Development and Evaluation of Rrna Targeted in Situ Probes and Phylogenetic Relationships of Freshwater Fungi

Development and evaluation of rRNA targeted in situ probes and phylogenetic relationships of freshwater fungi

vorgelegt von Diplom-Biologin Christiane Baschien aus Berlin

Von der Fakultät III - Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktorin der Naturwissenschaften - Dr. rer. nat. -

genehmigte Dissertation

Promotionsausschuss:

Vorsitzender: Prof. Dr. sc. techn. Lutz-Günter Fleischer Berichter: Prof. Dr. rer. nat. Ulrich Szewzyk Berichter: Prof. Dr. rer. nat. Felix Bärlocher Berichter: Dr. habil. Werner Manz

Tag der wissenschaftlichen Aussprache: 19.05.2003

Berlin 2003

D83

Table of contents

INTRODUCTION ...... 1

MATERIAL AND METHODS ...... 8

1. Used organisms ...... 8 2. Media, culture conditions, maintenance of cultures and harvest procedure...... 9 2.1. Culture media...... 9 2.2. Culture conditions ...... 10 2.3. Maintenance of cultures...... 10 2.4. Harvest procedure...... 10 2.4.1. Harvest of cultures ...... 10 2.4.2. Harvest of conidia ...... 11 2.4.3. Harvest of hyphal tips ...... 11 3. River sampling sites ...... 11 3.1. Elbe river, Germany ...... 11 3.2. Oberer Seebach, Austria...... 12 4. Sampling and monoconidial isolation strategies ...... 13 4.1. Foam ...... 13 4.2. Leaves and wood ...... 13 4.3. Water and river snow ...... 14 4.4. Sporocarps...... 14 4.5. Monoconidial isolations from cultures ...... 14 4.6. Trapping by surface exposure ...... 14 4.6.1. Polyethylene slides ...... 14 4.6.2. Leaves...... 15 4.6.3. Cellulose ...... 15 5. Germination experiments ...... 15 5.1. Exposure of conidia and spores in cages ...... 16 6. Phenotypic characterisation ...... 18 6.1. Morphological determination...... 18 6.2. Fluorescent stains ...... 18 6.2.1. Calcofluor white ...... 18 6.2.2. Live-Dead stain ...... 18 6.2.3. SYTO stains...... 18 6.2.4. Lectin stain...... 19 7. Genotypic characterisation...... 20 7.1. Extraction of genomic DNA...... 20 7.2. Amplification of the 18S rRNA genes...... 20 7.3. Amplification of the ITS1, 5.8S rRNA and ITS2 genes ...... 20 7.4. Amplification of the 28S rRNA genes...... 21 7.5. Ribosomal DNA sequencing ...... 21 7.5.1. Nucleotide sequence accession numbers ...... 23 7.6. Alignment of rDNA sequences...... 24 8. Modification of ARB software settings...... 24 8.1. Change of the reference organism and adjusting the 18S and the 28S rRNA secondary structure...... 24 8.2. Enlargement of the ARB database with fungal sequences...... 25 9. Reconstruction of phylogenetic trees ...... 25 9.1. Neighbour Joining ...... 25 9.2. Maximum Parsimony...... 25 9.3. Maximum Likelihood ...... 25 9.4. Bayesian analysis ...... 25 10. Fluorescence in situ hybridisation ...... 29 10.1. Fixation...... 29 10.1.1. Paraformaldehyde fixation ...... 29 10.1.1.1. Cultures, conidia, hyphal tips...... 29 10.1.1.2. Biofilms ...... 29 10.1.1.3. River snow ...... 29 10.1.1.4. Foam...... 29

I Table of contents

10.1.2. Ethanol fixation ...... 29 10.1.3. Methanol fixation...... 29 10.2. Permeabilisation...... 29 10.2.1. Permeabilisation by chitinase treatment...... 29 10.2.2. Permeabilisation by electroporation...... 30 10.3. Design and evaluation of specific oligonucleotide probes ...... 30 10.4. Hybridisation procedure ...... 31 10.4.1. Cultures, conidia, hyphal tips...... 31 10.4.2. Biofilms...... 32 10.4.3. River snow and foam ...... 32 10.5. Determination of hybridisation stringencies ...... 32 11. Microscopical analysis and documentation ...... 33 11.1. Phase contrast microscopy...... 33 11.2. Epifluorescence microscopy ...... 33 11.3. Confocal laser scanning microscopy ...... 33 11.3.1. Measurement of signal intensities and autofluorescence scan ...... 34

RESULTS...... 35

A Sampling, isolation, cultivation and identification of freshwater fungi...... 35

1. River Elbe...... 35 1.1. Fungal isolates from different substrata...... 36 1.2. Seasonal patterns ...... 37 1.3. Freshwater fungal conidia abundant in fixed foam samples...... 39 2. Oberer Seebach ...... 40 2.1. Fungal isolates from different substrata...... 40 2.2. Freshwater fungal conidia abundant in fixed foam samples...... 42

B Phylogenetic relationships of freshwater fungi...... 44

1. ARB modification...... 44 1.1. Databases of fungal sequences...... 44 2. Inference of phylogenetic relationships of freshwater fungi based on rDNA data ...... 44 2.1. Tetracladium marchalianum...... 44 2.2. Alatospora acuminata ...... 53 2.3. Tricladium splendens and Tricladium angulatum...... 57 2.4. Heliscus lugdunensis ...... 64 2.5. Anguillospora crassa and Anguillospora longissima...... 66 2.6. Lemonniera aquatica and Lemonniera terrestris ...... 73 2.7. Varicosporium elodeae and Anguillospora furtiva ...... 73

C Fluorescence in situ hybridisation (FISH) of freshwater fungi...... 75

1. Development of rRNA targeted oligonucleotide probes...... 75 1.1. Design and evaluation of oligonucleotide probes specific for the division Eumycota...... 75 1.1.1. Fungal probe FUN1429 ...... 75 1.1.2. Fungal probe MY1574 ...... 79 1.2. Design and evaluation of FISH probes for freshwater fungi specific on genus and species level ...... 79 1.2.1. Probe specific for the genus Tetracladium ...... 79 1.2.1.1. Probes specific for Tetracladium marchalianum...... 80 1.2.2. Probes specific for Alatospora acuminata ...... 81 1.2.3. Probe specific for Tricladium angulatum...... 82 1.2.4. Probe specific for Heliscus lugdunensis ...... 83 1.2.5. Probes specific for Anguillospora longissima ...... 83 1.3. Summary of all newly designed fungal oligonucleotide probes and their binding sites...... 86 2. Evaluation of factors influencing FISH, and improvement of fungal FISH signal detection...... 89 2.1. Influence of culture media...... 89 2.2. Influence of fixation methods ...... 89

II Table of contents

2.3. Freshwater fungi and inherent fungal autofluorescence...... 89 2.4. Comparison of epifluorescence and confocal laser scanning microscopy ...... 94 2.5. FISH signal intensities of different parts of the mycelium ...... 95 2.6. Influence of culture age...... 96 2.7. Permeabilisation...... 97 2.7.1. Permeabilisation of 2 and 8 weeks old cultures with chitinase treatment ...... 97 2.7.2. Permeabilisation by electroporation...... 98 2.8. Incubation time...... 99

D In situ detection of freshwater fungi ...... 100

1. Critical evaluation of fluorescent stains...... 100 1.1. Calcofluor White...... 100 1.2. Life-Dead stain ...... 100 1.3. SYTO stains ...... 100 1.4. Lectin stain ...... 101 2. FISH of freshwater fungi within environmental samples ...... 101 2.1. Biofilms on polyethylene slides ...... 101 2.2. Fungi directed FISH on leaves and cellulose...... 105 2.2.1. Leaves...... 105 2.2.2. Cellulose ...... 105 2.3. Fungal FISH of foam samples ...... 107 2.4. River snow...... 108 3. Investigation of the germination of conidia and spores within the Oberer Seebach by FISH.... 108

DISCUSSION ...... 112

A) Sampling and isolation of fungi in the river Elbe and the stream Oberer Seebach...... 112

1. Fungal assemblages in two different lotic habitats ...... 112 2. Sampling and isolation strategies ...... 115

B) Phylogenetic characterisation of freshwater fungi ...... 116

1. Tetracladium marchalianum...... 117 2. Alatospora acuminata...... 119 3. Tricladium splendens and Tricladium angulatum...... 120 4. Anguillospora longissima and Anguillospora crassa...... 122 5. Heliscus lugdunensis...... 124 6. Lemonniera aquatica and Lemonniera terrestris ...... 125 7. Varicosporium elodeae...... 126 8. Phylogeny reconstruction using rDNA data and different treeing methods ...... 126

C) Fluorescence in situ hybridisation of freshwater fungi ...... 127

1. Specificity of oligonucleotide probes for fungi...... 128 2. Factors influencing FISH of fungi ...... 131 2.1. Fixation of fungi and environmental samples intended for FISH ...... 131 2.2. Dealing with autofluorescence and the influence of the microscopic technique...... 132 2.3. Enhancement of freshwater fungal cell wall permeability for FISH probes ...... 134 2.4. Influence of different functional parts of the fungal mycelium on FISH signal intensities.... 136

D) In situ detection of freshwater fungi in the river Elbe and in the Oberer Seebach by FISH ...... 137

1. Estimation of fungal diversity and biomass...... 138 2. FISH of fungi in the lowland river Elbe and the alpine creek Oberer Seebach...... 139

Outlook ...... 141

III Table of contents

SUMMARY ...... 142

ZUSAMMENFASSUNG ...... 144

ACKNOWLEDGEMENTS...... 147

REFERENCES ...... 149

APPENDIX ...... 167

IV Abbreviations

ABBREVIATIONS

A adenine acc. no. GenBank accession number a.s.l. above sea level ATP adenosine triphosphate bp base pair(s) BLAST Basic Local Alignment Search Tool C cytosine CBS Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands CCMF- fungal strain from the Czech Collection of Microorganisms CFW Calcofluor White CLSM confocal laser scanning microscopy CTAB N-Cetyl-N,N,N-trimethyl-ammonium bromide Cy3 5,5’-disulfo-1,1’di(g-carbopentynyl)-tetramethyl- inolocarbocyanine Cy5 N,N'-biscarboxypentyl-5,5'-disulfonato-indodicarbocyanine Da Dalton, 1Da = approximately 1.66 x 10-24 grams DAPI 4’,6-diamidino-2-phenylindol dihydrochloride ddH2O double distilled water DOC dissolved organic carbon DOM dissolved organic matter DNA desoxynucleic acid EDTA ethylenediaminetetraacetic acid E-FISH electroporation fluorescence in situ hybridisation F Farad (standard unit of electrical capacity) FDA fluorescein diacetate Fig. Figure(s) FITC fluorescein isothiocyanate G guanine g gram GC guanine + cytosine h hour(s)

H2O water HEPES N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic acid IGS intergenic spacer ITS internal transcribed spacer k kilo (103) kb kilobase (=1000 bp) l litre Ln likelihood logarithmic likelihood LSU large subunit rRNA gene or 28SrRNA gene M molar m meter µ micro (10-6)

V Abbreviations

MCMC Markov chain Monte Carlo algorithm min minute(s) ML maximum likelihood treeing method mM millimol ms milliseconds (10-3 s) mtDNA mitochondrial DNA n nano (10-9) NA numerical aperture NJ neighbour joining treeing method NNI nearest neighbour interchange Ω Ohm (standard unit for electrical resistance) P statistical confidence interval p pico (10-12) PBS phosphate buffered saline PCR polymerase chain reaction PE(-HD) polyethylene (high-density) POM particular organic matter ppm parts per million R base A or G RAPD rapid amplification of polymorphic DNA rDNA ribosomal desoxynucleic acid RFLP restriction fragment length polymorphism RNA ribonucleic acid rpm rounds per minute rRNA ribosomal ribonucleic acid s second(s) SD standard deviation SDS sodium dodecyl sulfate SSU small subunit rRNA gene or 18S rRNA gene T thymine TE Tris EDTA Tris tris-(hydroxymethyl)-aminomethane Tween 80 polyoxyethylenesorbitan momooleate

Tm melting temperature U uracil U unit UV ultra violet V Volt v/v volume/volume W base A or T w/v weight/volume Y base C or T

