra biler. Selv om der vel ikke kan være så megen tvivl om at Lecania fuscella er blevet meget sjælden, kan man samtidig spørge, om det virkelig er noget at bekymre sig om. En lille uanselig lav, som kun eksperter kan finde og genkende, hvilken betydning har den? Der findes og anvendes mange meget subjektive måder at tage stilling til bevaring af arter, bl.a. om har de økonomisk værdi, og om mennesket etisk har lov til at bestemme, hvilke arter der skal udryddes for nu at nævne to yderligheder. En mere objektiv fremgangsmåde er at vurdere en arts økologiske værdi og biologiske særegenhed og dette kan systematikken hjælpe til med. Analyserer man hvor meget en art adskiller sig genetisk fra andre arter kan man anvende dette til at maximere den fylogenetiske diversitet, som man bevarer. Teorien bag dette er, at hvis man bevarer så megen genetisk diversitet som muligt har man større chance for at sikre en fortsat evolution. Grupper af arter som har stor genetisk diversitet har desuden teoretisk større mulighed for at kunne udvikle sig og overleve fremtidige miljøforandringer. Jeg beregnede den fylogenetiske diversitet for Lecania fuscella ud fra det stamtræ, jeg fik frem i artikel I (Fig. 5), og fandt ud af at arten øger slægtens fylogenetiske diversitet med hele 13 %. Hvis Lecania fuscella beskyttes er der altså en stor del af diversiteten i Lecania som beskyttes med den. Der er stadig mange uløste spørgsmål både med hensyn til systematik og økologi i lavslægten Lecania. Med denne afhandling har jeg både bidraget med nogle svar og skabt nye spørgsmål.

Figur 11. Tværsnit af et apothecium. Længst oppe ses hymeniet hvor man finder de sporeproducerende sække (asci) som indeholder sporer. Den allerøverste del af hymeniet, som ofte er pigmenteret, kaldes epihymeniet. Imellem sporesækkene vokser sterile svampehyfer (parafyser), og lige under hymeniet findes et sterilt lag af svampehyfer som kaldes hypotheciet. Hypotheciet er også tit pigmenteret. Det sporeproducerende hymenium er omgivet af en egenrand (excipulum) som kan indeholde fotobiontceller (et såkaldt lecanorint apothecium) eller være uden (et lecideint apothecium). Desuden ses en lille del af løvet (thallus) på begge sider af apotheciet. Løvet består af overbark, fotobiontlag og marv. Illustration af forfatteren.

Tvärsnitt genom ett apothecium. Längst upp syns hymeniet med sporsäckarna (asci). Inuti sporsäckarna syns sporer. Översta skiktet av hymeniet, som ofta är pigmente- rad, kallas epihymeniet. Mellan sporsäckarna växer sterila svamphyfer (parafyser), och strax under hymeniet finns ett sterilt skikt av svamphyfer (hypotheciet). Även hypotheciet är ofta pigmenterat. Det sporbildande hymeniet omsluts längst ut av en kant (excipulum) som antingen kan innehålla fotobiontceller (lecanorint apothecium) eller ej (lecideint apothecium). Dessutom syns en liten bit av bålen (thallus) på båda sidorna av apotheciet. Bålen utgörs av överbark, fotobiontskikt och märg. Il- lustration av författaren.

