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Phylogenetic Relationships and Species Richness of Coprophilous Ascomycetes

Phylogenetic Relationships and Species Richness of Coprophilous Ascomycetes

Phylogenetic Relationships and Richness of Coprophilous Ascomycetes

Åsa Nyberg Kruys

Department of Ecology and Environmental Science Umeå University Umeå 2005

AKADEMISK AVHANDLING

som med vederbörligt tillstånd av rektorsämbetet vid Umeå universitet för avläggande av filosofie doktorsexamen framläggs till offentligt försvar i Stora hörsalen, KBC, fredagen den 25 november 2005, kl. 9.00. Avhandlingen kommer att försvaras på engelska.

Examinator: Dr. Mats Wedin, Umeå Universitet

Opponent: Ass. Prof. Thomas Laessøe, Department of Microbiology, University of Copenhagen, Denmark.

ISBN 91-7305-949-8 © Åsa Nyberg Kruys Printed by Solfjädern Offset AB Cover: Ascus of antarctica with eight 13-celled ascospores. Design by Åsa and Nic Kruys. Organization Document name UMEÅ UNIVERSITY DOCTORAL DISSERTATION Department of Ecology and Environmental Science Date of issue SE-901 87 Umeå, Sweden November 2005

Author Åsa Nyberg Kruys

Title Phylogenetic relationships and species richness of coprophilous ascomycetes.

Abstract Coprophilous ascomycetes are a diverse group of saprobes, of which many belong to three families, , and , within the large order . The natural relationships and circumscription of these families are unclear, especially within the family Sporormiaceae, where the generic delimitation have been questioned. There is also a need to understand how different ecological processes affect species richness and occurrence of coprophilous ascomycetes in general. The aim of this thesis was therefore to test earlier classifications of coprophilous taxa within Pleosporales, using phylogenetic analyses of DNA sequences; and to study how the habitat, dung type and herbivores´ food choice may affect the species richness and species composition of coprophilous ascomycetes. A phylogenetic study shows that coprophilous taxa have arisen several times within Pleosporales. Sporormiaceae and Delitschiaceae are separate monophyletic groups and should continue to be recognized as two distinct families within Pleosporales. Phaeotrichaceae forms a monophyletic group, and is, unexpectedly, a strongly supported sister-group to , but if they belong to Pleosporales or not, remains unresolved. and , which previously had an unclear position in , are shown to be members of Pleosporales and should be treated as two separate families. The Eremodothis is, however, not related to Testudinaceae, but is nested within Sporormiaceae and should be transferred to . The natural relationships within Sporormiaceae are still not fully resolved and consequently, I suggest a rather conservative generic classification, accepting , , Westerdykella, as well as Sporormiella, despite that the latter is not conclusively well supported as monophyletic. Characters previously used in the and classification of Sporormiaceae, as choice of substrate, presence or absence of an ostiole, presence or absence of germ slits, and ornamentation, were all homoplastic and not very useful for circumscribing monophyletic groups. Field-studies of moose (Alces alces), mountain hare (Lepus timidus) and roe deer (Capreolus capreolus) dung resulted in several new species records, which suggests that coprophilous ascomycetes in boreal Sweden are poorly known. Fungal species richness and occurrence on moose dung varied significantly between habitats. Species diversity was negatively associated with amount of attack, and feeding either on the dung and/or the fungi may be an important factor explaining the observed pattern. Species richness of coprophilous fungi varied also significantly between different dung types. A study of moose, mountain hare, and roe deer dung did not show any consistent patterns in respect to the ´ digestive system. There was, however, a general strong positive relationship between the total number of ascomycete species and the number of plant species foraged by the three herbivores. Fungal species with large (≥ 50 µm) were over-represented on roe deer dung, and under-represented on moose dung, while the reverse was found for species with small spores (<10µm). This suggests that the foraging level of the herbivore, which in turn mirrors species-specific differences in spore dispersal of the fungi, may be an important factor in explaining species richness and diversity of the coprophilous community.

Key words: ß-tubulin, Bayesian analysis, dung, forage, fungi, moose, parsimony, phylogeny, Pleosporales, spore dispersal, Sporormiaceae

Language: English ISBN: 91-7305-949-8 Number of pages: 28 + 4 papers

Signature: Date: 25 Oktober 2005 List of papers

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

I. Kruys Å, Eriksson OE, Wedin M. Phylogenetic relationships of coprophilous Pleosporales (, Ascomycota), and the classification of some bitunicate taxa of unknown position. Manuscript.

II. Kruys Å, Wedin M. A study of the natural relationships and traditionally used taxonomic characters in Sporormiaceae (Pleosporales, Ascomycota), utilizing multi-gene phylogenies. Manuscript.

III. Nyberg Å, Persson I-L. 2002. Habitat differences of coprophilous fungi on moose dung. Mycological Research 106: 1360-1366.

IV. Kruys Å, Ericson L. Species richness of coprophilous ascomycetes in relation to variable food intake by herbivores. Submitted manuscript.

Paper III is published with the kind permission of the publisher. TABLE OF CONTENTS

1. INTRODUCTION ...... 5 1.1 Natural relationships of the coprophilus Pleosporales...... 6 1.2 Focus on Sporormiaceae...... 7 1.3 Species richness of coprophilous ascomycetes ...... 8 2. OBJECTIVES...... 9 3. MATERIAL AND METHODS ...... 10 3.1 Molecular methods...... 10 3.2 Field studies...... 10 4. MAJOR RESULTS AND DISCUSSION...... 11 4.1 Phylogenetic relationships of coprophilous families in Pleosporales..11 4.2 Phylogenetic relationships within Sporormiaceae...... 13 4.3 The effect of habitat on coprophilous fungi on moose dung ...... 16 4.4 Species richness in relation to variable food intake by herbivores ..17 4.5 New records of coprophilous ascomycetes in Sweden ...... 19 5. FINAL CONCLUSIONS...... 19 6. ACKNOWLEDGEMENTS...... 21 7. REFERENCES ...... 21 8. POSTSCRIPT ...... 27

APPENDICES: PAPER I-IV 1. INTRODUCTION

A dung pile is a tremendously rich substrate sparkling with life! Fungi, bacteria, , and mosses compete for space and nutrients, and interact in the degradation of the heterogeneous substrate. Coprophilous (dung-loving!) fungi are a diverse group of saprobes including taxa from most major fungal groups. This thesis will, however, focus on Ascomycota, which is the largest group of fungi with more than 32 000 species described (Kirk et al. 2001). The number of coprophilous ascomycetes has not been estimated, though. Coprophilous ascomycetes can be found on various dung types from all over the world, but are more frequent on dung of herbivores than carnivores (Lundqvist 1972, Richardson 2001). In addition, they have seldom been reported on reptile or amphibian dung, indicating that coprophily of fungi developed among the warm-blooded animals (Webster 1970). Some species are strictly coprophilous, others occur on different ephemeral substrates. The strictly coprophilous ascomycetes have a remarkable life-cycle, dung Æ plant Æ gut Æ dung (Fig. 1). Thus, they do not disperse between habitat patches (Wicklow 1992). Instead they have to pass the digestive system of an animal, and spore germination may even be triggered by gastric juices (Sussman & Halvorson 1966, Furuya & Naito 1980). The spores of coprophilous ascomycetes are often darkly pigmented, with thick walls, and are probably well protected against both gastric juices and the harmful UV-light of the sun (Ingold & Hudson 1993).

Fig. 1. The life-cycle of a strictly coprophilous pyrenomycete. A fruit-body that is produced on a dung pile, will discharge its spores when mature. The spores are often surrounded by mucilage or have gelatinous appendages, and attach easily to the plant parts on which they land. When a plant is foraged by a herbivore, the spores will follow and be transported through the digestive gut. Finally, when ending up in a new dung pile, the spores will germinate and produce new fruit- bodies. Drawing inspired by Bell (1983).