VI Introduction

INTRODUCTION

Freshwater lakes, rivers and streams play an important role in the supply of water for mankind. These aquatic environments often are very rich in a wide variety of organic matter, including decaying tree leaves and wood. Fungi are, along with bacteria, well known to be intensively involved in the degradation and conversion of biopolymers in aquatic systems (Bärlocher 1992). Also in drinking water distribution systems (Doggett 2000), cooling towers (Eaton 1976), dew and tree trunk water fungi are common members of the microbial biocoenosis of freshwater systems. As organisms, responsible for degradation and conversion of organic matter, fungi are equipped with a specific set of enzymes, especially for the degradation of cellulose and lignin (Suberkropp 1992). Although a number of pure culture-based studies attempted to describe the ecological role of freshwater fungi associated with the degradation of leaf material (e.g. Suberkropp & Klug 1980; Gessner 1989; Bärlocher 1991), the information about the spatial fungal distribution and metabolic activity in their aquatic microenvironments is still sparse (e. g. Shearer & Lane 1983b). This is mainly due to the difficulties in suitable cultivation approaches and the lack of appropriate in situ methods for detecting and monitoring fungal life in an ecosystem. Historically, Ingold (1942, 1943a, 1943b, 1943c, 1944) realised for the first time the existence of an extensive flora of hyphomycetes on submerged decaying leaves of dicotyledoneous trees and shrubs. He found, that asexual spores, the conidia, can be isolated from foam collected in clean, well aerated running waters. Before that, descriptions of waterborne spores such as the four armed conidia of Tetracladium spp. and Lemonniera aquatica were made by de Wildeman (1893, 1894, 1895). In the past, conidia were also often mistaken as the whole fungal thallus (Huber-Pestalozzi 1938) or confused with algae (Kol 1928). Traditionally, the term “freshwater fungi” (Thomas 1996; Goh & Hyde 1996) is used to describe fungi which complete their asexual life cycle in freshwater and disperse via water. Freshwater fungi include a phylogenetically heterogeneous group of fungi. Mainly based on morphological differences they are divided into three main ecological groups (Gams et al. 1998): (i) The zoosporic “water moulds“ mainly belonging to the Mastigomycotina live permanently or in stages of their life cycle in water (Hudson 1986; Fuller & Jaworski 1987). (ii) The group of aero-aquatic fungi (Webster & Descals 1981) is defined by their occurrence in lentic systems of stagnant waters such as lakes, ponds and ditches. Sporulation occurs on emerged leaves with propagules floating then on the water surface. (iii) In contrast the group of aquatic hyphomycetes, in the honour of the important researcher on their life cycles also called “Ingoldians“ (Webster & Descals 1981), can be found in lotic systems such as well aerated brooks, streams and rivers. Morphologically, aquatic hyphomycetes are characterised by their stauroform or scolecoform conidia released mostly from submerged conidiophores.

Since aquatic hyphomycetes are defined as ecological entity and not as a natural group of fungi, Ingold (1975) included species the conidia of which are frequently found in foam and which may be either facultative aquatic hyphomycetes or stream associated fungi with waterborne conidia. At present, more than 300 species are defined to be belonging to the ecological group of aquatic hyphomycetes (Marvanová 2001, pers. comm.).

1 Introduction

The classification of aquatic hyphomycetes is based on asexual (anamorph) genera. They are grouped by similarities in conidium morphology and development (Webster & Descals 1981). The majority of aquatic hyphomycetes are known only from their anamorphs. Often they show extraordinary mitotic macroconidia. However, several studies connecting anamorphs to their meiotic sexual states (teleomorphs) have confirmed that many anamorph-genera include taxa of diverse relationships (e.g. Webster 1961, Willoughby & Archer 1973, Webster & Descals 1979, Digby & Goos 1987). Up to now, 38 aquatic hyphomycetes with ascomycetous and seven with basidiomycetous teleomorphs are known (Nawawi et al. 1977; Marvanová & Stalpers 1987; Marvanová & Suberkropp 1990; Webster 1992; Marvanová 1997; Shearer: http://fm5web.life.uiuc.edu:23523/ascomycete/ default.html). In several cases not only macroconidial anamorphs, but also microconidial synanamorphs (a second morphologically distinct anamorph) are described (Webster 1992). In general, two types of microconidia are distinguished: (i) microconidia capable of germination and (ii) spermatia that do not germinate (Webster 1992). Classification and nomenclature of fungi suffers from the existence of anamorph, teleomorph and sometimes even synanamorph states in one fungus (Hennebert 1987). As recommended by Mason (1937) “one species, one name“ would be ideal. However, a dual nomenclature with different names for different morphs is commonly used. The detection of anamorph and teleomorph connections is often difficult and up to now mostly relying on culture dependent methods (e.g. Reynolds 1987). Molecular phylogenetic studies concerning several genera of aquatic hyphomycetes, which could help to evaluate taxonomic relationships have been missing so far. The present work combines for the first time taxonomic and molecular phylogenetic methods for the evaluation of several aquatic hyphomycete genera.

Investigations in the early nineteen-seventies of the last century showed that aquatic hyphomycetes are important components of microbial communities associated with the early decomposition of most organic materials, particularly allochthonous litter in freshwater streams (Kaushik & Hynes 1971; Willoughby & Archer 1973, Bärlocher & Kendrick 1974). They are involved in the mineralisation of leaf litter and its conversion into other pools of organic matter such as fine particular organic matter (POM) and dissolved organic matter (DOM) (Suberkropp & Klug 1980). Although aquatic hyphomycetes are taxonomically diverse, they convergently evolved conidia with stauroform and scolecoform (thread-like) shapes. Webster (1959a) and later Read (1990), Read, Moss and Jones (1992) studied the putative function of the shape of tetraradiate, with four arms diverging more or less from a common point, and sigmoid (worm-like) conidia. From these data, the authors concluded that tetraradiate and branched conidia have at least three points for the attachment to the substratum (e.g. leaves), whereas sigmoid conidia have only two points for attachment. Following initial contact with the substratum the conidia produce mucilage and some form appressoria during the germination process (Harrison et al. 1988). The next step in the life cycle of aquatic hyphomycetes is the formation of a septate mycelium invading the substratum. For asexual reproduction conidiophores grow out from the substratum and conidia are released into the water

2 Introduction body. In temperate climate, the number of conidia found in water peaks in autumn, early winter, and, to a lesser extent, also in early spring when submerged decaying leaves and organic input are most abundant in lotic systems (Webster & Descals 1981). Up to 103 - 104 conidia per litre of river water have been found. As mentioned above aquatic hyphomycetes can also have a sexual stage. Generally, it is found in moist but not submerged locations and could provide a terrestrial source of inoculum for the anamorphs occurring in water. But also the anamorphic state itself seems to be able to at least survive in terrestrial habitats such as soils and landplants (e.g. Gönczöl 1976; Bandoni 1981; Fisher & Petrini 1989). Nevertheless the complete life cycles of most aquatic hyphomycetes are not yet known. Bärlocher (1980, 1981, 1982a, 1985) and Suberkropp and co-workers (1976a, 1984, 1991) examined the dynamic and complex interactions between aquatic hyphomycetes and invertebrates. They showed that the enzymatic activities of aquatic hyphomycetes condition leaf material for the digestion by invertebrate shredders. Additionally, leaf inhabiting aquatic hyphomycete thalli themselves can serve as food source for invertebrates (Cummins & Klug 1979). The interactions between wood and aquatic hyphomycetes were investigated by Shearer and co- workers (e.g. 1983a, 1988, 1991). They assume that woody debris serves as habitat, reservoir, nutritional source, asexual and sexual reproduction site, competition site and agent for long distance dispersal.

Methods for examination of freshwater fungi in streams were mostly based on microscopic detection and identification of conidia trapped in foam, associated with decaying leaves and suspended in water. Commonly used techniques are the isolation of conidia by streaking out foam on slides covered with nutrient media, aerating leaves collected from streams in the laboratory inducing sporulation of leaf associated fungi and the subsequent transfer of conidia to defined culture media (Shearer & Lane 1983b; Descals 1997a). Determination of aquatic hyphomycetes relies on the distinctive conidial morphology and conidial development which in many cases allows identifying them down to the species level. However, the investigation of aquatic hyphomycetes communities requires not only isolation and description for taxonomical purposes, but also the quantification and species composition of the fungal biomass. Early attempts to estimate fungal biomass on decaying leaves were performed by direct microscopic examinations of cleared substrates (Iversen 1973; Bärlocher & Kendrick 1974). Traditionally, aquatic fungal communities have been characterised by concentrating conidia from stream and river water or conidia developed from incubated substrates on filters (Suberkropp & Klug 1976a; Bärlocher 1982b). Total conidium counts have been used for characterising fungal assemblages present in the water body or during various stages of decay.

Fungal communities are insufficiently described if culture dependent methods are used (Warcup 1957; Parkinson 1981). The spatial fungal distribution and even more the metabolic activity in the aquatic microenvironment cannot be accessed for the following reasons:

3 Introduction

• Conidia do not represent the metabolically active parts of a fungus, whereas the vegetative mycelium does. • Conidia and spores of many species can survive a long time after the mycelium they originated from ceased its activity. • Conidia may be transported downstream from other locations to the sampling site and remain dormant. • Often sporulation of fungi occurs after the conditions for balanced vegetative growth became unfavourable. Conidia numbers may not be correlated with growth (Smith 1978; Maharning & Bärlocher 1996). Since hyphae are insufficient for morphological identification and difficult to isolate, several methods for estimating fungal biomass have been attempted that utilise the metabolic activity of the mycelium.

More recently, indicator molecules have been used for the quantification of fungal biomass, in particular ATP and ergosterol (Newell 1992). The ergosterol method is essentially restricted on the detection of intact membranes of higher fungi and therefore accounts for a reasonably constant fraction of total fungal biomass. Measuring ergosterol levels in decaying leaves has therefore become the preferred method for estimating living fungal biomass. Different fluorescence based cell staining methods such as Calcofluor White, Fluorescein diacetate (FDA) (Söderström 1977) and lectin staining (Brul et al. 1997) have been used less intensively. However, neither microscopic examination, nor ergosterol measurement or fluorescent cell stainings provide sufficient information on the specific composition of the fungal community or its spatial organisation. Species specific detection of aquatic hyphomycetes has recently been performed by the use of immunoassays (Bermingham et al. 1995a, 1997, 2001). However, the need for highly specific antibodies requires pure cultures of the expected target species. Molecular tools for the identification and detection of microorganisms within their natural habitats promise to overcome the limitations of culture dependent methods. As early as in the nineteen sixties, Zuckerkandl and Pauling (1965) suggested to investigate macromolecules as “documents of evolutionary history”, paving the way for comparative sequence analysis of marker molecules. Since then sequence comparison has been used to infer phylogenetic trees reflecting the evolutionary relationships of organisms (Fitch & Margoliash 1967). With the development of PCR (polymerase chain reaction; Saiki et al. 1985, Saiki et al. 1988), many genes became accessible for phylogenetic reconstruction. In particular, the ribosomal DNA (rDNA) genes have been characterised (Hibbett 1992). Following the requirements as spelled out by Bruns et al. (1991), the rDNA genes are a good choice as phylogenetic marker molecule: • Ribosomal RNAs are ubiquitous molecules with the same function in all taxa. • Ribosomal DNA is a multiple copy gene that can easily be isolated and directly sequenced. • The mosaic pattern of conserved and variable regions in rDNA supplies enough consistency differences for comparisons at many taxonomic levels.

The improvement of sequencing techniques lead to a rapid increase in rRNA sequence data which are accessible in databases such as GenBank (http://www.ncbi.nlm.nih.gov/). Today (May 2002) more

4 Introduction than 34000 bacterial, and about 16000 fungal rRNA sequences are accessible in the GenBank sequence database. Increasing availability of small subunit (SSU) and to a lesser extend large subunit (LSU), and spacer regions (ITS; IGS) sequence data improves the inference of phylogenetic relationships (e.g. Woese et al. 1990; Van de Peer et al. 1997). The sequence databases provided the basis for the development of molecular tools for the investigation of the ecology of microorganisms. Within the last decade molecular techniques like extraction and analysis of DNA and RNA, PCR, gene clone libraries, RAPD (random amplification of polymorphic DNA), RFLP (restriction fragment length polymorphism) and denaturing gradient gel electrophoresis (DGGE) have become of major interest for microbial ecology studies. However, all methods based on total DNA extraction lack the information about the physiological status of the studied organisms. The detection of metabolically active bacteria within their natural environment was achieved using synthetic oligonucleotide probes for in situ hybridisation (Amann et al. 1995). In situ hybridisation allows the detection of RNA or DNA sequences directly in prokaryotic and eukaryotic cells by binding of oligonucleotide probes to their complementary target sequences (Amann et al. 1995). The construction of hybridisation probes requires the localisation of specific rRNA stretches (signatures), which are unique to different phylogenetic levels. For this purpose, aligned rRNA sequence data have to be screened and evaluated. The oligonucleotide probes are usually 15 to 20 nucleotides in length. Formerly, the probes have been labeled using radioactivity (Giovannoni et al. 1988). Nowadays they are usually labeled with haptens (Zarda et al.1991), enzymes (Schönhuber et al. 1997) or fluorochromes (DeLong et al. 1989), which are directly coupled to the nucleotides. Today, mostly fluorochrome-dye labeled oligonucleotide probes combined with epifluorescence microscopy are used because of their superior spatial resolution and their convenient detection. The oligonucleotide probe can be applied to cultured cells or environmental samples and has to penetrate the cell walls of the target cells. Specifically annealing of the probe with its complementary RNA or DNA sequence within the target cell results in a measurable detection signal. The probe signal is correlated with the ribosome content. The signal increases with numbers of ribosomes and rRNA in the target cell (DeLong et al. 1989; Kramer & Singleton 1992). Poulsen et al. (1993) showed that the ribosome content is correlated with growth activities of bacterial cells. Because slow growing or non growing but viable organisms have a lower rRNA content it is sometimes difficult to detect probe conferred hybridisation signals. Combining fluorescent in situ hybridisation (FISH) with advanced microscopically techniques such as confocal laser scanning microscopy (CLSM) increases the sensitivity of probe signal detection. Within the last decade probes were mostly applied to studies of bacterial cells. The potential of FISH challenged to adapt it to other than prokaryote organisms. Up to now design and evaluation of FISH- probes has been restricted to single celled eukaryotic organisms, such as protozoa and yeast-like organisms (Li et al. 1996; Brenner et al. 1998). Recent studies concerning the use of the FISH method for fungi, such as the detection of yeasts in yoghurt cultures (Kosse et al. 1997) or of yeast-like organisms such as Aureobasidium pullulans, a common phylloplane inhabitant with some filamentous structures (Li et al. 1997), have shown several limitations of this molecular tool. Thus many questions about the accessibility of fungi for fluorescent in situ hybridisation probes became apparent:

5 Introduction

- Can eumycotan (fungal) cell walls, which consist of several layers of carbohydrate polymers such as glucan and chitin, be properly permeabilised to allow the penetration of the FISH-probes ? - Does fungal inherent autofluorescence prevent the detection of FISH probe conferred signals ? - Is the resolution power of the different regions of the 18S or 28S rRNA gene sufficient for the design of specific phylogenetic oligonucleotides ? - Are the targeted regions within ribosomes accessible for binding with FISH probes ? - Is the probe conferred signal influenced by the physiological condition of the target cells either in culture or in environmental samples ? Answering these questions could increase the reliability and understanding how to interpret FISH data.

It is doubtful if the entire “staurosporous and scolecosporous fungi need to be considered as aquatic hyphomycetes” as it has been defined by Webster & Descals 1981. It is known that aquatic hyphomycetes are a phylogenetically heterogeneous group (Webster 1992). However, the phylogenetic placement of aquatic hyphomycetes within the classes and genera of the Ascomycota remained often unresolved. Especially phylogenetic relationships of anamorph species for which teleomorph states are unknown are unclear. Therefore one concern of the present study is to characterise the phylogenetic relationships of several frequently observed type species of aquatic hyphomycetes with different conidial shapes. Furthermore the phylogenetic affiliation of aquatic hyphomycetes with unknown teleomorphs should be investigated. The widely stated assumption that they are not a taxonomic group, but an ecological group now even has to be doubted. Some were never found in streams and others that have been found in streams, were never seen on leaves (Ludmila Marvanová, pers. comm.). Some are involved in leaf degradation, some in woody debris degradation and the ecological role in streams of many conidia found in foam is unclear, their natural substrate is not known. As mentioned before many stauroform and scolecoform conidia found in foam and running waters in temperate climate were observed in autumn, only. Their lifecycles eventually comprising anamorphic or synanamorphic and teleomorphic states are still unknown. It is suspected that the inoculum of aquatic hyphomycete conidia in streams in autumn and spring is derived from terrestrial teleomorphs. However the mycelia of germinated ascospores or basidiospores possibly developing the submerged anamorph state could never been observed in nature. Mycelia cannot be classified unless sporulation occurs or is induced in laboratory experiments. It is not clear if a development from the anamorph state back to the teleomorph state exists in nature. In order to increase the knowledge of aquatic hyphomycetes lifecycles, the objective of the present work was to evaluate and further develop the FISH method as a molecular tool for the in situ detection of the active mycelia of aquatic hyphomycetes in situ. Special emphasis should be given to the questions concerning the accessibility of fungi for in situ probing. The prerequisite for the development of phylogenetically specific rRNA targeted hybridisation probes for the use in aquatic fungal communities is the ribosomal DNA sequencing of fungi isolated from aquatic habitats. Pure cultures of aquatic hyphomycetes were obtained from Dr. Ludmila Marvanová from the Czech Collection of Microorganisms (CCM). To include environmental specimen for phylogenetic investigations and for development and evaluation of the FISH method sampling and isolation was conducted in the German lowland river Elbe and the Austrian alpine stream Oberer Seebach. In a first approach towards the

6 Introduction detection and identification of aquatic hyphomycetes with molecular tools the fungal isolates were to be sequenced with regard to their phylogenetic placement and evaluation of signatures suitable for subsequent probe design. For comparative sequence alignment and design of oligonucleotides the software environment for sequence data ARB (http://www.biol.chemie.tu-muenchen.de/pub/ARB) should be adapted to the work with fungi. Based on the information from sequence data oligonucleotides of different specificity should be designed followed by the evaluation and the application of these probes in pure cultures and on substrates of the natural habitats in order to detect and identify aquatic hyphomycetes in situ.

.

7 Material and Methods

MATERIAL AND METHODS 1. Used organisms Used fungal isolates, reference organisms and their sources are listed in Table 1.

Table 1: Reference organisms and sources (ATCC = American Type Culture Collection; Rockville, MD, USA; CCM = Czech Collection of Microorganisms; Brno, Czech Republic; Bärlocher = Collection of Professor Dr. F. Bärlocher, Mount Allison University, Sackville, Canada; Elbe, L = Personal isolates of C. B.; UTEX = UTEX Culture Collection of Algae, Austin, Texas, USA)

FUNGI SOURCE FUNGI SOURCE Alatospora acuminata Ingold CCM F-02383 Penicillium chrysogenum CBS 197.46 Alatospora acuminata CCM F-12186 Penicillium chrysogenum CBS 307.48 Alatospora acuminata CCM F-13089 Penicillium chrysogenum Elbe 107 Alatospora acuminata CCM F-37194 Pleospora herbarum CBS191.86 Alatospora acuminata CCM F-18799 Syncephalastrum racemosum CBS 557.81 Alatospora acuminata L 8, Austria Tetracladium marchalianum Elbe 50 De Wild. Alatospora acuminata L 27, Austria Tetracladium marchalianum CBS 979.87 Alternaria alternata CBS 154.31 Tetracladium marchalianum CCM F-312 Anguillospora crassa Ingold CCM F-07082 Tetracladium marchalianum CCM F-11391 Anguillospora crassa CCM F-13483 Tetracladium marchalianum CCM F-19399 Anguillospora crassa CCM F-15283 Tetracladium marchalianum CCM F-26199 Anguillospora crassa CCM F-15583 Tetracladium marchalianum CCM F-26299 Anguillospora crassa CCM F-05584 Tetracladium marchalianum CCM F-26399 Anguillospora furtiva Bärlocher Tetracladium maxilliforme CCM F-14286 Anguillospora furtiva L 16, Austria (Rostr.) Ingold Anguillospora longissima (Sacc. & CCM F-00980 Tetracladium setigerum (Grove) CCM F-20987 Syd.) Ingold Ingold Anguillospora longissima CCM F-11791 Tetracladium apiense CCM F-23199 Anguillospora longissima CCM F-11891 Tetracladium furcatum E. CCM F-11883 Descals Anguillospora longissima CCM F-42894 Tricladium angulatum Ingold CCM F-139 Anguillospora longissima CCM F-10691 Tricladium angulatum CCM F-01380 Aspergillus fumigatus ATCC 36607 Tricladium angulatum CCM F-14186 Aspergillus nidulans CBS 100.20 Tricladium angulatum CCM F-10200 Chrysosporium parvum CBS 237.77 Tricladium angulatum CCM F-10300 Helicodendron giganteum CBS 280.51 Tricladium splendens Ingold CCM F-12386 Helicomyces roseus CBS 102.76 Tricladium splendens CCM F-19087 Helicosporium phragmites CBS 286.54 Tricladium splendens CCM F-11989 Heliscus lugdunensis Sacc. & Therry CCM F-245 Tricladium splendens CCM F-16599 Heliscus lugdunensis CCM F-13783 Varicosporium elodeae W. Kegel CCM F-04276 Heliscus lugdunensis CCM F-05185 Varicosporium elodeae CCM F-00583 Heliscus lugdunensis CCM F-10699 Varicosporium elodeae CCM F-11783 Heliscus lugdunensis CCM F-12486 Varicosporium elodeae CCM F-29887 Heliscus lugdunensis L 5, Austria Varicosporium elodeae CCM F-17099 Heliscus lugdunensis Elbe 98 Verticillium lecanii CBS 126.27 Heliscus lugdunensis Bärlocher BACTERIA SOURCE Lemonniera terrestris Tubaki CCM F-125 Aquabacterium commune DSMZ11901 Lemonniera terrestris CCM F-11486 ARCHEA SOURCE Lemonniera aquatica DeWild CCM F-04480 Methanosarcina barkeri DSMZ 800 Leptosphaeria bicolor ATCC42652 ALGA SOURCE Neobulgaria premnophila CBS 154.31 Scenedesmus quadricauda UTEX 76 Oidiodendron tenuissimum CBS 238.31 Paecilomyces variotii CBS 265.35

8 Material and Methods

2. Media, culture conditions, maintenance of cultures and harvest procedure 2.1. Culture media Media were made up with 1 l distilled water unless otherwise indicated. For solid media, 15-20g l-1 agar (Difco, Augsburg, Germany) was added. The media were sterilised by autoclaving at 121°C for 20 min; pH was adjusted with HCl or NaOH before autoclaving. For suppression of bacterial growth, in some cases Chloramphenicol (0.1 mg / ml Ethanol, Sigma-Aldrich Schnelldorf, Germany) was added before autoclaving, or 16 ml Penicillin-Streptomycin-solution (16 mg PenicilliumG, 10 mg Streptomycin / ml 0.9% NaCl; Sigma-Aldrich) was added after autoclaving. If not stated otherwise, formulation of media corresponds to the descriptions given in Gams et al. 1998.

M20 Malt extract agar MEA Malt extract agar 2.5% powdered malt extract (Merck, 20g powdered malt extract 25g Darmstadt, Germany) pH 5.0-5.5 peptone 1g glucose 20g OA Oatmeal agar pH 5.6 oatmeal flakes (Kölln, Cologne, 20g Germany) were wrapped in cloth, brought YSSA Yeast soluble starch agar in pan and simmered for 2h; yeast extract (Serva, Heidelberg, Germany) 2g then squeezed and filtered soluble starch 10g through cloth dissolve all ingredients without agar, add tap water 1l agar, adjust to 1l, pH 6.0 pH 6.0 CMA cornmeal agar SNA Synthetic nutrient-poor agar Cornmeal extract (Difco, Detroit, Michigan, USA) 17g

KH2PO4 1g

KNO3 1g MEB Malt extract broth

MgSO4 x 7H2O 0.5g powdered malt extract 15g KCL 0.5g pH 5.0-5.5 saccharose 0.2g glucose 0.2g MEBmod modified malt extract broth pH 5.5 malt extract 15g peptone 0.5g WA Water agar casamino acids 0.5g Elbe river water or 1l Na-pyruvate 0.3g Oberer Seebach water or 1l Tween 80 (Sigma-Aldrich) 1ml tap water 1l pH 7.0,

9 Material and Methods

2.2. Culture conditions All fungal strains were grown on M20, MEA, OA and in MEB at 16-18 °C without agitation under 12 h daylight exposure, or incubated at 20 °C on a shaker, respectively. Aquabacterium commune was grown on modified R2A medium (0.5 g yeast extract, 0.5 g Difco proteose peptone no. 3, 0.5 g casamino acids, 0.5 g glucose, 0.1 % (vol / vol) Tween 80, 0.3 g sodium pyruvate, 0.3 g K2HPO4,

0.05 g MgSO4 · 7H2O, 1 l ddH2O, pH 7.2; Kalmbach 1998). To study conidiogenesis, pieces of pure fungal colonies were cut out and submerged in standing sterile distilled water incubated at 16 °C or transferred to SNA, WA, YSSA and / or CMA. To induce sporulation, a sporulation apparatus was employed similar to the one described by Iqbal & Webster 1973. This apparatus consisted of a sterile falcon tube instead of a parallel-sided Perspex box used by Iqbal and Webster. The falcon tube was filled with autoclaved distilled water or tap water and aerated through a small hole (1mm diameter) with a hypodermic needle (0.70 x 30 mm, Braun, Melsungen, Germany) employing a peristaltic aquarium pump (Tetratec, AP80, Tetra Werke, Melle, Germany). For fructification cultures were inoculated on autoclaved lupine stems or decayed Acer pseudoplatanus leaves, which were incubated for several weeks on M20 at 16°C. Defined mixed cultures were established by cutting out small circles (0.5-1 cm diameter) from pure cultures using a sterile scalpel. The circles from different pure cultures were transferred to one M20 agar plate or submerged in one flask containing MEB. For inoculation of different fungi on leaves, small amounts of pure cultures were transferred to one leaf using a sterile needle. Defined mixed cultures were prepared of Tetracladium marchalianum 26199, Anguillospora crassa 13483, Heliscus lugdunensis 12486 and Varicosporium elodeae 29887 (Mix I) and Tetracladium marchalianum 26199, Alatospora acuminata 02383 and Anguillospora longissima 00980 (Mix II) and grown on M20, MEB and on autoclaved Acer pseudoplatanus leaves. A defined mixed culture of Tetracladium marchalianum (CBS 979.87) and Aquabacterium commune was prepared by submerging pure culture circles of the fungus and adding 2 ml of a pure culture bacterial suspension (approximately 108 cells / ml) into a flask containing MEBmod and incubated at 20°C with agitation.