31 Svensk sammanfattning

Lavar Lavar är symbiotiska system, som utgörs av en svamp (mykobionten) och antingen en grönalg eller en cyanobakterie (fotobionten). En lav är alltså inte en organism, utan utgörs alltid av två olika organismer, svamp och fotobiont. I vanliga fall är en symbios något där båda parter har fördel av att leva ihop, men det finns undersökningar som pekar på att svampen ofta parasiterar på fotobionten i begränsad omfattning, och att fotobionten hindras från att fort- planta sig sexuellt inuti svampen. Varför lever då dessa två organismer ihop? Både svamp och fotobiont har trots allt fördel av samlevnaden. Svampen utnyttjar socker och andra kolhydrater som produceras av fotobionten via fotosyntes. I utbyte förses fotobionten med vatten och får skydd mot starkt ljus. Svampens kanske viktigaste funktion i detta sammanhang är förmodli- gen att den förser fotobionten med en miljö där den kan frodas på växtplatser (habitat) där den knappast skulle överleva frilevande, t.ex. på näringsfattig sten, torr bark, eller i öknar. En lav är uppbyggd av en bål (thallus) och fruktkroppar. Bålen omsluter fotobionten och den är oftast indelad i flera skikt: överbark, fotobiontskikt, märg och hos några lavar underbark. Bålen kan ha mycket olika utseende i olika lavar och av praktiska skäl urskiljs ofta lavarna enligt bålens form: bladlavar, som har breda, tillplattade bålflikar, busklavar, som har runda, grenade bålflikar och skorplavar, som växer som en skorpa över underlaget. Fruktkropparna kan också ha olika form och byggnad; ibland är de öppna och skållika (apothecium), ibland nästan slutna och urnformade (perithecium), och det finns gott om variation inom dessa två grundformer. Oavsett fruktkroppens form bildas sporerna inuti små säckar (asci – ental ascus). Några vanliga begrepp i lav-terminologin finns illustrerade i Figur 11. Om en lav ska förökas sexuellt, dvs. producera sporer, är det endast svampkomponenten som sprids. Sporerna måste då hitta rätt fotobiont om de ska utvecklas till en lav, något som inte är okomplicerat eftersom många fotobionter inte finns frilevande. Svampen löser ofta detta genom att utnyttja de alger som finns till hands för tillfället för att sedan stjäla rätt typ av fotobiont från andra lavar. Många lavar förökas även asexuellt genom att bilda isidier (små barkklädda bålutväxter som är uppbyggda av svamphyfer och alger) eller soredier (ett mjölliknande pulver som består av en lös vävnad av svamphyfer

32 och alger). På detta sätt behöver laven inte leta rätt på en fotobiont, eftersom båda komponenter finns med då den sprids.

Lavsystematik Svampriket, som är närmare besläktad med djur än vad det är med växter, indelas i sju fyla varav de fyra största är gisselsvampar (Chytridiomycota), kopplingssvampar (Zygomycota), sporsäcksvampar (Ascomycota) och basidsvampar (Basidiomycota). Fyla delas i sin tur i klasser, ordningar, familjer, släkten och arter. Indelningen är hierarkisk så att t.ex. en ordning kan innehålla fler familjer, som i sin tur kan innehålla fler släkten. Arten är den grundläggande nivån i hierarkin. Denna indelning av svampriket (och alla andra riken) baseras på systematiska grupper, dvs. att alla arter i en grupp härstammar från samma anfader. En sådan grupp där alla arter har samma närmaste anfader kallas monofyletisk, medan en grupp som innehåller arter som härstammar från flera olika anfäder sägs vara polyfyletisk. Den vetenskapliga disciplinen där man inde- lar arter enligt släktskap benämns systematik. Systematik utgörs av flera underdiscipliner: identifiering, där arten identifieras och beskrivs, taxonomi, där de beskrivna arterna namnges, och fylogeni, där de namngivna arterna indelas i systematiska grupper enligt släktskap. Släktskap kan utredas med hjälp av morfologiska (utseende) och molekylära karaktärer, det sistnämnda oftast med hjälp av DNA sekvenser. För att överskådliggöra släktskapen, konstruerar man stamträd, som beräknas med avseende på likheter och olik- heter mellan de ingående arterna. Beskrivning, namngivning och bestämning av släktskap hos lavar baseras på svampkomponenten. De flesta lavarna tillhör fylum sporsäcksvampar men det finns även lavar i fylum basidsvampar. Sporsäcksvamparna indelas i 61 ordningar varav 13 är enbart licheniserade, dvs. alla arter i ordningen är lavar. Mer vanligt är att licheniserade och icke-licheniserade svampar finns i samma grupp. Eftersom licheniserade svampar finns i så många olika grupper, inklusive basidsvamparna, är det otänkbart att de har samma närmaste anfader. Man kan därför dra slutsatsen att symbiosen mellan svamp och fotobiont har uppkommit parallellt åtskilliga gånger i olika grupper under evolutionen. När man började indela arter i systematiska grupper fanns inga andra verktyg än de morfologiska karaktärerna. Därför tillade man t.ex. lavens växtform stor betydelse, och de systematiska grupperna fick omfatta antingen bladlavar, busklavar eller skorplavar men aldrig en blandning av dessa former. Efterhand har man dock insett att växtformen utvecklas som respons på miljömässig påverkan och inte alltid på grund av släktskap. Genom möj- ligheten att undersöka det genetiska släktskapet genom DNA-analyser har vi upptäckt flera exempel på familjer där både blad-, busk- och skorplavar in-