5 Coprophilous ascomycetes are advantageous organisms to study; a great range of various morphologically and systematically different taxa can appear on a fresh dung pile within few weeks, there is also no shortage of dung in nature, and a majority of the species are possible to grow in vitro. Consequently, coprophilous fungi have been subject to early and extensive studies (Massee & Salmon 1901, Lundqvist 1972, Richardson 2001). The dark, thick-walled spores can be found in ancient soil samples and sediments, and are like pollen grains, useful tools in paleoecological studies (Hausmann et al. 2002, van Geel et al. 2002, Burney et al. 2003). A number of the coprophilous ascomycetes, such as Podospora anserina and Sordaria macrospora are utilized as model systems for genetic, biochemical and molecular studies (Esser 1974, Silliker et al. 1996, Nowrousian et al. 2005), and have therefore been investigated very thoroughly. However, saprobic ascomycetes in general are poorly known organisms, the circumscription of many groups are unclear and they often lack good morphological features to distinguish them. Phylogenetic information based on DNA sequences revolutionised the knowledge of the classification of Ascomycota during the 1990´s (as continuously summarised in Systema Ascomycetum between 1983-1998 by Eriksson or Eriksson & Hawksworth, and later in Myconet 1997-2005 by Eriksson & Winka, Eriksson or Eriksson et al.). However, several groups of coprophilous ascomycetes are still in need of basic molecular systematic information. Unclear circumscriptions of taxa have also complicated ecological studies of coprophilous ascomycetes, and there is a need to reveal distribution patterns and how different ecological processes affect species richness and occurence.

1.1 Natural relationships of the coprophilus Pleosporales

Coprophilous ascomycetes are found in numerous non-related taxa. However, a large group belong to the bitunicate order Pleosporales in Dothideomycetes. Pleosporales (sensu Eriksson 2005) includes 13 families, most of the members are saprobes or parasites on vascular plants, but three of the families are solely or predominantly coprophilous, viz. Delitschiaceae, Phaeotrichaceae and Sporormiaceae. These families have often been assumed to be closely related, due to fimicolous lifestyle and shared combination of morphological characters (Munk 1957, Luttrell 1973, Hawksworth et al. 1983). Sporormiaceae is characterized by dark brown, septate spores with germ slits (Fig. 2). Several of the genera in Sporormiaceae are morphologically very similar and the circumscriptions of these are unclear (cf. treatments in Arenal et al. 2004, Doveri 2004). Barr (2000) has re-circumscribed Sporormiaceae and excluded and Semidelitschia. Delitschiaceae resembles the Sporormiaceae in spore characters (dark brown, septate, germ slits; Fig. 2), but differs in features of the ostiole, endotunica and the ocular chamber (Barr 2000). Delitschiaceae is, however, still treated as a synonym of Sporormiaceae in Kirk et al. (2001). Phaeotrichaceae was described to accommodate the single genus , characterized by dark brown, septate spores with terminal germ pores (Cain 1956). The ascomata are setose cleistothecia, but Cain (1956) pointed out that it otherwise had several similarities with the setose perithecial genus (Fig. 2). This genus was placed in Sporormiaceae by Munk (1957), but Lundqvist (1964) suggested it to be transferred to Phaeotrichaceae, a measure accepted by Luck-Allen

6 (1970), Eriksson (1981, in Eriksson & Hawksworth 1988), Parguey-Leduc (1974) and Barr (2000). Trichodelitschia has very characteristic, bitunicate asci. Dehiscence is fissitunicate and the ectotunica slides down to the base of the ascus and becomes strongly wrinkled. The taxonomical position and circumscription of Delitschiaceae, Phaeotrichaceae, and Sporormiaceae have varied over time and previous molecular studies included a very limited number of taxa, making detailed sister-group relationships difficult to infer. Several authors have also suggested a close relationship between one or more of these three families, and members of Zopfiaceae and Testudinaceae (von Arx & Müller 1975, Malloch 1981, von Arx 1981). Members of Zopfiaceae and Testudinaceae have mainly been isolated from soil. In the case of Zopfiaceae, this has mostly been on or in association with roots. A majority of the species in Zopfiaceae and all Testudinaceae have non-ostiolate ascomata and brown, smooth to variously ornamented, usually septate spores (Hawksworth 1979, Hawksworth in Eriksson & Hawksworth 1987). Both families have at present an unclear position in the classification of Ascomycota (Kirk et al. 2001, Eriksson 2005).

Fig. 2. Spores of Trichodelitschia (Phaeotrichaceae), Delitschia (Delitschiaceae), Sporormiella (Sporormiaceae), (Zopfiaceae) and (Testudinaceae). The drawings are not proportional in size.

1.2 Focus on Sporormiaceae

Sporormiaceae is the largest family among coprophilous fissitunicate ascomycetes. Most members of the family are strictly coprophilous, but some occur on other substrates, e.g. wood, soil, and plant debris. In the recent treatment by Barr (2000), the family comprises eight genera; Chaetopreussia, Pleophragmia, Preussia, , Sporormia, Sporormiella, Spororminula, and Westerdykella. Sporormia, Sporormiella, and Preussia are considered to be closely related and are easily confused by their shared morphological features. The type genus Sporormia is characterized by globose pseudothecia, which open with an ostiole. The spores are typically joined together in a bundle within one common gelatinous sheath, when released from the ascus (Ahmed & Cain 1972, Dissing 1992). Germ

7 slits, otherwise common and characteristic for the family, are absent in the type species S. fimetaria. Sporormiella, the most species-rich genus in the family, includes species with ampulliform ascomata and spores with distinct germ slits (Ahmed & Cain 1972). Preussia differs morphologically from 4-celled Sporormiella species only by the ascomata being non-ostiolate (Cain 1961, Barr 2000). The taxonomic importance of the ostiole has, however, been questioned (von Arx 1973, Guarro et al. 1997). Von Arx & van der Aa (1987) instead used substrate choice as a diagnostic character when separating Preussia and Sporormiella. Preussia, in their sense, included species on plant debris, wood or soil, while Sporormiella was restricted to comprise only strictly coprophilous species. However, several Sporormiella species are not only generalists among coprophilous substrates, but may occur on wood and soil, according to Guarro et al. (1997). Westerdykella, Pycnidophora, and Chaetopreussia form, like Preussia, non-ostiolate ascomata (Clum 1955, Stolk 1955, Locquin-Linard 1977). The spores of Westerdykella, Pycnidiophora and Chaetopreussia separate at a very early stage in the ascus and the lack of germ slits is also considered a taxonomically important character (von Arx & van der Aa 1987, Barr 2000). Pycnidiophora has been included in Westerdykella by several authors (Cejp & Milko 1964, von Arx & Storm 1967, von Arx 1981). Both von Arx & van der Aa (1987) and Barr (2000), however, suggested that species with smooth spore walls should be included in Pycnidiophora, while Westerdykella should be restricted to the type W. ornata, the only species with ornamented spores. Barr (2000) has also suggested a close relationship between Pleophragmia, Sporormia, and the monotypic Spororminula, because of their ostiolate ascomata, and the spores that lack germ slits, and should be triangular in transverse view. The classification of taxa within Sporormiaceae is apparently based on a comparatively small number of easily observed ecological and morphological characters. Several of these traditionally used characters have, however, been claimed to be poor predictors of phylogenetic relationships, both in Sporormiaceae as well as in other groups of Ascomycota (von Arx 1973, Guarro et al. 1997, Dettman et al. 2001, Hansen et al. 2002), and their applicability for circumscribing genera, need to tested with molecular methods.