2.3. Maintenance of cultures Fungal cultures were maintained in M20 agar slants sealed with parafilm (American National Can, Neenah, USA) at 4 °C with periodic transfer every 3 months.

2.4. Harvest procedure 2.4.1. Harvest of cultures Pure cultures and Mix I and Mix II were harvested after 1, 2 and 8 weeks. Mycelium was scraped from the agar surface using a sterilised spatula. Submerged mycelia grown in MEB were harvested by filtering through a sieve (mesh size 2mm). The mixed culture of Tetracladium marchalianum and Aquabacterium commune was harvested after 3 days by microfiltration through cellulose nitrate membranes (45 mm diameter, 0.2 µm pore size, Schleicher & Schuell, Dassel, Germany) by using a vacuum filtration unit (Stericup TM, Millipore, Eschborn, Germany). Following harvest, cultures were washed twice with sterile phosphate buffered saline (PBS: 0.05 M Na2PO4, 0.15 M NaCl, pH 7.2) by centrifugation at 8000 rpm for 5 min to eliminate medium residues. After removing the PBS the

10 Material and Methods cultures were either fixed as described under 10.1., or stored at –20 °C until DNA-extraction without further treatment.

2.4.2. Harvest of conidia Conidia were harvested from the sporulation apparatus (2.2.) with a sterile Pasteur pipette 2-5 days after air bubbles had formed foam at the falcon tube wall. Following harvesting, conidia were either fixed as described in 10.1., or used immediately as inoculum or for subsequent germination experiments (5.).

2.4.3. Harvest of hyphal tips Hyphal tips (10 – 100 µm length) were dissected using a sterile scalpel directly from growing pure cultures employing phase contrast microscopy at a magnification of 400 X.

3. River sampling sites Sampling and exposition experiments were carried out in two different river systems in Germany and Austria.

3.1. Elbe river, Germany The river Elbe is one of the large river systems in middle Europe. At two sampling sites close to Magdeburg (Germany), bank vegetation is characterised by Alder, Poplar and Willow trees as well as meadows. The physical and chemical parameters of Elbe river water were analysed at the Landesamt für Umweltschutz Sachsen-Anhalt and are summarised in Table 2. Sampling was done every three months through 1999, 2000 and 2001 at two different sampling sites, left Elbe bank km 318 (ferry and station of the Landesamt für Umweltschutz Sachsen-Anhalt) and right downstream Elbe bank at km 322. The sampling sites were characterised by a saprobic index of 2.2 at km 318 and 2.12 at km 322.

11 Material and Methods

Table 2: Physical and chemical parameters of river Elbe water at two sampling sites obtained from the Landesamt für Umweltschutz Sachsen-Anhalt. (mean values ± standard deviation of 12 month of the year 2000) Parameter (unit) km 318 km 322 Temperature (°C) 12.0 ± 6.8 12.8 ± 7.0 Q discharge (m3 s-1) 510 410 -1 O2 (mg l ) 10.7 ± 1.4 11.1 ± 1.4 Oxygen saturation index (%) 99 ± 15.9 105 ± 21.2 CODa (mg l-1) 22.5 ± 7.8 23.5 ± 9.1 pH 8.2 ± 0.4 8.3 ± 0.6 -1 NH4-N (mg l ) 0.17 ± 0.2 0.13 ± 0.2 1 NO3-N (mg l- ) 4.2 ± 0.9 3.8 ± 1.0 -1 NO2-N (mg l 0.03 0.02 TINb mg l-1) 4.41 ± 1.1 3.97 ± 1.1 -1 total PO4 (mg l ) 0.74 ± 0.3 0.73 ± 0.2 2- –1 SO4 (mg l ) 160 ± 27.5 113 ± 10.4 Na+ (mg l–1) 101 ± 37.1 50 ± 10.8 Ca 2+ (mg l-1) 130 ± 35.3 77 ± 8.8 Mg2+ (mg l-1) 18.9 ± 3.3 13 ± 1.1 K+ (mg l-1) 8.4 ± 1.8 6.3 ± 0.8 Cl- (mg l-1) 218 ± 84.3 94 ± 21.9 Conductivity (µS cm-1) 1367 ± 400.6 798 ± 155.5 Hardness (°dH) 6 ± 0.8 5.2 ± 0.7 DOCc (mg l-1) 4.8 ± 0.8 4.9 ± 0.9 TOCd (mg l-1)l 7.8 ± 2.8 8.4 ± 2.4 As (µg l-1) 2.6 ± 0.6 3.1 ± 0.8 Cu (µg l-1) 4.9 ± 1.1 4.7 ± 1.3 Fe (µg l-1) 404 ± 175.1 452 ± 274.4 3 Chlorophyll tot (mg/m ) 77.6 (25.10.00) 39.7 (25.10.00) EDTA (µg l-1) 11.1 ± 2.9 16.9 ± 5.4 Heavy metals: in the liquid phase hardly in the liquid phase hardly Zn, Cd, Pb, Hg, Ni, Cr, Mn, measurable, but abundant measurable, but abundant in in sediments sediments a COD, chemical oxygen demand b TIN, total inorganic nitrogen c DOC dissolved organic carbon d TOC total organic carbon

3.2. Oberer Seebach, Austria In autumn 2000 and summer 2001, sampling and exposition experiments were carried out within the study area (RITRODAT, 615 m a.s.l., 47°51´N, 15°04´E) of the Oberer Seebach at the Biological Station Lunz, Austria. The Oberer Seebach is a approximately 15 m wide second order limestone gravel stream located roughly 150 km south-west of Vienna on the northern limestone fringe of the eastern Alps. The carstic catchment of 20 km is uninhabited. The riparian vegetation is dominated by Fraxinus excelsior L., Acer pseudoplatanus L., Salix caprea L., Fagus sylvatica and Picea abies. The stream water is chemically well-buffered and without seasonal patterns (Wagner & Bretschko 2002). Characteristic abiotic data are set out in Table 3. Sampling was performed along the entire 100 m stretch of the RITRODAT area. Exposure experiments were carried out in the RITRODAT area at pool sites (approximately 0.6 m deep) with slow current velocity (slow current sites 1R1, 1C1.2, 2C1.1) and at riffle sites with fast current velocity (fast current sites: 15F3.3, 16F3.3, 17G2.2).

12 Material and Methods

Table 3: Physical and chemical parameters of Oberer Seebach water at the RITRODAT study area (obtained from Dr. M. Leichtfried 2001 (pers. comm.))

Long term means Parameter (unit) (1989/1990) ± standard deviation Temperature °C 6.8 pH 8.1 ± 0.1 Q discharge (m3 s-1) 0.72 -1 O2 (mg l ) 11.1 ± 0.2

O2 saturation (%) 100 Conductivity (µS cm-1) 216 ± 6 Cl- (mg l-1) 1.4 Ca2+ (mg l-1) 40.9 ± 1,2 Na+ (mg l-1) 0.69 K+ (mg l-1) 0.39 2- -1 SO4 (mg l ) 5.3 Mg2+ (mg l-1) 0.63 ± 0.49 -1 Ptotal (mg l ) 0.012±0.03 TINa (mg l-1) 1.01±0.238 - -1 NO3 (mg l ) 1.54 DOCb (mg l-1) 1.36 ± 0.08 a TIN, total inorganic nitrogen b DOC dissolved organic carbon

4. Sampling and monoconidial isolation strategies 4.1. Foam Foam collected from the river Elbe and the Oberer Seebach was transferred into sterile screw-capped jars and immediately mixed with a formaldehyde-alcohol-acetic acid (10.1.) solution for fixation. Fungal isolations from foam were performed directly in the field by streaking a loopfull of foam onto object slides with a thin layer of 2% malt extract agar with or without Chloramphenicol (1 mg ml-1). The slides were subsequently incubated at 15 °C for 12-24 h. Single germinating conidia were transferred onto fresh 2% MA.

4.2. Leaves and wood Leaves and twigs collected in the river Elbe and the Oberer Seebach were transferred into dry sterile screw-capped jars or plastic bags and returned to the laboratory under cool conditions. The leaves or twigs were washed under tap water, rinsed with distilled water, and incubated in Petri dishes with distilled water or submerged in sterile 50 ml centrifuge tubes (Labcon, San Rafael, USA) aerated by air flowing through hypodermic needles. The incubated samples were observed daily. Developing conidia were picked using a very fine glass needle and transferred to M20 agar.

13 Material and Methods

4.3. Water and river snow Water samples were taken 0.4 – 0.5 m below the water surface with a bucket on every sampling event and four 250 ml aliquots of each sample were filtered through membrane filters (0.45 µm pore size, 45 mm diameter, NC 45; Schleicher & Schuell, Dassel, Germany). The filters were dried, stained with cotton blue in lactic acid (Merck) and then the entire surface was examined for conidia using phase contrast microscopy. In order to examine the macroscopic aggregates (expected size range: 0.5-5 mm, river snow, Neu 2000) for the occurrence of conidia, 1 l water samples were centrifuged at 8000 rpm for 5 min. Resulting pellets were resuspended in 10 ml filter sterilised river water (0.2 µm cellulose nitrate filters, Schleicher & Schuell, Dassel, Germany). Aliquots of 1 ml were either examined immediately by phase contrast microscopy or fixed for FISH as described under MM 10.1.. Additionally, 1 ml of the resuspended concentrated river snow was plated on M20, MEA, YSSA and WA agar, respectively and incubated at 16 °C. Developing mycelia were transferred to M20 medium.

4.4. Sporocarps To examine potential anamorph – teleomorph connections basidio – and ascocarps were collected from the banks of the river Elbe and the Oberer Seebach. For single spore isolations basidiocarps were exposed over sterile slides. After release of basidiospores, spore prints were diluted in sterile ddH2O and were streaked out on slides covered with M20 agar. Single spores were picked with a very fine needle and transferred to fresh M20 agar. Single ascospores were obtained by squeezing a single ascus until ascospores were released and transferring single spores with a very fine needle to fresh M20 agar. Developing mycelia were either transferred to M20 or OA agar supplemented with autoclaved lupine stems and incubated for several months at 16 °C and 20 °C for fructification or covered with sterile ddH2O and incubated at 16 °C to induce the development of a possible anamorph state. If basidio- or ascospores were not available, hyphal tips of 10 to 100 µm length were directly dissected from the hymenium of basidiocarps and from ascocarps using a sterile scalpel.

4.5. Monoconidial isolations from cultures To obtain pure cultures of monoconidial origin, cultures were examined under a dissecting microscope and single conidia were picked using a very fine glassneedle and transferred to fresh M20 medium. Very small conidia were isolated by flooding the culture with distilled water to get a conidia dilution. The dilution was then streaked out on slides covered with M20 agar and single conidia were picked with a very fine needle and transferred to fresh M20 agar.

4.6. Trapping by surface exposure In order to bait aquatic hyphomycetes, different surfaces were exposed at defined sampling sites for attachment of conidia.

4.6.1. Polyethylene slides Polyethylene (PE-HD) slides were exposed for 2-4 months throughout the years 1999 (26.5.-2.9.1999; 2.9.-5.11.1999; 5.11.1999-29.2.2000), 2000 (1.3.-19.6.2000; 19.6.-1.8.2000; 1.8.-1.11.2000;

14 Material and Methods

1.11.2000-14.3.2001) and 2001 (14.3.-14.6.2001; 14.6.-29.9.2001; 29.9.-14.12.2001) in the river Elbe at the station of the Landesamt für Umweltschutz Sachsen-Anhalt. Within the study area RITRODAT of the Austrian Oberer Seebach, PE slides were exposed for 2 weeks (28.9.-11.10.2000) and for 3 months (28.9.-20.12.2000) in autumn 2000, over winter in 2000/2001 (11.10.2000-15.2.2001) and for 2 weeks from 2.8.-16.8. 2001. Exposition of PE slides within the RITRODAT took place at the sampling sites 1R1 with slow current and 17G2.2 with faster current. Following harvest of four replicates, the PE slides were fixed immediately as described under 10.1. for examination by fluorescent stains (7.) and FISH (10.).