33 går. Ett exempel är familjen Ramalinaceae, som det lavsläkte jag har stude- rat, Lecania, hör till. Ramalinaceae omfattade tidigare enbart busklavar – släktet har namn efter Ramalina, brosklavar på svenska – men en molekylär undersökning av släktskap mellan denna familj och Bacidiaceae, en familj som omfattade skorplavar, avslöjade att de är så nära släkt med varandra att det var omöjligt att urskilja två familjer. Eftersom Ramalinaceae var det äldsta namnet, är det enligt systematiska regler det som är giltigt, och familjen omfattar nu därför både busk-, blad- och skorplavar.

Lecania och min forskning Lecania är ett släkte av små, föga iögonfallande skorplavar. Bålen är oftast tunn och/eller gråaktig, fruktkropparna är bruna–brunsvarta apothecier som i allmänhet är mindre än 1 mm. Det finns ungefär 40 arter spridda i världen, mest i tempererade områden. De olika Lecania-arterna växer på sten, bark och ved. Släktet har i hög grad förbisetts av systematiker, sannolikt för att det är så anonymt och dessutom inte helt lätt att identifiera. Av den anled- ningen har hela släktet aldrig behandlats i modern tid. Släktet har definierats på basis av relativt få morfologiska karaktärer, t.ex. den skorpformade bålen, lecanorina apothecier (se Fig. 11 för förklaring), två–fyrcelliga sporer, och en ascustopp som har ett specifikt utseende som syns om den behandlas med först kaliumhydroxid och sedan jod. Om man baserar släktskap på en eller få karaktärer riskerar man dock att se likheter mellan karaktärsdrag som har uppkommit på olika sätt, dvs. att de härstammar från olika anfäder. Det är därför bättre att basera sina släktskapsrevisioner på många karaktärer och mycket gärna både morfologiska och molekylära karaktärer. På det sätt minskas risken att icke-jämförbara likheter får för stor vikt. Innan jag påbörjade min forskning fanns det som redan nämnts inte mycket information om släktet. Ett par morfologiska revisioner av stenlevande arter från specifika geografiska områden, och ett par molekylära un- dersökningar av familjen där Lecania bara fanns representerad av en till två arter. Det var därför omöjligt att veta något om släktskap inom gruppen och följaktligen fanns det anledning att misstänka, att den grupp arter man traditionellt hänförde till Lecania inte var en monofyletisk grupp. I artikel I un- dersökte jag det molekylära släktskapet mellan 25 arter från Lecania och 21 arter från andra släkten som tidigare undersökningar hade visat var närbe- släktade med Lecania. Undersökningen visade att Lecania är polyfyletiskt och att nio av de arterna som traditionellt inkluderats i släktet enligt DNA sekvenserna inte hör dit (se Fig. 5). Jag fann även en monofyletisk grupp bestående av 16 Lecania-arter, inklusive typarten Lecania fuscella, som släk- tets beskrivning baserats på. Jag föreslår därför att Lecania endast ska omfatta dessa 16 arter plus eventuellt ytterligare arter som inte ingick i vår analys. Jag konstaterade också att Lecania sambucina inte är synonym med Lecania