1.3 Species richness of coprophilous ascomycetes

Species richness and community composition of coprophilous ascomycetes vary with abiotic and biotic factors. Temperature and moisture affect growth rate, fruiting and species richness of coprophilous fungi (Wicklow & Moore 1974, Harrower & Nagy 1979, Yocom & Wicklow 1980, Kuthubutheen & Webster 1986). Intra- and interspecific interactions occur at the scale of the individual dung pile and have been shown to affect fungal development, as well as species composition (Ikediugwu & Webster 1970, Wicklow & Hirschfield 1979, Weber and Webster 1998). Coprophagous arthropods can graze on fungal mycelia or indirectly affect fruiting by hastening the decay process (Lussenhop et al. 1980, Wicklow & Yocom 1982, Stevenson & Dindal 1987). These factors are expected to vary in nature, e.g. depending on the habitat and the season, however, the majority of previous studies have been conducted in vitro. Also, most studies of coprophilous ascomycetes have focused on dung of domesticated animals (Dickinson & Underhay 1977, Wicklow & Hirschfield 1979) and rabbits in grasslands (Yocom & Wicklow 1980, Angel &

8 Wicklow 1983), while dung from wild boreal animals, and especially forest-living species, has been much less studied. Many coprophilous ascomycetes are most common on only one or a few dung types (Lundqvist 1972) and dung from closely related animals generally show a similar species composition (Richardson 2001). Angel & Wicklow (1983) found that the fungal community varied more between dung types (rabbit and cattle dung) than between various grassland habitats. This suggests that the digestive system of the herbivore may be another important factor for species richness, as differences in digestion will affect both the passage of the spores through the digestive gut, and the dung quality, e.g. consistency, moisture and nutrient content. Studies addressing the importance of different digestive systems have been limited, though. However, Wicklow et al. (1980) compared dung of a single rabbit and a single sheep, fed with hay from the same bale, and found marked differences in species compositions, differences that they attributed to differences in digestion. Alternatively, differences in species richness and composition may mirror differences in feeding habits and/or food choice between herbivores. Ebersohn and Eicker (1992) in their study on coprophilous fungi on African herbivores concluded that feeding habits and food choice were more important than the animals´ digestive system for the species composition. Dung from generalist herbivores, feeding upon a large number of plant species and within a large height interval, was generally more species rich and also showed the highest species diversity. In contrast, dung from specialist grazers foraging within a narrow height interval was characterised by fewer species and lower species diversity. However, whether this reflects a wider variation in food quality per se, or that generalist feeders while foraging both lower and taller plants are inoculated with higher amounts of spores, or spores from more species, remains unclear. That spore dispersal may be one important factor in explaining species composition is supported by the fact that the the elephant, which showed the broadest foraging level, feeding from grass root to tree top level, also had the highest species diversity of fungi. In another study, Bell (1975) observed that the coprophilous flora of the tree-feeding brush-tailed opossum showed a predominance of small, hyaline spores in opposite to ground-dwelling herbivores. Thus, there is some support that the coprophilous community mirrors the foraging level of the herbivore.

2. OBJECTIVES

The main objectives with this thesis was to gain more knowledge in a difficult group of coprophilous ascomycetes with unclear natural relationships, and to study a number of factors that may have an effect on the species richness and species composition of these organisms. To reveal these topics I, therefore, aimed to:

ß study phylogenetic relationships between coprophilous families in Pleosporales, and test their earlier classifications (paper I). ß clarify the circumscriptions of taxa within Sporormiaceae, and test the homology of traditionally used characters (paper II). ß study the effect of habitat on species richness of coprophilous fungi (paper III). ß investigate how dung type and food choice may affect species richness and species composition of coprophilous ascomycetes (paper IV).

9 3. MATERIAL AND METHODS

3.1 Molecular methods

I utilized several independent genomic markers for the phylogenetic studies (paper I, II). Nuclear ribosomal genes (e.g. nSSU and nLSU) are commonly used in fungal phylogenetics, as the high number of copies and the availability of general primers (White et al. 1990, Gargas & DePriest 1996) make them easy to amplify. Ribosomal encoding genes can also be amplified from the mitochondrial genome (e.g. mtSSU), although in fewer copies. Nuclear protein-coding genes (e.g. ß- tubulin) are becoming increasingly used and have several advantages compared to ribosomal genes. Since they code for proteins, the alignment is likely to contain less ambiguity due to codon constraint, and both nucleotides and amino acids can be analysed. However, only nucleotide sequences were analysed in these studies. In order to reveal phylogenetic relationships on the family level within Pleosporales, I amplified nSSU rDNA, nLSU rDNA, and mtSSU rDNA (paper I). The more variable gene partitions, nITS-LSU, mtSSU and ß-tubulin, were used to resolve relationships on the generic level within Sporormiaceae (paper II). For the sequencing of ß-tubulin, I designed a new primer, BT1903R (paper II). The data sets were analysed with parsimony (paper I, II) and Bayesian (paper II) methods (Kitching et al. 1998, Ronquist 2004, Huelsenbeck et al. 2001). Alternative tree topologies (paper I) were investigated with constrained parsimony analyses followed by the Shimodaira-Hasegawa test (Shimodaira & Hasegawa 1999).

3.2 Field studies

The field work conducted for paper III and IV were done in boreal forest in Västerbotten and Ångermanland in northern Sweden. To study the effect of habitat on species richness and occurrence of coprophilous fungi I used fresh moose (Alces alces) dung from a nearby moose farm, where moose calves were kept in a natural pasture and all were fed the same diet (paper III). Due to the homogenous origin, composition and age of the dung, it was assumed that the dung piles had the same inoculum of species at the start of the experiment. The dung piles were placed along line transects in three different habitats. As previous studies have frequently shown that temperature and humidity are key factors for fungal communities, the following habitat types were chosen: one sunny and dry habitat (pine forest), one sunny and wet habitat (mire) and one shady and mesic habitat (spruce forest). The water content of the dung was used as a measurement of habitat moisture, and the number of holes visible on the surface of the dung, which could be attributed to insect larvae, was used as an estimate of insect attack. The percent cover of discomycetes was visually estimated in field, while the species number of fungi was determined in laboratory. Differences in species richness and composition of coprophilous ascomycetes between dung types (paper IV) was studied by sampling dung of three herbivores which differ with regard of their digestive system; two ruminants, moose and roe deer (Capreolus capreolus), and one hindgut fermentor, mountain hare (Lepus timidus). Winter dung was sampled from three areas in the coastal region near Umeå. The three areas have a constant occurrence of the three herbivores, and hence they were expected to have a similar species composition of coprophilous

10 fungi. Sampling of dung piles took place on one occasion in late winter after which the dung piles were inoculated in vitro. This was done in order to minimize effects on species composition depending on humidity and temperature (Yocom and Wicklow 1980), occurrence of various arthropods (Stevenson & Dindal 1987), as well as age of the dung and season (Angel and Wicklow 1983, Richardson 2001). Moose, mountain hare, and roe deer forage within the same habitat, although within different height intervals. During the winter season, all three animal species are browsers and forage mainly on woody plants. In order to describe the food choice of the three herbivores in each area, I followed their tracks and scored visible signs of recent foraging.