4.6.2. Leaves Freshly fallen leaves of Acer pseudoplatanus, Acer campestre, Fagus sylvatica and Salix caprea were collected from the Oberer Seebach in autumn 2000 (26.9.). In order to exclude contamination prior to exposure, all leaves were sterilised by autoclaving and incubated in Petri dishes. Examination for fungal growth took place after 2 and 5 days employing a dissecting microscope. Exposure of 0.48 and 0.23 g sterile leaf material was performed in sterile Polyamide mesh bags (0.25 mm mesh size) at the RITRODAT sampling sites 2C1.1 (slow current) and 16F3.3 (fast current). The leaf packs were harvested after 2 weeks and either fixed immediately (10.1.) or incubated in moist chambers for fungal isolations as described under 4.2..

4.6.3. Cellulose Autoclaved pieces of native cellulose (4.5 X 4.5 cm, Filtropa, Maastricht, Netherlands) were exposed at the RITRODAT sampling sites 2C1.1 (slow current) and 16F3.3 (fast current) on 29.9.2000. Harvest was performed after 10 days and the cellulose packs were either fixed immediately or incubated in moist chambers for further examination.

5. Germination experiments In order to examine the germination of conidia and spores under natural conditions, conidia, basidiospores, ascospores and hyphal tips were exposed in cages (Fig. 1) within the RITRODAT area of the Oberer Seebach in Austria in autumn 2000. Conidia were obtained by the use of a sporulation apparatus as described above. Basidiospores, ascospores and hyphal tips were collected as described for single spore isolations. Conidia, spores and hyphal tips were counted under 400 X and 1000 X magnification employing phase contrast microscopy.

15 Material and Methods

5.1. Exposure of conidia and spores in cages A number of 10 to 1000 spores ml-1 and conidia were put in a cage (Fig. 1) between two Cellulose acetate membranes (Millipore GmbH, Eschborn, Germany) for exposure. In order to avoid loss of conidia or spores, the pore dimensions of the cage membranes were adjusted to the spore size. Cage membranes for conidia had a pore size of 5 µm, whereas the pore size of cage membranes for basidiospores, ascospores and hyphal tips ranged from 1.2 – 3.5 µm. The cage membranes were clamped between two hose connections which were screwed together and bond to a stiff metal wire for fixation at the sampling site. The cages were exposed at slow and fast current sites for 2, 5, and 14 days in 5, 10 and 25 cm water depth as indicated in Table 4. The fungal species used for the germinating experiments are also indicated in Table 4. Following harvest, the cage membranes were fixed as described under 10.1.1.2.. Germinating conidia and spores were counted directly on the cage membranes. Hyphal tips were measured directly on the membranes and branching, new septa and elongation > 100% were counted as growth.

Metal wire for fixation at sampling site

Hose connections screwed together

Conidia between two cellulose acetate membranes

2 Cellulose acetate membranes

Figure 1: Schematic view of a cage used for the exposure of conidia, spores and hyphal tips in running waters

16 Material and Methods

Table 4: Species, number of conidia and spores exposed, and exposition time and site (slow and fast current, see 3.2.) in the RITRODAT area of the Oberer Seebach in autumn 2000 Conidia or spores Exposition- time Species Exposition-site ml-1 (days) Heliscus 680 2C1.1 slow 2 lugdunensis Heliscus 680 15F3.3 fast 2 lugdunensis Varicosporium 320 2C1.1 slow 2 elodeae Varicosporium 320 15F3.3 fast 2 elodeae Tetracladium 1000 2C1.1 slow 2 marchalianum Tetracladium 1000 15F3.3 fast 2 marchalianum Nectria cinnabarina 1000 2C1.1 slow 2 Nectria cinnabarina 1000 15F3.3 fast 2 Anguillospora crassa 10 2C1.1 slow 2 Anguillospora crassa 10 15F3.3 fast 2 Heliscus 680 2C1.1 slow 5 lugdunensis Heliscus 680 15F3.3 fast 5 lugdunensis Varicosporium 400 2C1.1 slow 5 elodeae Varicosporium 400 15F3.3 fast 5 elodeae Tetracladium 1000 2C1.1 slow 5 marchalianum Tetracladium 1000 15F3.3 fast 5 marchalianum Nectria cinnabarina 1000 2C1.1 slow 5 Nectria cinnabarina 1000 15F3.3 fast 5 Anguillospora crassa 10 2C1.1 slow 5 Anguillospora crassa 10 15F3.3 fast 5 Melastiza scotica 100 2C1.1 slow 5 Melastiza scotica 100 15F3.3 fast 5 Climacocystes hyphal tips 15F3.3 fast 14 borealis Climacocystes hyphal tips 1C1.2.slow 14 borealis Undetermined hyphal tips 15F3.3 fast 14 aphyllophorales Undetermined hyphal tips 1C1.2 slow 14 aphyllophorales Baeospora 300 15F3.3 fast 14 myriadophylla Baeospora 300 1C1.2. slow 14 myriadophylla Gymnopilus sp. 50 15F3.3 fast 14 Gymnopilus sp. 50 1C1.2. slow 14 Bjerkandera adusta hyphal tips 15F3.3 fast 14 Bjerkandera adusta hyphal tips 1C1.2. slow 14 Entoloma hirtipes 1000 15F3.3 fast 14 Entoloma hirtipes 1000 1C1.2. slow 14 Xylaria longipes 1000 15F3.3 fast 14 Xylaria longipes 1000 1C1.2. slow 14

17 Material and Methods

6. Phenotypic characterisation 6.1. Morphological determination Fixed foam samples (4.1) were examined for conidia at magnifications of 400 X, 630 X and 1000 X. Wet (autoclaved tap water) mounts of fungal isolates were examined under phase contrast microscopy. Conidia were measured at magnifications of 400 X, 630 X and 1000 X. Conidial development was observed on M20 agar slides by phase contrast microscopy at a magnification of 400 X. Drawings were performed by the use of a Zeichenokular P8x (Zeiss, Jena, Germany).

6.2. Fluorescent stains For visualisation of fungal samples different fluorescent stains were evaluated and applied. Excitation and emission wavelengths of the used fluorescent stains are indicated in Table 5.

6.2.1. Calcofluor white Fixed PE slides and leaves, cellulose and cage membranes were stained with 0,5 mM solution of Calcofluor White M2R (4,4'-bis[4-anilino-6-bis(2-ethyl)amino-s-triazin-2-ylamino]-2,2'-disulfonic acid, Molecular Probes Europe, Leiden, The Netherlands). The targets of Calcofluor White M2R are Chitin, cellulose and carboxylated polysaccharides. Following incubation in the dark for 5 min the PE slides, leaves cellulose and cage membranes were observed with epifluorescence microscopy or confocal laser scanning microscopy (CLSM).

6.2.2. Live-Dead stain Life-dead staining was performed using the Live/Dead Yeast Viability kit (Molecular Probes), employing epifluorescence microscopy. The FUN 1 component of the kit is supposed to stain Cylindrical Intra Vacuolar Structures (CIVS) which are formed in metabolically active fungal cells 30 to 60 min after the treatment. In FUN 1 treated cells, CIVS have a distinct orange-red fluorescence when excited by light of a wavelength from 470 nm to 590 nm. Samples of Penicillium chrysogenum (CBS197.46), Tetracladium marchalianum, Heliscus lugdunensis and Varicosporium elodeae, grown on M20 medium, were harvested after four weeks. 50 % of the harvested cultures were killed by autoclaving. Aliquots of living and dead fungal material was smeared on different glass slides and treated with 1 ml of the FUN 1 component of the Live/Dead Yeast Viability kit in 10mM Na-HEPES buffer. Incubation took place for 30 min up to 60 min at 30 °C in the dark, followed by immediate mounting with the antifading reagent Citifluor AF2 (Citifluor Ltd., London, UK).

6.2.3. SYTO stains Seven different SYTO stains (Molecular Probes Europe, Leiden, The Netherlands) were evaluated for nuclear, mitochondrial or cytoplasmic staining of Penicillium chrysogenum (CBS197.46), Tetracladium marchalianum, Heliscus lugdunensis and Varicosporium elodeae. The fungi were grown on M20 medium. Following harvest by scraping the mycelia from the agar surface, medium residues were removed by centrifugation at 8000 rpm for 5 min. Living and dead fungal material was smeared onto

18 Material and Methods glass slides and incubated with SYTO dyes 9, 11, 13, 21, 24, 64 and 84 according to the manufacturers’ recommendations. The slides were examined with epifluorescence and CLSM.

6.2.4. Lectin stain Fixed (10.1.) fungal material of Penicillium chrysogenum (CBS197.46), Tetracladium marchalianum, Heliscus lugdunensis and Varicosporium elodeae was stained with wheat germ agglutinin (WGA) commercially labelled with Alexa 350 (formerly called AMCA-S, Molecular Probes Europe, Leiden, The Netherlands). WGA is a lectin obtained from Triticum vulgaris with a known sugar specificity to NN’-diacetylchitobiose and NN’N‘‘-triacetylchitobiose. The WGA Alexa conjugate was dissolved in PBS (pH 7.4) to a final concentration of 1mg/ml and stored at –20°C. Dilutions of 20, 30 and 50 µg/ml were used to Figure out the optimal lectin concentration for staining. The optimal working concentration was determined by epifluorescence microscopy and CLSM and defined as strong fluorescence signal of the fungal material without unspecific background staining. Prior to microscopical examination, the fungal samples with lectin solution were incubated for 10 to 30 min in the dark. Alternatively, the lectin solutions were added to fungal material which was suspended in 10 mM potassium phosphate (Kpi) buffer (pH 7.8), (Brul et al. 1997) with incubation for 30 min at 37 °C in the dark before microscopical examination.

Table 5: Specificity, excitation (ex) and emission (em) wavelengths of fluorescent stains used for fungal stains

Fluorescent stain Specificity Ex (nm) Em (nm) Chitin, cellulose, Calcofluor White M2R carboxylated 365 435 polysaccharides and ? FUN 1 in living cells CIVS 570-590 SYTO 11 Nucleic acids 508 527 SYTO 13 Nucleic acids 488 509 SYTO 21 Nucleic acids 494 517 SYTO 24 Nucleic acids 490 515 SYTO 64 Nucleic acids 599 619 SYTO 84 Nucleic acids 567 582 NN’-diacetylchitobiose and WGA-Alexa350 346 442 NN’N‘‘-triacetylchitobiose

19 Material and Methods

7. Genotypic characterisation 7.1. Extraction of genomic DNA Genomic DNA was isolated from fungal species grown on M20 plates or in MEB by using the FastDNASPIN kit for soil in conjunction with the FastPrep FP120 instrument (Qbiogene, Heidelberg, Germany) according to the manufacturer’s instructions. For some fungal isolates of which the FastPrep instrument method failed, a modified protocol for DNA extraction according to Gardes et al. 1991 with suggestions from Dr. Elke Lieckfeldt was used: Frozen fungal material (1-2 g) was mixed with 300 µl CTAB buffer (100 mM Tris pH 9, 1.4 M NaCl, 20 mM EDTA, 2 % CTAB (N-Cetyl-N,N,N-trimethyl-ammonium bromide, Merck), 0.2 % freshly added mercaptoethanol) in an 1 ml centrifuge tube. The fungal material was then softened by freezing and thawing three times and ground with a sterile spatula. Following incubation at 65°C for 1h, an equal volume of chloroform (Merck) was added. After vortexing, the tube was centrifuged for 15 min at 13000 rpm at room temperature. The supernatant was transferred to a new tube and DNA was precipitated with 600µl ice-cold isopropanol for 1h at –20 °C. Following centrifugation the resulting pellet was washed twice with 70% ethanol and resuspended in 100 µl autoclaved distilled water.

7.2. Amplification of the 18S rRNA genes 18S rRNA sequences were amplified with the primers NS1 (5´ GTAGTCATATGCTTGTCTC 3´) and NS8 (5´ TCCGCAGGTTCACCTACGGA 3´) published by White et al. 1990. PCR mixtures contained PCR buffer (10 mM Tris/HCl, 50 mM KCl, pH 8), 200 µM of each desoxynucleotide, 1.5 mM magnesium chloride, 20 pM of each primer, 50-200 ng of genomic DNA and 2.5 U of Taq polymerase (Boehringer, Mannheim, Germany). The PCR was performed in a personal cycler (Primus 25 HE, MWG-Biotech, Ebersberg, Germany) with an initial denaturation step for 2 min at 95 °C, followed by 35 cycles consisting of 30 s of denaturation at 94 °C, 1 min 30 s annealing at 55 °C and 1 min 45 s extension at 72 °C. The extension was followed by a final extension for 10 min at 72 °C. The PCR products were purified with the QIAquick purification kit (Qiagen, Hilden, Germany).