34 cyrtella som man tidigare har trott, utan är en egen art. Två nya, obeskrivna Lecania-arter ingick i analysen (sp. 1 och sp. 2, Fig. 5). En av dessa (sp. 1) beskrivs formellt och namnges (Lecania belgica) i artikel II. Eftersom de stenlevande arterna i Lecania redan har beskrivits och revide- rats ett par gångar morfologiskt, valde jag att koncentrera min nästa under- sökning (artikel III) på en grupp som inkluderar både trädlevande och stenlevande arter – Lecania cyrtella-gruppen. Arterna i denna grupp är lika varandra morfologiskt, och flera av dem har ofta förväxlats med varandra. Jag beskrev därför arternas morfologiska drag noggrant och gjorde dessutom en nyckel så det går att åtskilja arterna. För att befästa mina morfologiska resultat analyserade jag flera individer från varje art och resultatet gav stöd för alla arter som jag hade urskiljt morfologiskt (Fig. 6 & Fig. 7). Två nya, obeskrivna arter (den ena sp. 2 från artikel I, Lecania leprosa, den andra en ny art från nordvästra USA, Lecania madida) beskrivs formellt och namnges i artikel III. Två andra arter som ingick i undersökningen, Lecania prasinoides och Lecania proteiformis, beskrevs i slutet av 1800-talet/början av 1900- talet, men har av någon anledning aldrig riktigt accepterats som arter. Denna undersökning visar dock att båda bör accepteras i Lecania som egna arter. En av dem (Lecania prasinoides) har tidigare bara påträffats i Ryssland och Östeuropa, men jag kunna konstatera att den även finns i de Baltiska stater- na, Norden och västra Kanada. Som redan nämnts var det mycket viktigt att ha med släktets typart, Leca- nia fuscella, i den fylogentiska undersökningen. Om den inte hade ingått skulle jag inte kunna ha avgjort, huruvida den monofyletiska gruppen av Lecania-arter verkligen skall kallas Lecania, eftersom släktnamnet alltid följer typarten, och det skulle vara omöjligt att veta om typarten skulle ham- na i samma grupp. Ska man extrahera DNA från en lav är det oftast viktigt att materialet som man använder är färskt – företrädesvis inte mer än 3–5 år gammalt. DNA bryts ned ganska snabbt i många lavar, och då blir det mycket svårt att få fram ett resultat. Jag var därför tvungen att få tag på färskt material, men det var inte alltid lätt. Även om jag kontaktade lichenologer överallt var det endast enstaka som kunna hjälpa mig med Lecania fuscella. Denna art verkar vara sällsynt på de flesta ställen, och detta bekräftas om man kontrollerar uppgifter om förekomst och utbredning. Arten är rödlistad i Sverige, Danmark och Tyskland och mycket sällsynt eller försvunnen i många andra länder. Jag ville därför leta upp lokaler i Sverige, där arten tidigare hade funnits för att se om den fortfarande fanns och för att öka kun- skapen om ekologin – dvs. artens val av omgivningar och miljö. Jag under- sökte tolv gamla lokaler men återfann Lecania fuscella bara på ett ställe (artikel IV). Jag undersökte även artsammansättningen av andra lavar, både i de gamla insamlingarna och på de lokalerna jag undersökte. Det visade sig att de flesta andra lavarna i de gamla insamlingarna var ganska eller mycket vanliga; av de 44 arter jag identifierade i de gamla insamlingarna var det endast nio (förutom Lecania fuscella som inte återfanns i 11 insamlingar)