4. MAJOR RESULTS AND DISCUSSION

4.1 Phylogenetic relationships of coprophilous families in Pleosporales

The coprophilous families Delitschiaceae (Fig. 3; clade H) and Sporormiaceae (Fig. 3; clade F) clustered on separate branches in a strongly supported clade, corresponding to Pleosporales, and they were not closely related (paper I). Coprophilous taxa have apparently arisen several times within Pleosporales, as in a number of other fungal groups. Delitschiaceae forms a distinct monophyletic group, well supported also by a constrained-topology analysis (p<0.001), and should be accepted as a separate family. The bootstrap support for the monophyletic Sporormiaceae is just below the level of significance (bs 68). This clade also includes one species that has been accommodated in Testudinaceae, Eremodothis angulata. It has one-celled spores with four blunt ends, projecting in caltrop spine directions, but without germ pores (Udagawa & Ueda 1981), which makes it morphologically distinct from other Testudinaceae. Eremodothis shares the lack of ostiole and germ slits with Westerdykella, to which it is apparently most closely related, but it has eight one-celled spores, contrary to the rest of Westerdykella. Trichodelitschia grouped with Phaeotrichum with high support, and this confirms the opinion of Lundqvist (1964), Eriksson (1981), and Barr (2000). An unexpected result was the close relationship between Phaeotrichaceae (Fig. 3; clade Q) and Venturiaceae (Fig. 3; clade P), which are strongly supported sister-groups on a branch outside Pleosporales. This position of Venturiaceae is not in agreement with some previous molecular studies (Silva-Hanlin & Hanlin 1999, Olivier et al. 2000, Eriksson & Hawksworth 2003). However, this result is also supported by morphological traits, since Venturiaceae and Phaeotrichaceae both have setose ascomata and coloured, one-septate spores (Eriksson 1981). Phaeotrichaceae and Venturiaceae both belong to the class Dothideomycetes, but if they belong to the order Pleosporales or not, is unclear. A constrained-topology analysis did not reject such an alternative (p=0.10-0.57). All taxa included in this study that have an unclear position in Eriksson (2005), were nested within Dothideomycetes. Zopfiaceae and Testudinaceae belong to Pleosporales, but they do not form a monophyletic group and should be treated as two separate families. Zopfiaceae is polyphyletic, the type Zopfia is sister-group to Delitschiaceae (with no support), while Didymocrea was nested within a mixed clade (Fig. 3; clade B), consisting of one member of each of the families Melanommataceae, , and Tubeufiaceeae. All members of the

11 Testudinaceae, except for Eremodothis angulata, form one monophyletic clade (Fig. 3; clade G), which also includes Verruculina enalia (now in ).

99 Arthonia dispersa 99 Combea mollusca 100 Dendrographa leucophaea Schismatomma decolorans salicis A: Arthopyreniaceae 58 Bimuria novae-zelandiae 100 Didymocrea sadasivanii Letendraea helminthicola B: Cucurbitariaceae, Melanommataceae, Curreya pityophila !!!!!Tubeufiaceeae and Zopfiaceae 64 Clathrospora diplospora 74 52 Lewia infectoria 100 Pleospora herbarum C: Diademaceae and Cochliobolus heterostrophus 99 Pyrenophora tritici-repentis 98 Leptosphaeria cf. macrospora Leptosphaeria doliolum 63 83 Ophiobolus herpotrichus D: , Phaeosphaeria avenaria !!!!!, Setomelanomma holmii !!!!!and genera of uncertain position Shiraia bambusicola Byssothecium circinans Herpotrichia diffusa E: , 85 62 Herpotrichia juniperi Pleosporales 100 Melanomma pulvis-pyrius !!!!Melanommataceae, Lophiostoma macrostomum !!!!and siparia 99 Eremodothis angulata 100 Westerdykella dispersa 68 Westerdykella cylindrica F: Melanommataceae, Sporormiaceae, 71 60 Preussia terricola 100 Sporormia lignicola !!!!!and Testudinaceae 100 Trematosphaeria heterospora nicotiae Verruculina enalia 98 bilgramii G: Didymosphaeriaceae Neotestudina rosatii !!!!!and Testudinaceae 99 Delitschia didyma Delitschia winteri H: Delitschiaceae and Zopfiaceae Tubeufia helicoma I: Tubeufiaceeae 100 Botryosphaeria dothidea 63 Botryosphaeria ribis J: Botryosphaeriaceae Guignardia bidwelli !!!!and Mycosphaerellaceae Delphinella strobiligena 100 100 Dothidea sambuci Stylodothis puccinioides K: Dothideales Sydowia polyspora Elsinoë veneta Myriangium duriaei Piedraia hortae L: Myriangiales and Piedraiaceae Microxyphium citri Mycosphaerella populorum M: Capnodiales, Mycosphaerellaceae, Mycosphaerella punctiformis !!!!!!and Pseudoperisporiaceae Raciborskiomyces longisetosum Hysteropatella clavispora N: Hysteriales Farlowiella carmichaeliana O: Hysteriales 100 Metacoleroa dickiei 82 Spilocaea oleaginea 100 69 chlorospora P: Venturiacceae Venturia inaeqalis 100 Phaetrichum benjaminii Trichodelitschia munkii Q: Phaeotrichaceae 100 Capronia mansonii 93 Ceramothyrium carniolicum EUROTIOMYCETES incl. 100 Eurotium herbariorum CHAETOTHYRIOMYCETES Spiromastix warcupii 100 Cladonia rangiferina Xanthoria parietina LECANOROMYCETES Hypocrea lutea Xylaria hypoxylon SORDARIOMYCETES

Fig. 3. A strict consensus of the 30 most parsimonious trees, based on nSSU, nLSU and mtSSU rDNA sequences. Numbers represent bootstrap support. Bold lines represent branches with 100% support. Arrows indicate the extent of the clades and bold family names indicate clades including the type species of the family. Clades discussed in paper I are marked with letters A-Q.

12 4.2 Phylogenetic relationships within Sporormiaceae

Within Sporormiaceae (paper II), Westerdykella (including Pycnidiophora), Sporormia, Preussia (including Sporormiella alloiomera), and several groups within Sporormiella are significantly supported as monophyletic (Figs. 4, 5). However, the circumscription of the difficult Sporormiella–Preussia complex is not revealed with convincing support. Several characters traditionally used as diagnostic features within Sporormiaceae are here shown to be poorly correlated with monophyletic groups. The absence of germ slits is not a homologous feature, and I found no support for a close relationship between Sporormia and the monotypic Spororminula, as suggested by Barr (2000). The results do not support a separation between Pycnidiophora and Westerdykella based on spore ornamentation, as suggested by von Arx & van der Aa (1987) and Barr (2000). The type of Pycnidophora, P. dispersa (=Westerdykella dispersa), is nested within the Westerdykella clade, close to the type of Westerdykella, W. ornata. Pycnidiopora should, therefore, remain as a synonym of Westerdykella. The members of Preussia and Sporormiella included in this study are intermixed in the tree, and at present, I know few morphological features that are useful for circumscribing the well supported clades presented here. My results confirm the findings of Guarro et al. (1997), namely that the presence or absence of the ostiole is not a useful character when delimiting Preussia and Sporormiella, because non-ostiolate species are mixed with ostiolate species with a distinct neck. Further, the results do not support a distinction between Preussia and Sporormiella with regard to substrate choice, as suggested by von Arx & van der Aa (1987). For practical reasons, one could therefore argue that Preussia and Sporormiella should be treated as one genus. However, there is no support for one major monophyletic group including both Preussia and Sporormiella, and a majority of the Preussia species (including the type P. funiculata) still comprise a very well supported clade, which could be treated as Preussia in a restricted sense. Therefore, I suggest a conservative approach and continue to treat the two genera as separate, at least as an ad interim solution until the knowledge of the relationships within the Preussia- Sporormiella complex has been improved. One character that may be of importance for the circumscription of some of the Sporormiella-Preussia groups is the shape of the ascus, especially if used in combination with other features, such as the shape of the spores.