7.3. Amplification of the ITS1, 5.8S rRNA and ITS2 genes The internal transcribed spacer regions of the rDNA gene cluster consisting of ITS1, the 5.8S rDNA gene and ITS2, was amplified with the primers SR6R (5´ AAGWAAAAGTCGTAACAAGG 3´, Vilgalys, http://www.botany.duke.edu/fungi/mycolab/primers.html) which consisted of a conservative sequence within the small subunit (18S rDNA gene) and LR1 (5´ GGTTGGTTTCTTTTC 3´, Vilgalys et al. 1990) in the large subunit (28S rDNA gene). PCR mixtures contained the same concentrations of PCR buffer, desoxynucleotides, magnesium chloride, primers and Taq polymerase as given above, but 20- 100 ng genomic DNA. PCR was performed with an initial denaturation step for 2 min at 94 °C, followed by 40 cycles consisting of 1 min denaturation at 94 °C, 45 s annealing at 46 °C and extension at 72 °C for 2 min. The final extension was performed for 5 min at 72 °C. Alternatively, the primers ITS1F (5´ CTTGGTCATTTAGAGGAAGTAA 3´, Gardes et al. 1993) and ITS4 (5 ´TCCTCCGCTTATTGATATGC 3´, White et al. 1990) were used changing the annealing

20 Material and Methods temperature to 50 °C. The PCR products were purified with the QIAquick purification kit (Qiagen, Hilden, Germany).

7.4. Amplification of the 28S rRNA genes The first two thirds of the 28S rDNA gene were amplified with the primers 5.8SR (5 ´TCGATGAAGAACGCAGCG 3´, Vilgalys et al. 1990) and LR5 5´ TCCTGAGGGAAACTTCG 3´, Vilgalys et al. 1990). The PCR mixtures contained the same concentrations of ingredients as described for the amplification of the 18S rRNA sequences. PCR was performed with an initial denaturation step for 2 min at 95 °C, followed by 35 cycles consisting of 30 s at 92 °C, 1 min 30 s annealing at 56 °C an extension at 72 °°C for 1 min 45 s. The final extension was performed for 10 min at 72 °C. The PCR products were purified with the QIAquick purification kit (Qiagen, Hilden, Germany).

7.5. Ribosomal DNA sequencing Automatically sequencing of the purified ribosomal DNA PCR products was done with the BigDye Terminator Ready Reaction kit (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany) according to the manufacturer’s protocol. The 10 µl cycle sequencing reaction mixture contained 50-250 ng DNA, 4µl BigDye and 1 pM primer. Primers used for the sequencing of the different regions of the rDNA gene cluster are summarised in Table 6. DNA sequencing was performed in a personal cycler (Primus V1.01, MWG-Biotech). The templates were subjected to an initial denaturing step of 1 min at 96 °C followed by 2 min at 94 °C. The thermal profile consisted of 35 cycles of 30 s at 94 °C, 1 min 30 s at 55 °C and 2 min at 72 °C. Final extension was performed at 72 °C for 9 min. The samples were held at 4 °C until 10 µl were mixed with 10 µl of precipitation buffer (150mM sodium acetate, 25mM sodium EDTA, 0.1 mg/ml dextran blue) and 80 µl ethanol (96%). Precipitation was reached by centrifugation at 15 °C for 30 min. The DNA pellets were washed with 100 µl ethanol (70%), centrifuged again and dried at room temperature. Sequences were generated with an ABI373-sequencer (Perkin-Elmer Applied Biosystems, Foster City, USA) and analysed with the sequence analysis software version 3.3. at the Sequenzierservice Dr. Martin Meixner (Berlin, Germany). The resulting sequences were assembled, corrected and exported as consensus text with Auto Assembler version 2.1 (Perkin-Elmer).

21 Material and Methods

Table 6: Primers used for sequencing of the SSU (small subunit 18S), LSU (large subunit 28S) and ITS regions of the rDNA gene cluster

Name Sequence (5´-3´) Target region a Reference NS1 GTAGTCATATGCTTGTCTC SSU 20-38 White et al. 1990 NS2 GGCTGCTGGCACCAGACTTGC SSU 573-553 NS3 GCAAGTCTGGTGCCAGCAGCC SSU 553-573 NS4 CTTCCGTCAATTCCTTTAAG SSU 1150-1131 NS5 AACTTAAAGGAATTGACGGAAG SSU 1128-1150 NS6 GCATCACAGACCTGTTATTGCCTC SSU 1436-1412 NS7 GAGGCAATAACAGGTCTGTGATGC SSU 1413-1436 NS8 TCCGCAGGTTCACCTACGGA SSU 1788-1769 nu-SSU-402-5´ CCGGAGAAGGAGCCTGAGAAAC SSU 381-402 Gargas et al. 1996 nu-SSU-819-5´ GAATAATAGAATAGGACG SSU 802-819 nu-SSU-852-3´ CGTCCCTATTAATCATTACG SSU 871-852 nu-SSU-305-3´ TCGAAAGTTGATAGGGCAG SSU 323-305 ITS1F CTTGGTCATTTAGAGGAAGTAA SSU Gardes et al. 1993 SR6R AAGWAAAAGTCGTAACAAGG SSU 1747-1766 Vilgalys www 5.8SR TCGATGAAGAACGCAGCG 5.8S 34-51 Vilgalys et al. 1990 5.8S CGCTGCGTTCTTCATCG 5.8S 51-35 Vilgalys et al. 1990 ITS4 TCCTCCGCTTATTGATATGC LSU 60-41 White et al. 1990 LR1 GGTTGGTTTCTTTTCCT LSU 73-57 Vilgalys et al. 1990 LROR ACCCGCTGAACTTAAGC LSU 26-42 LR21 ACTTCAAGCGTTTCCCTTT LSU 424-393 LR2R AAGAACTTTGAAAAGAG LSU 374-389 LR3 CCGTGTTTCAAGACGGG LSU 651-635 LR5 TCCTGAGGGAAACTTCG LSU 964-948 aSaccharomyces cerevisiae numbering

22 Material and Methods

7.5.1. Nucleotide sequence accession numbers The sequences of the different regions of the rRNA gene of 38 aquatic hyphomycete strains were submitted to GenBank. The accession numbers are indicated in Table 7.

Table 7: Accession numbers obtained from GenBank for fungal strains sequenced in this study GenBank accession numbers Species Strain SSU ITS LSU Anguillospora crassa CCMF-05584 AY204574 AY204579 - Anguillospora crassa CCMF-15283 AY204575 AY204581 - Anguillospora crassa CCMF-15583 AY204577 AY204582 - Anguillospora crassa CCMF-13483 AY204576 - - Anguillospora crassa CCMF-07082 AY204578 AY204580 - Anguillospora longissima CCMF-00980 AY204598 AY204592 AY204597 Anguillospora longissima CCMF-10691 - AY204593 - Anguillospora longissima CCMF-11791 - AY204594 - Anguillospora longissima CCMF-11891 AY204599 AY204595 - Anguillospora longissima L22 AY204600 AY204596 - Anguillospora furtiva L16 AY204601 - - Alatospora acuminata CCMF-02383 AY204583 AY204587 - Alatospora acuminata CCMF-13089 AY204584 AY204589 - Alatospora acuminata CCMF-37194 AY204585 AY204590 - Alatospora acuminata CCMF-12186 - AY204588 - Alatospora acuminata L8 AY204586 AY204591 - Heliscus lugdunensis L5 AY204602 - - Heliscus lugdunensis CCMF-245 AY204603 - - Heliscus lugdunensis ELBE98 AY204604 - - Lemonniera aquatica CCMF-04480 AY204605 - - Lemonniera terrestris CCMF-11486 AY204606 - - Lemonniera terrestris CCMF-125 AY204607 - - Tetracladium marchalianum CCMF-19399 AY204613 AY204620 - Tetracladium marchalianum CCMF-26199 AY204614 AY204621 AY204612 Tetracladium marchalianum CCMF-26299 AY204615 AY204622 - Tetracladium marchalianum CCMF-26399 AY204616 AY204623 - Tetracladium marchalianum ELBE90 AY204617 - - Tetracladium marchalianum ELBE50 AY204618 AY204624 - Tetracladium marchalianum L27 AY204619 AY204625 - Tricladium angulatum CCMF-01380 AY204573 AY204608 - Tricladium angulatum CCMF-139 AY204627 AY204610 - Tricladium angulatum CCMF-14186 AY204628 AY204611 - Tricladium angulatum CCMF-10200 AY204626 AY204609 - Tricladium splendens CCMF-11989 AY204629 AY204633 - Tricladium splendens CCMF-12386 AY204630 AY204634 - Tricladium splendens CCMF-16599 AY204631 AY204635 - Tricladium splendens CCMF-19087 AY204632 AY204636 -

23 Material and Methods

7.6. Alignment of rDNA sequences All sequences were aligned with various sequences using the Fast Aligner V1.03. of the ARB software package involving the secondary structures of helices, loops and bulges. All alignments were examined and manually optimised according to primary and secondary structure similarities.

8. Modification of ARB software settings ARB (from arbor, Latin: tree) is a package of software tools for building, handling and using databases of sequences and associated information. ARB incorporates an alignment feature (Fast Aligner V1.03.) and tools for phylogeny reconstruction employing neighbour joining, parsimony and maximum likelihood algorithms. Features for searching of optimal trees and confidence tests are included in the software package. ARB was developed by Strunk et al. (1999) at the Department of Microbiology at the Technical University of Munich (free download at: http://www.biol.chemie.tu-muenchen.de/pub/ARB/). Additionally to the software package, a database of rDNA sequences can be obtained from the above mentioned web link. The original sequence database consists mainly of bacterial 16S rDNA sequences and to a much lesser extent of fungal data. For the work with fungal sequences the database was extended with fungal rDNA sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov/) and the alignment settings were adapted to the work with fungi as described below.

8.1. Change of the reference organism and adjusting the 18S and the 28S rRNA secondary structure The ARB Fast Aligner does incorporate secondary structure information of the rRNA molecule by evaluating base pairing at secondary structure positions. In ARB, secondary structure information is controlled by the sequence administrated information (SAI) (“SAI:HELIX” and “SAI: HELIX_NR”) laid down in the reference organism within the ARB EDITOR (version 4). In order to adapt the alignment features of ARB to the work with fungal sequences, the bacterial reference organism Escherichia coli was exchanged for baker’s yeast Saccharomyces cerevisiae, widely used in mycological research. Therefore the 18S rDNA sequence of S. cerevisiae (GenBank accession number: JO1353) was copied to SAI and saved as new reference organism. Helix information was manually adjusted by following the secondary structure model for the nuclear SSU rRNA of S. cerevisiae (http://bioc- www.uia.ac.be/u/yvdp; Wuyts et al. 2000). The same procedure was performed for alignments of 28S rDNA sequences employing the data and the secondary structure model of the LSU rRNA of S. cerevisiae (accession number: JO1355) published by Ben Ali et al. 1999. Because of the high variability of ITS1 and ITS2 sequences, no secondary structure model was adjusted for the alignment of these sequences.

24 Material and Methods

8.2. Enlargement of the ARB database with fungal sequences In order to enlarge the data set of fungal sequences, 18S rDNA, 28S rDNA and ITS1, 5.8S rDNA, ITS2 sequences were downloaded from GenBank, imported in ARB and aligned before integration into the database. Starting in September 1999, the original extant data set of the ARB program was enlarged by including sequences of so far missing taxa of Zygomycota, Ascomycota, Basidiomycota and anamorphic genera. During 2000 and 2001, new fungal sequences from GenBank were monthly imported into the ARB database.

9. Reconstruction of phylogenetic trees All sequences were used as queries in BLAST Search (Altschul et al. 1997) with normal stringencies. The top hits scoring sequences from the BLAST searches were included into phylogenetic analysis. Ambiguously aligned characters were excluded prior to phylogenetic analysis.

9.1. Neighbour Joining 56 to 68 sequences of selected orders (Table 8) were included into the phylogenetic analysis on the basis of a dissimilarity matrix for phylogenetic placement of aquatic hyphomycetes within Eumycota. Basidiomycetes were chosen as outgroup. Distance matrices were calculated from the aligned sequences and corrected for superimposed substitutions at single positions employing the algorithm of Jukes & Cantor (1969). On the basis of the corrected matrices, phylogenetic trees were reconstructed by the neighbour joining method of Saitou & Nei (1987). Branch support was tested by 1000 replications based on bootstrapped data sets.

9.2. Maximum Parsimony Heuristic searches of most parsimonious trees (Swofford et al.1996) were performed with ARB_PARS. Gaps were treated as missing data and only parsimony informative sites were used employing different filter sets prepared manually within the ARB editor. Resulting most parsimonious tree topologies were optimised by nearest neighbour interchange and globally KL. Trees were rooted to Basidiomycetes and branch lengths were calculated using the corresponding tool of the ARB program. Robustness of branch lengths was evaluated by 1000 bootstrap replicates. Sequences included into the analysis are indicated in Table 8.