35 som inte återfanns. Varför har då Lecania fuscella försvunnit om de flesta andra lavarna finns kvar? Min teori är att surt regn har påverkat arten nega- tivt, antingen genom försurning av barken på de träd där laven växer, eller mer direkt genom giftiga effekter. De senaste årtionden har försurningen och det sura regnet avtagit avsevärt, men ändå tycks inte arten återhämta sig. Detta kan bero på olika orsaker. Om laven helt har försvunnit från ett områ- de kan den inte sprida sina sporer på nytt. Det är också möjligt att den inte tål konkurrens från den gruppen lavar som gynnas av de ökade halter när- ingsämnen som sprids från t.ex. lantbruk och bilavgaser. Trots att man kan konstatera att Lecania fuscella har blivit mycket säll- synt, måste man samtidigt ifrågasätta vilken betydelse det har. En liten, föga iögonfallande lav som bara experter kan hitta och identifiera, vad spelar den för roll? Det finns och används många subjektiva sätt att ta ställning till be- varande av arter, bl.a. huruvida de har ekonomisk värde, och om människan etisk får bestämma vilka arter som får utrotas. En mer objektiv metod har utarbetats som värderar artens ekologiska värde och biologiska unikhet och här kan systematiken spela en viktig roll. Analyserar man hur mycket en art skiljer sig genetisk från andra arter, kan man använda detta för att maximera den fylogenetiska mångfald som skyddas genom att skydda en viss art. Teo- rin bakom detta är, att om man bevarar så mycket genetisk mångfald som möjligt har man större chans att säkra den fortsatta evolutionen. Grupper av arter med stor genetisk mångfald har dessutom teoretiskt större chans att utveckla sig och därmed överleva framtida miljöförändringar. Jag beräknade den fylogenetiska mångfalden för Lecania fuscella med hjälp av det stamträd som framlades i artikel I (Fig. 5), och kom fram till att arten utökar släktets fylogenetiska mångfald med 13 %. Om Lecania fuscella skyddas är det där- för en ganska stor mängd av släktets unikhet som skyddas. Det finns fortfarande många olösta frågor med avseende på både systematik och ekologi i lavsläktet Lecania. Med denna avhandling har jag både bidragit med några svar och skapat nya frågeställningar.

36 Acknowledgements

There are many people without whom this thesis would never have seen the light of day, thank you so much all of you. I would like to start by apologiz- ing if I for some reason have left you out, it doesn’t mean that I’m not grateful just forgetful! Roger Gran was the person who introduced me to the fascinating world of cryptogams and their potential for nature conservation evaluations. Thank you for taking out a couple of days of your busy schedule and magically showing me an entirely new world. I would like to thank my supervisor Leif Tibell for the help I have re- ceived. I have benefited from your comments on my manuscripts and thesis. My other supervisor Stefan Ekman has been in Norway during most of my studies. Nevertheless, you always answered my, at times, stupid questions often within minutes of sending off a mail. You’re always there and always take the time to help. Thank you so much, I couldn’t have done this without you. Also sincere thanks to Wenche and your daughters for welcoming me into your home during my visits to Bergen. I am grateful to Bruce McCune for taking the time to offer suggestions concerning the field work for my fourth paper. Furthermore, Bruce, Pat and their daughter Sarah are warmly thanked for welcoming me into their family during my visit to the Pacific Northwest. You made me feel at home in Cor- vallis despite the short time I spent there. Many heartfelt thanks to the international lichenological community who actually thinks it’s great that someone works with Lecania!!! I have so much enjoyed meeting all of you at excursions, conferences, courses, and dissertations, and I am always swept off my feet by the warmth and interest I’ve been met with. And of course, I am deeply indebted to everyone who has provided lichen specimens from private as well as public herbaria. Without these I would never have completed my research. Bengt Oxelman, Katarina Andreasen, and Mats Thulin are warmly thanked for valuable comments on my thesis. Through her associate profes- sor presentation and subsequent discussions, Katarina inspired me to include measures of phylogenetic diversity in my fourth paper. Christina Wedén came to my rescue with indispensable comments on my last manuscript and thesis summary during the summer when everybody else was away on vacation. Thank you very much Christina!