13 Sporormiella heptamera 61 Sporormiella octomera 100 The Sporormiella 100 Sporormiella affinis vexans clade Sporormiella vexans Sporormiella leporina 52 Sporormiella dakotensis Sporormiella longisporopsis The Spororminula clade Spororminula tenerifae 68 Sporormiella irregularis 100 Sporormiella tetramera Sporormiella dubia The Sporormiella irregularis clade Sporormiella splendens 99 Sporormiella borealis Sporormiella intermedia Sporormiella bipartis The Sporormiella "Sporormia" lignicola intermedia clade Sporormiella minipascua 55 100 Sporormiella australis Sporormiella minima Preussia isomera Sporormiella antarctica Sporormiella septenaria Preussia fleischhakii Preussia funiculata 100 Preussia terricola The Preussia clade Preussia typharum 67 Preussia vulgaris Sporormiella alloiomera 100 Pycnidiophora dispersa CBS 297.56 62 Pycnidiophora dispersa CBS 508.75 100 Westerdykella multispora The Westerdykella 77 Westerdykella ornata clade 55 Westerdykella cylindrica Westerdykella nigra 100 Sporormia fimetaria 2302-c Sporormia fimetaria 81.194 The Sporormia clade 100 Preussia terricola CBS 527.84 The Sporormiella 100 Sporormiella megalospora megalospora clade 99 Curreya pityophila Pleospora herbarum 100 Herpotrichia juniperi Melanomma pulvis-pyrius Trematosphaeria heterospora Lepidosphaeria nicotiae Verruculina enalia Fig. 4. A strict consensus tree of the 49 most parsimonious trees from an analysis based on ITS-nLSU rDNA, mtSSU rDNA, and ß-tubulin sequences. Bootstrap values above 50% are shown above branches. Branches with ≥70% bootstrap support (level of significance) are marked in bold.

14 86 Preussia terricola Preussia.typharum 99 Preussia funiculata The Preussia clade 100 Preussia vulgaris Preussia fleischhakii Sporormiella alloiomera 67 100 Pycnidiophora dispersa CBS 297.56 100 Pycnidiopora dispersa CBS 508.75 Westerdykella multispora 100 The Westerdykella clade 67 68 Westerdykella ornata Westerdykella cylindrica 100 Westerdykella nigra 67 100 Sporormia fimetaria 2302-c Sporormia fimetaria 81.194 The Sporormia clade 100 Preussia terricola CBS 527.84 The Sporormiella Sporormiella megalospora megalospora clade 100 Sporormiella borealis 88 94 Sporormiella intermedia 94 Sporormia "lignicola" Sporormiella antarctica 91 Sporormiella bipartis 60 The Sporormiella Sporormiella minipascua intermedia clade 100 Preussia isomera 100 100 Sporormiella australis Sporormiella minima 100 Sporormiella septenaria 85 Sporormiella heptamera 99 Sporormiella octomera 100 The Sporormiella Sporormiella affinis vexans clade 100 Sporormiella vexans 100 Sporormiella leporina 90 97 Sporormiella dakotensis 100 Sporormiella longisporopsis The Spororminula clade Spororminula tenerifae 98 100 Sporormiella irregularis Sporormiella tetramera 100 100 The Sporormiella Sporormiella dubia irregularis clade Sporormiella splendens Trematosphaeria heterospora 100 Curreya pityophila 100 Pleospora herbarum 100 Herpotrichia juniperi Melanomma pulvis-pyrius Lepidosphaeria nicotiae Verruculina enalia 50 changes

Fig. 5. A fifty percent majority-rule consensus tree of the 45 000 sampled trees with best likelihood from a bayesian analysis, based on ITS-nLSU rDNA, mtSSU rDNA, and ß-tubulin sequences. Branches with ≥95% posterior probability support (level of significance) are marked in bold.

15 4.3 The effect of habitat on coprophilous fungi on moose dung

The results in paper III revealed significant differences in occurrence and species richness of coprophilous fungi, between the three habitats (Fig. 6). As I used uniform moose dung, assumed to have the same inoculum of species, the importance of the habitat for the community composition is clearly demonstrated. This is also in accordance with earlier studies of coprophilous fungi in grasslands (Lussenhop et al. 1980, Yocom & Wicklow 1980). In this experiment, dung from the most shady habitat, the spruce forest, contributed most to the observed differences. It had the significantly highest average water content, the significantly highest number of insect holes, and the significantly lowest number of fungal species, as well as percent cover of discomycetes. The species poor coprophilous flora was opposite to that I expected as fungi are mostly associated with moist habitats, and experiments in vitro have found an increased species richness of coprophilous fungi when provided with freely available water (Kuthubutheen & Webster 1986).

45 b Sampling 1 40 7 c 35 a Sampling 2 6 a 30 e c 5 25 d 4 20 f 3 15 b 10 2 No. of fungal species

Water content/total weight (%) 5 1 0 0 Pine forest Spruce forest Mire Pine forest Spruce forest Mire a. Habitat type b. Habitat type

14 7 b 12 a 6 10 a 5 a 8 4 6 3 b a 4 2 No. of insect holes 1

Discomycete cover (%) 2 0 0 Pine forest Spruce forest Mire c. Pine forest Spruce forest Mire d. Habitat type Habitat type

Fig. 6. The parameters measured on moose dung in three habitat types in northern Sweden: water content (a), the number of fungal species (b), percent cover of discomycetes (c), and number of insect holes on the surface (d). The figure has been altered compared to paper III.

16 There was a significant negative correlation between water content and number of species of fungi, as well as cover of discomycetes, whereas there was a significant positive correlation between water content and number of insect holes. Furthermore, a negative correlation, although not significant, was found between the number of insect holes and number of fungal species, and between number of insect holes and the cover of discomycetes. This suggests a negative effect of insects on the fungal community, but the lack of significance may be due to differences in the insect assemblage between the three studied habitats, since different insect species may have different impact on the fungi. Especially larval dung can contribute to crumbling and disruption of the dung (Lussenhop et al. 1980, Stevenson & Dindal 1987) and the dung in the spruce forest, was considerably more disintegrated than the dung in the other two habitats. Previous studies have also shown that coprophagous insects not only feed on remaining plant debris, but also graze on fungal mycelium and fruit bodies (Wicklow 1979, Wicklow & Yocom 1982).

4.4 Species richness in relation to variable food intake by herbivores

I found that species richness differed significantly between various dung types, both for the total number of species and the mean number of species per sample, as well as between localities (paper IV). The highest species richness was found for roe deer, while the other ruminant, moose, did not differ from mountain hare, a hindgut fermentor. Thus, the data did not show any consistent patterns in respect to the animals´ digestive system, which is in accordance with a study by Ebersohn and Eicker (1992). For roe deer I found a higher species richness and species diversity in the two areas where graminoids, rich in cellulose, constituted 1/3 of the bites, compared to the third area where browsing constituted 96% of the bites. For both moose and mountain hare the lowest species richness was observed where the bites were dominated by a few deciduous species rich in phenolic compounds (Salix spp., Betula pubescens). These observations, although based on correlative data, suggest that differences in plant chemistry may be of importance for the species composition. The high species richness on roe deer dung correlated also to a much wider food choice by roe deer (Fig. 7). For moose and mountain hare the most striking pattern was that the lowest number of coprophilous species was observed at the same locality, where these two herbivores showed the lowest food diversity. Together, these patterns suggest that a more species rich food intake may be one explanation for higher species richness of coprophilous ascomycetes (cf. Ebersohn and Eickner 1992). However, an increase in the number of foraged species also correlates both to an increased variation in the chemical composition of the forage, and foraging of low-growing as well as taller plant species. Thus the data does not allow an evaluation of the relative importance of food quality or foraging level for species richness. Nor is it possible to exclude the possibility that plant chemistry matters. I found also that the coprophilous community on roe deer and moose dung differed with regard to spore size. Species with large spores (> 50 µm) were over- represented on roe deer dung and under-represented on moose dung, while species with small spores (< 10 µm) showed the reverse pattern (Fig. 8). These patterns also correspond with moose foraging on taller plants, while roe deer at all localities foraged within a broader height interval. For mountain hare the deviations between

17 observed and expected frequencies of species in different spore size classes showed no clear pattern. However, the species found on mountain hare dung were either in association with roe deer or occurred on all three substrate types, while none of the species was associated with only mountain hare and moose. All toghether, this suggests that the species composition mirrors the foraging level of the herbivore and thus differences in spore dispersal (cf. Bell 1975). If species with large spores are characterized by an inferior dispersal ability, one would expect them to be more frequent on dung of mountain hare. However, at the time of sampling, mountain hare foraged mostly deciduous shrubs protruding above the snow package, which may be the reason why this was not the case.