9.3. Maximum Likelihood Maximum likelihood inference (Felsenstein 1981) was performed with various taxa (Table 8) employing the program fastDNAml (Olsen 1994) implemented in ARB. The ratio of transition to transversion type substitutions was set to 2.0. Searches were performed with random sequence addition and 100 replicates, use of empirical base frequencies and equal rates for variable sites. Branch support was tested with 1000 replications on bootstrapped data sets.

9.4. Bayesian analysis Bayesian inference of phylogeny is a statistical method, based on Markov chain Monte Carlo (MCMC) sampling procedures (Huelsenbeck 2001). The MCMC method samples trees in proportion to their

25 Material and Methods probability of occurrence, evaluated from the aligned DNA sequences, under a certain model of gene- sequence evolution. The Markov chain samples from the joint probability density of trees, branch lengths and substitution parameters. The proportion of the time any single tree is found during the sampling process is an approximation of the posterior probability of the tree. Hereby the proposed trees with the highest likelihood (according to the used phylogenetic model) are accepted leading to a convergence of the Markov chain indicated by a stable Ln L. In the present study the software program MrBayes (Huelsenbeck & Hall, http://morphbank.ebc.uu.se/mrbayes/) was used for Bayesian inference of phylogeny. 18S rDNA sequences of 56 taxa (Table 8) were included into the analysis. Non-informative sites within the alignment were excluded resulting in 294 phylogenetically informative positions used. The general- time reversible model of gene-sequence evolution combined with gamma rate heterogeneity was used to estimate the likelihood of each tree (Hillis et al. 1996). Additionally MrBayes program parameter were set to estimate base frequencies and save branch lengths. 2 million trees were generated using the MCMC procedure, sampling every hundredths tree. Out of 20000 resulting trees the first 139 trees were removed in order to avoid including trees before convergence of the Markov chain. The remaining trees of the plateau were used to evaluate a consensus tree employing PAUP 4.0b2 (Swofford 1998).

Table 8: List of sequences obtained from GenBank included in phylogenetic analyses (The classification is according to Eriksson 2002):

GenBank accession numbers Class / Orders Species SSU ITS LSU Eurotiales Monascus purpureus M83260 AF458472 AF222496 Eurotiales Paecilomyces variotii Y13996 AF033395 AF222501 Onygenales Onygena equina U45442 - - Onygenales ? Geomyces pannorum var. pann. AB015785 AF307760 AB040703 Onygenales ? Pseudogymnoascus roseus AB015778 AF062819 AB040690 Onygenales ? Myxotrichum deflexum AB015777 AF062814 AB040689 Onygenales ? Oidiodendron tenuissimum AB015787 AF062808 AB040706 Mycosphaerella mycopappi U43449 - U43480 Mycosphaerella berberidis - AF362062 - Dothideomycetes et Cucurbitara elongata U42482 - - Chaetothyriomycetes Hortaea werneckii Y18693 AJ238468 - incertae sedis Aureobasidium pullulans M55639 AF423114 AF0502395 Phoma like coelomycete - AJ310558 - Guignardia philoprina - AB041243 - Pleosporales Herpotrichia juniperi U42483 - - Pleosporales Pleospora betae U43466 - - Pleosporales Pleospora papaveraceae - AF102888 - Pleosporales Pleospora herbarum - - AF382386

26 Material and Methods

Continuing Table 8 SSU ITS LSU Pleosporales Ophiobolus herpotrichus U43453 - U43471 Pleosporales Leptosphaeria contecta - AF181702 - Pleosporales Leptosphaeria doliolum U43457 U04207 U43475 Pleosporales Kirschsteiniothelia aethiops AY016344 - AY016361 Pleosporales Kirschsteiniothelia elaterascus AF053728 - - Pleosporales Kirschsteiniothelia maritima AF053756 - - Pleosporales Massarina bipolaris AF164365 AF383956 - Pleosporales Massarina papulosa AF383961 Pleosporales Massarina ramunculicola AF383962 Pleosporales Massarina armatispora AF383955 Pleosporales Massarina corticola AF383957 Pleosporales Massarina fronisubmersa AF383960 Pleosporales Massarina rubi AF383964 Pleosporales Massarina walkeri AF383965 Pleosporales Massarina eburnea AF383959 Pleosporales Massarina australiensis AF164364 - - Pleosporales Lophiostoma caulium AF383953 Pleosporales Lophiostoma vagabundum AF383954 Pleosporales Curvularia eragrostidis - AF163077 AF163938 Rhytismatales Spathularia flavida Z30239 AF433155 AF433144 Rhytismatales Cudonia confusa Z30240 - - Rhytismatales Cudonia lutea - AF433151 AF433139 Rhytismatales Rhytisma acerinum - - AF356696 Helotiales ericoid mycorrhizal sp. Sm5 - AY046400 - Helotiales ericoid mycorrhizal sp. Sd9 AF269067.1 Helotiales Ectomycorrhizal ARONR1165 AJ430411 Helotiales Helotiales sp. ARON3063.S AJ292198.1 Helotiales Axenic ectomycorrhizal isolate AJ430405 Helotiales Cistella grevillei - U57089 - Helotiales Fabrella tsugae AF106015 U92304 AF356694 Helotiales Hymenoscyphus fructigenus U57430 AJ0396 - Helotiales Hymenoscyphus virgultorum Z81382 - Z81410 Helotiales Hymenoscyphus sp. GU30 - AF252836 - Helotiales Hymenoscyphus ericae 6 - AF252851 - Helotiales Hymenoscyphus ericae 7 - AF252835 - Helotiales Mollisia cinera - AJ430222 - Helotiales Mollisia minutella - AJ430223 - Helotiales Lachnum bicolor AJ430394 Helotiales Loramyces juncicola AF203464 - - Helotiales Phacidium infestans - U92305 - Helotiales ? Zalerion varium - AF169303 - Leotiales Leotia lubrica L37536 - - Leotiales Leotia viscosa - - AF113737

27 Material and Methods

Continuing Table 8 SSU ITS LSU Leotiales Bulgaria inquinans AJ224263 - - Leotiales ? Chalara constricta CBS 731.92 AF222454 Leotiales ? Chalara microchona CBS 867.73 AF222467 Leotiales ? Chalara longipes CBS 875.85 ÂF222465 Leotiales ? Salal root associated fungusUBC - - AF300724 Leotiales Leotiales sp. Bjelland 61 - AY011014 - Erysiphales Blumeria graminis L26253 AB000935 AB022362 Erysiphales Phyllactinia guttata AF021796 AF011315 - Cyttariales Cyttaria darwinii U53369 - - Microascales Microascus cirrosus M89994 - AF275539 Microascales Petriella setifera U43908 AF043596 AF027664 Microascales Graphium penicillioides AB007654 AB038431 AF222500 Halosphaeriales Halosarpheia retorquens AF050486 - - Halosphaeriales Halosarpheia viscosa - AF422979 AF396874 Hypocreales Hypomyces chrysospermus M89993 AF054844 AF160233 Hypocreales Hypocrea lutea D14407 AF275339 U00739 Hypocreales Giberella pulicaris AF081467 U85540 U85523 Hypocreales Nectria cinnabarina AB003949 AF163025 L36625 Hypocreales Geosmithia putterillii D88318 AF033384 D88326 Sordariales Neurospora crassa X04971 - M38154 Sordariales Sordaria firmicola X69851 - AF1323305 Sordariales Chaetomium elatum M83257 - - Sordariales Chaetomium globosum - AF423116 AF286403 Sordariales Kinochaeta ivoriensis AB003787 - - Sordariomycetes Phialophora sp. Elec-N-14.PN AJ278753 - Tetracladium marchalianum - AF411022 - Tetracladium marchalianum - AF411023 - Tetracladium marchalianum AF411024 - Tetracladium maxilliforme AF388577 AF411027 - - Tetracladium apiense AF388575 AF411025 - - Tetracladium furcatum AF388578 AF411026 - - Dactylaria dimorphospora U51980 Orbiliales Monacrosporium doedycoides AJ001994 U51969 - Orbiliales Arthrobotrys superba AJ001989 - - Orbiliales Orbilia delicatula U72603 - - Orbiliales Orbilia fimicola AF006307 - - Orbiliales Arthrobotrys oligospora AJ001987 - - Saccharomycetales Saccharomyces cerevisiae JO1353 Saccharomycetales ? Antarctic yeast CBS 893.1 AY040649 Basidiomycetes Bulleromyces albus X60179 - AF075500 Basidiomycetes Hydnum repandum AF026641 AF347095 - Basidiomycetes Peniophora nuda AF026586 - -

28 Material and Methods

10. Fluorescence in situ hybridisation 10.1. Fixation 10.1.1. Paraformaldehyde fixation 10.1.1.1. Cultures, conidia, hyphal tips Cultures, conidia and hyphal tips were harvested as described under 2.4. and fixed with freshly prepared 3.7% paraformaldehyde solution in PBS for 1-4 h at 4 °C in the dark. The paraformaldehyde- fixed thalli were washed twice with PBS and stored at -20 °C in a storage buffer consisting of a 1:1 (vol/vol) mixture of PBS and 96% ethanol.

10.1.1.2. Biofilms Immediately after recovery, PE slides, leaves, cellulose and cage membranes were submerged in 3.7% paraformaldehyde solution and incubated for 1h at 4°C. The biofilms were washed twice with PBS, air dried and stored at room temperature.

10.1.1.3. River snow 1 ml aliquots of concentrated river snow samples (4.3.) were centrifuged at 8000 rpm for 5 min. The pellets were resuspended in 1 ml 3.7% paraformaldehyde solution and incubated at 4 °C. The pellets were washed twice with PBS, resuspended in a 1:1 mixture of PBS to ethanol and stored at –20 °C.

10.1.1.4. Foam Foam was sampled as described under 4.1., and immediately mixed with an amount of approximately one tenth of the sample volume of a 18:1:1 solution of ethanol (70%) paraformaldehyde (37%) and concentrated acetic acid (glacial, 100 %). 1 ml aliquots were taken from the bottom of the jar and fixed as described for river snow.

10.1.2. Ethanol fixation Cultures sampled as described under 2.4. were fixed with a 1:3 PBS ethanol (96 %) solution, incubated at 4 °C for 1 to 4 h and stored at –20 °C.

10.1.3. Methanol fixation Cultures were mixed with a 3:1 methanol acetic acid mixture and incubated for 1 to 4 h. The methanol fixed thalli were washed twice with PBS and stored at -20 °C in a storage buffer consisting of a 1:1 (vol/vol) mixture of PBS and 96% ethanol.

10.2. Permeabilisation 10.2.1. Permeabilisation by chitinase treatment Fixed fungal cultures, conidia, hyphal tips and river snow samples were washed twice with autoclaved distilled water, and placed in small pieces or as aliquots (15 µl) on Teflon coated glass slides (Marienfeld, Bad Mergentheim, Germany) and air dried. The samples were subsequently covered with 15 µl permeabilisation buffer (1 x PBS, 1% SDS, pH 5.5) and 15 µl chitinase solution (Sigma Aldrich)

29 Material and Methods at a final concentration of 1 mg per ml. The slides were incubated at 20 °C for either 2, 4, 6, 10, 15 or 20 min, rinsed gently with distilled water and air dried. Biofilms were overlaid with permeabilisation buffer / chitinase solution as described above, gently rinsed with distilled water after incubation as described above and air dried.

10.2.2. Permeabilisation by electroporation.

For permeabilisation by electroporation, fixed cultures were washed with double distilled H2O by centrifugation at 8000 rpm for 5 min. The cleaned fungal samples were subsequently mixed in a sterile

1 ml vial with 250 ng oligonucleotide probe in 50 µl double distilled H2O. Fungal sample and oligonucleotide probe solution were transferred to a cuvette (Bio-Rad, Hercules, CA, USA) with 0,2 cm gap. For electroporation a Gene Pulser Pulse controller (Bio-Rad) was used. The electroporation procedure was run at 20 ± 3 °C, voltage 1,5 kV, capacity 25 µF, resistance 200 Ω (Pulse controller). Electrical pulsing took place for 1,34 - 4,18 ms. Following electroporation, the water was replaced by 50µl hybridisation buffer and hybridisation took place as described below.