37 My fellow PhD students and post-docs – prior and present – Andreas, Anja, Anneleen, Annika, Björn-Axel, Božo, Cajsa, Catarina, Christina, Dick, Donatha, Frida, Hege, Henrik, Hugo, Jesper, Johan, Johannes, Karolina, Katinka, Kristina, Magnus, Maria, Mattias, Per, Per, Samira, Sanja, Sunniva, Åsa – thank you for support, answered questions, interesting discussions, cakes, company at lunch breaks, whisky-tastings, parties, blueberry picking, ice-cream-excursions to Victoria, and everything else we’ve done during my years here. Special thanks to my office-mates for putting up with me. Kristina and I shared office during my first and her last year. Part of this year was rather confusing for both of us I believe, but you always took the time for answer- ing my questions, and we did have fun in between the stress. You are a very welcoming person. Anneleen moved into ‘my’ office a year or so ago (how long is it really?) – we have shared a lot of good laughs, and I love your good sense of humor and your ability to laugh, smile, and make fun of (al- most) everything. A minor downside to sharing office with you is that Dutch guy who keeps calling all the time, but I guess you can’t help being popular (Hugo, I didn’t mean that, it’s been fun having you around!) Elisabeth, I’m afraid you don’t qualify under fellow PhD students or post- docs since you had already finished before I started. Nevertheless, you were the first person who really made me feel welcome here. You are always (most of the time) happy and smiling, and we have shared many a good meal, whisky, lots of gossip, laughs, and heartaches. Thank you so much for living in Uppsala, not to mention all the times you’ve helped out with practi- cal stuff especially with the lay-out of the thesis cover. And I can’t mention Elisabeth without thinking of Arvid; my time in Uppsala would have been so much more boring without silly walks, head-banging, ice-cream-begging, and all the other things you and I have done in an attempt to drive your mother crazy. Did we succeed??? Nahid, our fabulous lab-technician who is extremely competent and furthermore a very likeable person – thank you very much for always taking the time and helping out! Afsaneh, thank you for your ready smile and interest in my progress. Ulla, you have brightened my stay with your good sense of humor and nailing comments. Agneta, always helpful, always ready to offer support, thank you! It is always possible to find a helping hand at the herbarium. Numerous are the times Anders Nordin has helped with Latin diagnoses, species deter- minations, or interpretation of cryptic herbarium labels. Roland Moberg has offered his support from the day I came. Barbro Lidfeldt Rahm always lends a hand interpreting complicated customs regulations and makes shipments of herbarium material go smoothly. Svengunnar Ryman has patiently assisted every time ‘unidentifiable’ fungi turned up during teaching sessions. Thank you so much all of you!

38 My choirs, Munviga and Friskören, made my stay in Uppsala a lot more fun. I have enjoyed singing with all of you. The following funds made my research possible: Helge Ax:son Johnsons Stiftelse, Sernanders stiftelse, Stiftelsen Anna Maria Lundins Stipendiefond, Sederholms nordiska resestipendium, Gertrud Thelins resestipendium, Bjärka-Säbys stipendiestiftelse, Kungliga Vetenskapsakademien, and Bertil Lundmans fond.

My family of course, there’s a lot of them, and I’m very happy I’ve got them. My brothers and sisters: Ulf (and family), Vibeke (and family), Helle (and family), Dorte, Bo (and family), Jette, Leif (and family), and Hanne (and family) – it feels good to know that you are there and that you care. I’m sure that you have wondered many times what the h… I was doing up here in Uppsala, here’s your chance to find out! Jette and Dorte have followed my progress more closely. They have been forced (or chosen?) to listen to re- ports of my work and life in the north and also to complaints when things weren’t going my way. Thank you for your patience and interest. My parents – sometimes I can’t help thinking how proud my dad Hen- ning, who unfortunately didn’t live to see this thesis, would have been. My mom Toni never for one second ceased to encourage me, believe in me, and patiently listen to complaints when times were tough – how could I possibly give up with support like that? I just can’t thank you enough, but I hope you know already!

Cameron – you light my way…

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Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 333

Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science and Technology, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology”.)

ACTA UNIVERSITATIS UPSALIENSIS Distribution: publications.uu.se UPPSALA urn:nbn:se:uu:diva-8183 2007