Fig. 7. The number of coprophilous ascomycetes plotted against the number of foraged plant species. A linear regression analysis revealed a general strong positive relationship between the two variables.

Fig. 8. The observed (black dots) and expected (dashed line) occurrence of ascomycete species of different spore size classes, for mountain hare, moose, and roe deer. Data is pooled for the three localities.

18 4.5 New records of coprophilous ascomycetes in Sweden

In total, 61 species of ascomycetes were encountered in the two field studies (paper III and IV). Five to six species represent undescribed species, Coniochaeta sp., Sporormiella sp., Trichodelitschia sp., and possibly also Sporormiella cf. ovina in paper IV, as well as two species of Sporormiella in paper III. Fifteen species were new to the province of Västerbotten, and 28 species were new to the province of Ångermanland. Five species were new records on mountain hare dung, 14 species were new on roe deer dung, and 19 species were new on moose dung. Cercophora gossypina (paper IV) has only been found twice in Sweden (Eriksson 1992), while Thelebolus caninus (paper III, IV) has been found in the country but the frequency and distribution is unknown (Hansen and Knudsen 2000). Apparently, the coprophilous ascomycetes in boreal Sweden are poorly known, in particular when considering the number of encountered species and the limited sampling effort in paper III and IV. The highest species richness was found on roe deer dung (paper IV). For the most species rich group, the pyrenomycetes, 39 species were found on roe deer dung, 21 on moose dung and 19 on mountain hare dung (paper IV). A comparison between this data and the number of coprophilous pyrenomycetes previously reported from Sweden by Eriksson (1992) shows the opposite pattern, namely 15, 29 and 52 species, respectively. These opposite patterns are likely to mirror earlier sampling efforts and show the difficulties involved in comparisons between different data sets.

5. FINAL CONCLUSIONS

This thesis clarifies the natural relationships of coprophilous families within Pleosporales, in particular of Sporormiaceae. It contributes with important pieces to the whole Pleosporales-puzzle, and sheds new light also on the relationships of other taxa within this large order. Sporormiaceae and Delitschiaceae are separate monophyletic groups within Pleosporales and should continue to be recognized as two distinct families. Phaeotrichaceae forms a monophyletic group, and is, unexpectedly, a strongly supported sister-group to Venturiaceae, but if they belong to the order Pleosporales still remains unclear. The two families Testudinaceae and Zopfiaceae, which previously had an uncertain position in Ascomycota, are demonstrated to be members of Pleosporales and should be treated as two separate families. Zopfiaceae is polyphyletic, while a majority of the members of Testudinaceae form one clade. The monotypic genus Eremodothis is, however, not related to Testudinaceae, but is nested within Sporormiaceae and should be transferred to the genus Westerdykella. Several questions still remain to be solved though; the circumscription of Pleosporales is not clear, a number of clades within Pleosporales do not have a natural classification, and several taxa placed with uncertain position in Dothideomycetes/Chaetothyriomycetes in Eriksson (2005), still need to be investigated. The relationships between genera within Sporormiaceae are still not fully resolved and I therefore, suggest a rather conservative generic classification of the family, including the well supported and monophyletic Sporormia (excluding S.

19 lignicola), Preussia (excluding P. isomera, but including Sporomiella alloiomera), and Westerdykella (including Pycnidiophora and Eremodothis). I also suggest accepting Sporormiella, despite not being conclusively well supported as monophyletic, at least as a preliminary solution. Sporormiella should then include the monotypic Spororminula, but exclude Sporormiella alloiomera. Characters previously used in the taxonomy and classification of Sporormiaceae, as the choice of substrate, the presence or absence of an ostiole, the presence or absence of germ slits, and spore ornamentation, were all homoplastic and not very useful for circumscribing monophyletic groups. I believe, however, that characters of the ascus may be important for the circumscription of some of the Sporormiella–Preussia groups. A number of features may be useful for circumscribing smaller clades if used in combination with other characters, as for instance the shape of the spore combined with the shape of the ascus. I believe that an extended search for useful morphological characters is needed though, preferably in combination with physiological and molecular studies. Future molecular studies of Sporormiaceae should also include more taxa. The two genera Chaetopreussia and Pleophragmia remain to be investigated, and considering that I found several undescribed Sporormiella species in the field studies, an extended search may result in even more taxa, which could help to resolve the phylogeny of Sporormiaceae in the future. Several new records of coprophilous ascomycetes were observed in the two field studies, in spite of a limited sampling effort. This suggests that the coprophilous ascomycetes in boreal Sweden are poorly known, and it is likely to assume that more species can be revealed in the future. The species richness is, however, affected by a number of ecological factors. When comparing moose dung of uniform age and composition and thus assumed to have the same inoculum of species, the fungal species richness and occurrence varied significantly between habitats. There was a highly significant negative correlation between water content of the dung and number of species of fungi, as well as percent cover of fungi, which is opposite to what I expected. However, species diversity was also negatively associated with amount of insect attack, which suggests that insects have a large impact on the fungal community, and insects feeding either on the dung and/or the fungi may be an important factor for species richness. Species richness of coprophilous fungi varies also significantly between different dung types. A study of the coprophilous ascomycetes on moose, mountain hare, and roe deer dung did not show any consistent patterns in respect to the animals´ digestive system, since the highest species richness was found for roe deer, while the other ruminant, moose, did not differ from mountain hare, a hindgut fermentor. However, there was a general strong positive relationship between the total number of ascomycete species and the number of plant species foraged by the three herbivores. Whether this mirrors the number of forage species per se, or changed chemical composition of forage, or foraging of both low- and tall-growing plants remains unclear as all these parameters were confounded. However, species of different spore sizes differed in their occurrence. Species with large spores (≥ 50 µm) were over-represented on roe deer dung, and under-represented on moose dung, while the reverse was found for species with small spores (<10µm). This suggests that the foraging level of the herbivore, which in turn mirrors species-specific differences in spore dispersal of the fungi, may be the most important factor in explaining species richness and diversity of the coprophilous community.

20 6. ACKNOWLEDGEMENTS

Financial support for the studies in this thesis was kindly provided by the Swedish Research Council (NFR B5101-20005187, VR 629-2001-5756, VR 621-2002-349, VR 621-2003-3038), the Kempe Foundation (JCK2026), Magn. Bergvalls foundation, the Royal Swedish Academy of Sciences (Stiftelsen Th Kroks donation), and the Gunnar and Ruth Björkman Foundation. I am grateful to Nils Lundqvist for numerous specimens contributed to this study. The Directors and Curators of cited herbaria and museums are acknowledged for the loan of specimens. Many thanks also to Mary Berbee who kindly allowed us to use her extractions of five taxa, and to Katarina Winka who contributed with the extraction of one taxon. The staff at Umeå Plant Science Centre assisted with sequencing, and Carin Olofsson is thanked for invaluable laboratory assistance. Mats Wedin, Lars Ericson and Nic Kruys are acknowledged for valuable comments on earlier versions of this thesis.

7. REFERENCES

Ahmed SI, Cain RF. 1972. Revision of the genera Sporormia and Sporormiella. Canadian Journal of Botany 50: 419-477.

Angel K, Wicklow DT. 1983. Coprophilous fungal communities in semiarid to mesic grasslands. Canadian Journal of Botany 61: 594-602.

Arenal F, Platas G, Pelaez F. 2004. Variability of spore length in some species of the genus Preussia (Sporormiella). Mycotaxon 89: 137-151.

Arx JA von. 1973. Ostiolate and nonostiolate pyrenomycetes. Proceeding Koninklijke Nederlandse Akademie van Wetenschappen, Series C 76: 289-296.

Arx JA von. 1981. The genera of fungi sporulating in pure culture, 3rd ed. Cramer.

Arx JA von, Aa HA van der. 1987. Spororminula tenerifae gen. et sp. nov. Transactions of the British Mycological Society 89: 117-120.

Arx JA von, Müller E. 1975. A re-evaluation of the bitunicate Ascomycetes with keys to families and genera. Studies in Mycology 9: 1-159.

Arx JA von, Storm PK. 1967. Über einige aus dem Erdboden isolierte, zu Sporormia, Preussia und Westerdykella gehörende Ascomyceten. Persoonia 4: 407- 415.

Barr ME. 2000. Notes on coprophilous bitunicate ascomycetes. Mycotaxon 76: 105- 112.

Bell A. 1975. Fungal succession on dung of the brush-tailed opossum in New Zealand. New Zealand Journal of Botany 13: 437-462.

21 Bell A. 1983. Dung fungi: an illustrated guide to coprophilous fungi in New Zealand. Viktoria University Press, Wellington.

Burney DA, Robinson GS, Burney LP. 2003. Sporormiella and the late Holocene extinctions in Madagascar. Proceedings of the National Academy of Sciences of the United States of America 100: 10800-10805.

Cain RF. 1956. Studies of coprophilous ascomycetes II. Canadian Journal of Botany 34: 675-687.

Cain RF. 1961. Studies of coprophilous ascomycetes. VII. Preussia. Canadian Journal of Botany 39: 1633-1666.

Cejp K, Milko AA. 1964. Genera of the Eurotiaceae with 32 ascospores – I. Westerdykella. Ceska Mykologie 18: 82-84.

Clum FM. 1955. A new genus in the Aspergillaceae. Mycologia 47: 899-901.

Dettman JR, Harbinski FM, Taylor JW. 2001. Ascospore morphology is a poor predictor of the phylogenetic relationships of Neurospora and Gelasinospora. Fungal Genetics and Biology 34: 49-61.

Dickinson CH, Underhay VHS. 1977. Growth of fungi in cattle dung. Transactions of the British Mycological Society 69: 473-477.

Dissing H. 1992. Notes on the coprophilous pyrenomycete Sporormia fimetaria. Persoonia 14: 389-394.

Doveri F. 2004. Fungi Fimicoli Italici: A guide to the recognition of basidiomycetes and ascomycetes living on faecal material. Associazione Micologica Bresadola, Trento.

Ebersohn, C. and Eicker, A. 1992. Coprophilous fungal species composition and species diversity on various dung substrates of African game animals. Botanical Bulletin of Academia Sinica 33: 85-95.

Eriksson OE. 1981. The families of bitunicate ascomycetes. Opera Botanica 60: 1- 220.

Eriksson OE. 1992. The non-lichenized pyrenomycetes of Sweden. Btj tryck, Lund.

Eriksson OE. 2005. Outline of Ascomycota – 2005. Myconet 11: 1-113.

Eriksson OE, Hawksworth DL. 1987. Notes on ascomycete systematics. Nos 225- 463. Systema Ascomycetum 6: 111-166.

Eriksson OE, Hawksworth DL. 1988. Notes on ascomycete systematics – Nos 552- 727. Systema Ascomycetum 7: 59-101.

22 Eriksson OE, Hawksworth DL. 2003. Saccharicola, a new genus for two Leptosphaeria species on sugar cane. Mycologia 95: 426-433.

Esser K. 1974. Podospora anserina. In: King RC (ed) Handbook of Genetics. Vol. 1. Plenum Press, New York, pp 531-551.

Furuya K, Naito A. 1980. Stimulation of ascospore germination by phenolic compounds in members of the Sordariaceae. Transactions of the Mycological Society Japan 21: 77-85.

Gargas A, DePriest PT. 1996. A nomenclature for fungal PCR primers with examples from intron-containing SSU rDNA. Mycologia 88: 745-748.

Geel B van, Buurman J, Brinkkemper O, Schelvis J, Aptroot A, Reenen G van, Hakbijl T. 2003. Environmental reconstruction of a Roman Period settlement site in Uitgeest (The Netherlands), with special reference to coprophilous fungi. Journal of Archaeological Science 30: 873-883.

Guarro J, Abdullah SK, Gene J, Al-Saadoon AH. 1997. A new species of Preussia from submerged plant debris. Mycological Research 101: 305-308.

Hansen L, Knudsen H. 2000. Nordic Macromycetes vol. 1. Ascomycetes. Helsinki University Printing House.

Hansen K, Laessøe T, Pfister DH. 2002. Phylogenetic diversity in the core group of Peziza inferred from ITS sequences and morphology. Mycological Research 106: 879-902.

Harrower KM, Nagy LA. 1979. Effects of nutrients and water stress on growth and sporulation of coprophilous fungi. Transactions of the British Mycological Society 72: 459-462.

Hausmann S, Lotter AF, Leeuwen JFN van, Ohlendorf Ch., Lemcke G, Grönlund R, Sturm M. 2002. Interactions of climate and land use documented in the varved sediments of Seebergsee in the Swiss Alps. The Holocene 12: 279-289.

Hawksworth DL. 1979. Ascospore sculpturing and generic concepts in the Testudinaceae (syn. Zopfiaceae). Canadian Journal of Botany 57: 91-99.

Hawksworth DL, Sutton BC, Ainsworth GC. 1983. Ainsworth & Bisby´s Dictionary of the Fungi. CAB, Surrey.

Huelsenbeck JP, Ronquist F, Nielsen R, Bollback JP. 2001. Bayesian inference of phylogeny and its impact on evolutionary biology. Science 294: 2310-2314.

Ikediugwu FEO, Webster J. 1970. Antagonism between Coprinus heptemerus and other coprophilous fungi. Transactions of the British Mycological Society 54: 181- 204.

23 Ingold, CT, Hudson HJ. 1993. The Biology of Fungi. 6th ed. Chapman & Hall, London.

Kirk PM, Cannon PF, David JC, Stalpers JA. 2001. Ainsworth and Bisby´s Dictionary of the Fungi. CAB International.

Kitching IJ, Forey PL, Humphries CJ, Williams DM. Cladistics, the Theory and Practice of Parsimony Analysis. 2nd ed. Oxford University Press.

Kuthubutheen AJ, Webster J. 1986. Water availability and the coprophilous succession. Transactions of the British Mycological Society 86: 63-76.

Locquin-Linard M. 1977. Á propos des genres non ostiolés placés dans la famille des Microascaceae. Revue de Mycologie 41: 509-523.

Luck-Allen ER. 1970. A new species of Trichodelitschia. Nova Hedwigia 19: 305- 309.

Lundqvist N. 1964. The genus Trichodelitschia in Sweden. Svensk Botansk Tidskrift 58: 267-272.

Lundqvist N. 1972. Nordic Sordariaceae s. lat. Symbolae Botanicae Upsalienses 20: 1-374.

Lussenhop J, Kumar R, Wicklow DT, Lloyd JE. 1980. Insect effect on bacteria and fungi in cattle dung. Oikos 34: 54-58.

Luttrell ES. 1973. Loculoascomycetes. In: Ainsworth GC, Sparrow FK, Sussman AS (eds.) The Fungi. Academic Press, New York, pp 135-219.

Malloch D. 1981. The plectomycete centrum. In: Reynolds DR (ed.) Ascomycete Systematics, The Luttrellian Concept. Springer-Verlag. New York, pp 73-91.

Massee G, Salmon ES. 1901. Researches on coprophilous fungi. Annals of Botany 15: 313-357.

Munk A. 1957. Danish pyrenomycetes: a preliminary flora. Dansk Botanisk Arkiv 17: 1-491.

Nowrousian M, Ringelberg C, Dunlap JC, Loros JJ, Kück U. 2005. Cross-species microarray hybridization to identify developmentally regulated genes in the filamentous fungus Sordaria macrospora. Molecular Genetics and Genomics 273: 137-149.

Olivier C, Berbee ML, Shoemaker RA, Loria R. 2000. Molecular phylogenetic support from ribosomal DNA sequences for origin of Helminthosporium from Leptosphaeria-like loculoascomycete ancestors. Mycologia 92: 736-746.

Parguey-Leduc A. 1974. Les asques et L´ontogénie des périthèces chez les Trichodelitschia. Bulletin de la société mycologique de France. 90: 101-120.

24 Richardson MJ. 2001. Diversity and occurrence of coprophilous fungi. Mycological Research 105: 387-402.

Ronquist F. 2004. Bayesian inference of character evolution. Trends in Ecology and Evolution 19: 475-481.

Shimodaira H, Hasegawa M. 1999. Multiple comparison of log-likelihoods with applications to phylogenetic inference. Molecular Biology and Evolution 16: 1114- 1116.

Silliker ME, Liotta MR, Cummings DJ. 1996. Elimination of mitochondrial mutations by sexual reproduction: two Podospora anserina mitochondrial mutants yield only wild-type progeny when mated. Current Genetics 30: 318-324.

Silva-Hanlin DMW, Hanlin RT. 1999. Small subunit ribosomal RNA gene phylogeny of several loculoascomycetes and its taxonomic implications. Mycological Research 103: 153-160.

Stevenson BG, Dindal DL. 1987. Functional ecology of coprophagous insects: A review. Pedobiologia 30: 285-298.

Stolk AC. 1955. Emericellopsis minima sp.nov. and Westerdykella ornata gen.nov., sp.nov. Transactions of the British Mycological Society 38: 419-424.

Sussman AS, Halvorson HO. 1966. Spores, their dormancy and germination. Harper & Row, New York and London.

Udagawa S, Ueda S. 1981. Eremodothis angulata and Apodus oryzae, two rare Plectomycetes from marine sludges. The Journal of Japanese botany 56:289:294

Weber R, Webster J. 1998. Stimulation of growth and reproduction of Sphaeronaemella fimicola by other coprophilous fungi. Mycological Research 102: 1055-1061.

Webster J. 1970. Coprophilous fungi. Transactions of the British Mycological Society54:161-180.

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis N, Gelfand J, White T (eds), PCR Protocols, A guide to Methods and Applications. Academic Press, San Diego, pp 315-322.

Wicklow DT. 1979. Hair ornamentation and predator defence in Chaetomium. Transactions of the British Mycological Society 72: 107-110.

Wicklow DT. 1992. The coprophilous fungal community: an experimental system. In: Carrol GC, Wicklow DT (eds), The Fungal Community, its Organization and Role in the Ecosystem. Marcel Dekker, New York, pp 715-728.

25 Wicklow DT, Angel K, Lussenhop J. 1980. Fungal community expression in lagomorph versus ruminant feces. Mycologia 72: 1015-1021.

Wicklow DT, Hirschfield BJ. 1979. Evidence of a competitive hierarchy among coprophilous fungal populations. Canadian Journal of Microbiology 25: 855-858.

Wicklow DT, Moore V. 1974. Effect of incubation temperature on the coprophilous fungal succession. Transactions of the British Mycological Society 62: 411-415.

Wicklow DT, Yocom DH. 1982. Effect of larval grazing by Lycoriella mali (Diptera:Sciaridae) on species abundance of coprophilous fungi. Transactions of the British Mycological Society 78: 29-32.

Yocom DH, Wicklow DT. 1980. Community differentiation along a dune succession: an experimental approach with coprophilous fungi. Ecology 61: 868- 880.

26 8. POSTSCRIPT

Att snickra ihop denna avhandling, har varit ett långt snubblande, där jag ofta tänkt efter, just efter, men med hjälp av följande personer har jag ändå mirakulöst lyckats hålla mig på benen.

Mats, du har varit min vardags-handledare som med gott mod löst många problem längs vägen. Du har fått mig att inse storheten med systematiken, och att du dessutom tappert stått ut med mitt bristande lav-intresse och min förmåga att tycka tvärtemot, är jag otroligt glad för! Ove, du är en skicklig lärare som fångade upp mig som student och sedan dess har du varit min källa till mycket information och inspiration. Allt jag har lärt mig om de förunderliga ascomyceterna kommer ju i grunden från dig, och när du inte varit på plats så har jag hittat de mest knepiga referenserna i din bokhylla. Lasse, utan din entusiasm och uppmuntran hade den här avhandlingen varken påbörjats eller avslutats, du har också haft ett imponerande tålamod i fält, det var inte bara en gång mina markeringar regnade bort…

Ett speciell tack vill jag ge till Katarina W, för att du har lärt mig labba, hjälpt mig med undervisning, dragit med mig på iksu-träning och förklarat det här med fylogenetiska analyser ett oändligt antal gånger. Du har helt enkelt varit en skön kombination av extra-handledare och god vän. Tack också till min medförfattare Inga-Lill P, för ett fint samarbete. Jag hoppas att vi får chansen att klappa söta älgkalvar igen! Nils Lundqvist, du har hjälpt mig med många kollekter och varit en ovärderlig guide när jag gått vilse i bestämningsnycklarna. Hans G, utan din hjälp med statistiken hade jag faktiskt inte blivit klar.

Med Anna C & Elisabeth W har jag delat roliga kurser, kämpiga boktentor, lavexq i snöyra… tillsammans har vi stått starka när ekorrsamlandet fångat vår handledare i ett maniskt grepp! Anna du har blivit en jättebra kompis under dessa år och den som otroligt nog orkat läsa alla mina manus! Carin O, du har hjälpt mig med labbandet, men lika viktigt är att du alltid haft tid för prat och godis, tack för alla pauser. Våra postdocs, Per I och Heidi D har bidragit med kul sällskap och många nya ideer. Jag vill också tacka Karin D för snygga sekvenser, och Ulla C-G som peppat mig det sista året.

En av de mest lärorika erfarenheterna från doktorandtiden är den undervisning jag varit inblandad i, därför vill jag passa på att tacka systematikkollegorna som snöat in på djur, Anders N & Johannes B, samt Åsa H, du har med ett otroligt tålamod bankat in lite storsvampar i mitt huvud. Fast jag är fortfarande inte övertygad om varför man ska äta dom.

Institutionen är ju en stor, och dynamisk arbetsplats där man hela tiden lär känna nya personer (det liknar successionen av svampar på en stor spillningshög, faktiskt). Alla bidrar på sitt lilla sätt till att det är en trivsam stämning, men jag vill särskilt tacka alla doktorander, ni är för många att nämnas vid namn, men jag giller er alla skarpt!

Som doktorand flyter ju fritid och jobb lätt ihop och några av dom som genom jobbet, även lyst upp min tillvaro utanför, är: Lena D, ditt sällskap har varit ovärderligt, en enkel fika eller en kväll i din kala, kalla källare, alltid lika kul! Tina

27 N, tack för alla glada stunder, som på blåsiga klippor i Bodö eller en horisontell flygelsång. Tuss Z, tack för avslappnande middagar och hetsiga spelkvällar i vintermörkret. Mattias E & Anna-Maria E, Henrik & Kristina H, Jocke S & Hanna O, Johan & Eva O, och Pernilla & Bent C, tack för alla trevliga tillställningar.

Marie, Gunilla och Annette, ni fick mig genom grundutbildningen och har varit fenomenalt sällskap under roliga hajker och tradiga föreläsningar. Tack för att ni då och då påminner mig om vilket sjukt kul projekt jag haft.

Mamma och pappa, er outtröttliga hjälp med läx-traglandet under första halvan av skoltiden har gjort att jag på ren tjurighet orkat lika länge till. Tack för att ni alltid ställer upp och på ett övertygande sätt uppmuntrar de flesta av mina ideér. Mikke, ständig storebror som hetsar mig till de flesta stordåd och mindre katastrofer, tack för år av sparring.

Nic, du förgyller allt, hela mitt hjärta till dig!

Nu ska jag ut i solen.

/ åsa

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