10.3. Design and evaluation of specific oligonucleotide probes The construction of a specific hybridisation probe requires the localisation of a distinct rRNA sequence (“signature”) that is unique to certain organisms on a defined phylogenetic level. For this purpose, potential signatures for fungal in situ probes on different phylogenetic levels were evaluated using the Probe_Design tool of the ARB software package. Employing the Probe_Match tool of the ARB software, newly designed fungal FISH probes were compared to the available data set of 1700 complete and partial fungal 18S rRNA sequences, and 700 complete and partial 28S rRNA sequences, respectively. The specificity check was supplemented by data analysis of additional prokaryotic, archaeal and eukaryotic ribosomal gene sequences - within the ARB database and by searches in GenBank - that may coincidentally have the identical nucleotide sequences. In order to evaluate the phylogenetical resolution power, different fungal target sequences on division, genus, species and strain level were investigated. To complement the computer aided probe design, the specificity of the newly developed probes was verified under in situ conditions by the reproducible and discriminative staining of target and non-target species. Non-target organisms comprised different fungal species and single representatives of the Bacteria, Archaea and other Eucarya. The newly designed fungal probes FUN1429 and MY1574 intended for the in situ detection of a wide range of Eumycota, were tested by performing in situ hybridisations of pure and mixed cultures of freshwater fungi with the probes EUK516 (Amann et al. 1995, Eucarya probe) EUB338 (Amann et al. 1995, Bacteria probe) and ARCH915 (Stahl & Amann 1991, Archaea probe). The probe EUB338, which has a sequence complementary to a highly conserved region of the 16S rRNA in the domain Bacteria, served as a negative control for non-specific binding for all hybridisations of target organisms with the fungal probes FUN1429 and MY1574. The universal probe EUK516, which has a sequence complementary to most Eucarya was used as a positive control. To test non-specific binding of the

30 Material and Methods probes FUN1429 and MY1574 with non-target organisms, the domain specific probes EUK516, EUB338 and ARCH915 were used as positive controls.

For the construction of specific oligonucleotide probes on genus, species or strain level for Tetracladium, Alatospora, Anguillospora, Heliscus and Tricladium, the database was scanned for signatures i. e. other fungi should be discriminated by at least one mismatch in the target region. Additionally the target region of the probe should be located in a region of the 18S or the 28S rRNA molecule suitable for in situ hybridisations. Potential candidates for analytical in situ probes were compared with the ARB database by using the Probe_Match tool of the ARB software to search for organisms with complete homologies within the target sequences. In situ discrimination testing was performed against Aquabacterium commune and fungi displaying one or two mismatches within the target region, if available. The probes FUN1429, MY1574 and EUK516 were used as positive control. The probe EUB338 was used as positive control for Aquabacterium commune. All hybridisation probes were labelled at the 5’ terminus with a fluorescent marker, either with Oregon Green 488, with Alexa Fluor 350, or with the indocarbocyanine dyes Cy3 or Cy5. For simultaneous application of probes FUN1429, MY1454 and EUK516, EUB338 and ARCH915, alternating labelling with Oregon Green and Cy3 was employed. All probes were synthesised and labelled by Metabion (Planegg, Germany) and stored in ddH2O at –20 °C.

10.4. Hybridisation procedure Working solutions of probes had a concentration of 50 ng DNA per µl. The hybridisation buffer containing 0.9 M NaCl, 20 mM Tris/HCl (pH 7.2), 0.03% SDS, and 20, 35, 40 or 50 % formamide and the fluorescent probe were gently mixed in a ratio of 10:1 (vol/vol) to get a final oligonucleotide concentration of 5 ng per µl. Each sample was immobilised on the slide and was covered with 10 µl of probe containing hybridisation buffer. For hybridisation, slides were placed in sampling tubes and incubated at 46 °C in the dark for 1.5 to 16 h. Following hybridisation the slides were washed with prewarmed washing buffer (20 mM Tris/HCl, 0.01% SDS, and 250, 88, 62.4 or 31.4 mM NaCl, corresponding to 20, 35, 40 and 50 % formamide stringency) for 20 minutes at 46 °C, rinsed with ddH2O, air dried and mounted with the antifading reagent Citifluor AF 2 (Citifluor Ltd., London, UK).

10.4.1. Cultures, conidia, hyphal tips A small amount of fixed cultures, conidia or hyphal tips was placed on each cavity of a Teflon-coated microscopic slide (Marienfeld, Bad Mergentheim, Germany) and dried at 46 °C. The slides were subjected to treatment with increasing concentrations of ethanol (50, 80 and 96 %, 3 min each), 10 µl of probe containing hybridisation solution was placed on each cavity, incubated, washed and mounted with Citifluor.

31 Material and Methods

10.4.2. Biofilms In order to decrease inherent autofluorescence signals, leaves and cellulose were subjected to treatment with 100 mM Tris-HCl buffer (pH=8) with 50 mM sodiumborohydrid (NaBH4) for 30 min. Dry fixed PE slides, parts of leaves, cellulose and cage membranes were subjected to ethanol treatment by submerging into the ethanol series as described for cultures. The PE slides were partitioned into 2 sections with strong adhesive tape, enabling hybridisations with different probes on one slide. 50 µl of hybridisation solution was placed on each partition and incubated as described above. Leaves, cellulose and cage membranes were mounted on glass slides with 20 µl of hybridisation solution, overlaid with 50µl of hybridisation solution and incubated as described above. Leaves, cellulose and membranes were washed by gently submerging in a glass Petri dish containing prewarmed (46 °C) washing buffer.

10.4.3. River snow and foam 15 µl of fixed river snow or foam was placed on each cavity of Teflon-coated slides. Following drying at 46 °C, dehydration, incubation and washing took place as described for cultures.

10.5. Determination of hybridisation stringencies Hybridisations with Cy3- or Oregon Green-labelled probes were performed at the recommended stringency; 20-40 % for EUB338, EUK516 and 20 % formamide for probe ARCH915. Hybridisation stringencies for the newly designed division, genus, species and strain specific probes were adjusted by the stepwise addition of formamide in hybridisations against selected reference strains displaying one or two or more mismatches within the target region. All fungal probes were labelled with CY3 for the determination of hybridisation stringencies. Probe FUN1429 was tested against the fungus Aspergillus nidulans, the alga Scenedesmus quadricauda and the bacteria Aquabacterium commune and Methanosarcina barkeri. Probe MY1524 was tested against Verticillium lecanii, Syncephalastrum racemosum and Aquabacterium commune and Methanosarcina barkeri. Genus and species specific probes for Tetracladium (marchalianum) were tested against Epicoccum nigrum, Tricladium angulatum strain CCM F-10200 and Tetracladium setigerum, T. furcatum, T. apiense and T. maxilliforme. Probes for Tricladium splendens were tested against Neobulgaria premnophila, whereas probes for Tricladium angulatum were tested against Tricladium splendens. Probes for Alatospora acuminata strains were tested against Alternaria alternata and Geomyces pannorum, and the strains were tested against each other. Anguillospora longissima probes were tested against Leptosphaeria bicolor and Pleospora herbarum. Probes for Heliscus lugdunensis were tested against Trichoderma viride. The formamide concentrations necessary to discriminate non-target organisms for fungal probes are given in Table 31 in the results section. For evaluation of unspecific staining and autofluorescent cells, control hybridisations with CY3-labelled probe non-FUN were performed.

32 Material and Methods

11. Microscopical analysis and documentation 11.1. Phase contrast microscopy Phase contrast microscopy was performed with a Zeiss Axiolab (Jena, Germany) equipped with 10 X 0.25 NA, 40 X 0.65 NA, 63 X 1.25 NA and 100 X 1.3 NA objectives. Micrographs were taken with a Carena SRH 1001 camera equipped with Ilford 400 DECTA professional black and white films.

11.2. Epifluorescence microscopy Epifluorescence microscopy was performed with a Zeiss Axioplan II (Oberkochen, Germany) fitted with a 100 W high-pressure bulb and Zeiss light filter set no. 01 for DAPI (excitation 365 nm, dichroic mirror 395 nm, suppression 397 nm), no. 09 for Oregon Green 488 (excitation 450-490 nm, dichroic mirror 510 nm, suppression 520 nm) and HQ light filter 41007 (AF Analysentechnik, Tübingen, Germany) for Cy3-labelled probes (excitation 535-550 nm, dichroic mirror 565 nm, suppression 610- 675 nm).

11.3. Confocal laser scanning microscopy Within 1999 and 2000 confocal laser scanning microscopy (CLSM) was performed with a TCS 4D (Leica, Heidelberg, Germany) attached to an inverted microscope and equipped with an argon-krypton and UV laser. Fungi were observed with 20 x 0.6 NA, 40 x 0.75 NA, 63 x 1.2 NA and 100 x 1.4 NA lenses. The microscope was used in the single or multi channel mode to record the reflection signal, stained and hybridised fungi, environmental samples and autofluorescence of fungi and phototrophic organisms.

The following settings for excitation and detection of the emission signals were used:

Table 9: Excitation [nm] Emission [nm] signal 351-364 435 Calcoflour white 346 442 Alexa Fluor 350 labelled probe 488 520 Oregon Green labelled probe 568 565, 615 CY3 labelled probe 647 670 CY5 labelled probe

For presentation of micrographs the standard software ScanWare version. 5.1A (Leica) was used. The images were printed with Photoshop version 5.5 (Adobe, San Jose, USA) on a UP-D8800 digital colour printer (SONY, Japan).

Within 2001 laser microscopy was performed on a TCS SP/MP (Leica) setup with three visible lasers Ar (458 nm, 476 nm, 488 nm, 514 nm), Kr (568 nm) and He/Ne (633 nm) (Omnichrome, Chino, USA. The SP (spectral photometer) feature allowed adjustment of sliders on the detector side. Images were obtained with 40 x 0.8 NA, 63 x 0.9 NA and 63 x 1.2 NA water immersible lenses (Leica). Image

33 Material and Methods processing and three-dimensional reconstructions were done using the standard software package TCS NT version 1.6.587 (Leica, Heidelberg, Germany) delivered with the instrument and IMARIS version 3.06 (Bitplane, Zürich, Switzerland).

11.3.1. Measurement of signal intensities and autofluorescence scan Microscope TCS 4D: To obtain optimum dynamic range of the 8 bit photomultiplier which provides brightness values from 0 to 255 (= arbitrary units are given in percentages of maximum brightness values), image analysis of hybridised fungi were normalised to the results of epifluorescence observations of each individual fungal species in three steps: (i) whole cell hybridisations of all fungi were used to determine the lowest and highest fluorescence intensities for the specimen (threshold setting for fluorescence intensity) according to the corresponding light source (laser) settings (ii) gain and offset of the photomultiplier were adjusted to use nearly the complete dynamic range. These settings were used for all following fluorescence measurements (iii) the signal to noise ratio was improved by the application of an eightfold sampling of the identical microscopic field and subsequent use of the average pixel fluorescence intensity

Microscope TCS SP/MP: The photomultiplier voltage needed to detect probe conferred signals above autofluorescence level was determined and whole cell hybridisations of all fungal samples were used to determine the lowest and highest fluorescence intensities for all attempts (threshold setting for fluorescence intensity). Intensities were then converted into arbitrary units given as percentages of the highest value. In order to evaluate the inherent autofluorescence signals emitted from the different fungal species, hybridisation fluorescence intensity and autofluorescence intensity were simultaneously measured performing a scan over the visible light spectrum (300 to 600 nm wavelength) using confocal laser scanning microscopy. Fungal samples without FISH assay, but with hybridisation buffer and/or with permeabilisation buffer and sodiumborohydrid were used as negative controls. Statistical analysis (standard deviation (SD), Student’s t-test) and Gaussian curve fits were done with Microsoft® Excel 97 SR-2 for Windows software program.

34 Results A

RESULTS

A Sampling, isolation, cultivation and identification of freshwater fungi

Fungal species were isolated from the German river Elbe and the Austrian Oberer Seebach employing different sampling and isolation strategies. The occurrence of freshwater fungi was studied on different substrata, such as leaves, wood, alder roots, foam samples and by examination of water and river snow samples. Freshwater fungal species were characterised with regard to their qualitative abundance and phylogenetic affiliation. The fungal species and the environmental samples obtained from the river Elbe and the Oberer Seebach were used to design and evaluate group-, genus- and species specific fungal FISH probes.

1. River Elbe Sampling was performed in three months intervals throughout the years 1999, 2000 and 2001 at two different samplings sites located at KM 322 and FERRY at the river Elbe (3.1. Material and Methods). In 1999 four sampling campaigns yielded 100 fungal isolates in pure cultures. Morphological characters allowed identifying 65 cultures. Of the remaining 35 isolates, four isolates were determined by sequencing of the 18S rDNA gene as Plectosporium tabacinum. The morphological determination failed completely for 31 isolates because their mycelia remained sterile. The 69 isolates that were identified comprised 28 different species. Four different species belonged to the group of aquatic hyphomycetes as defined by Webster & Descals 1981. The aquatic hyphomycetes isolated in 1999 were Clavariopsis aquatica (1 isolate), Heliscus lugdunensis (3 isolates), Tetracladium marchalianum (4 isolates) and Tricladium splendens (1 isolate). In 2000 four sampling campaigns conducted at two sampling sites resulted in 80 isolates, which could be obtained in pure cultures. 58 morphologically characterised isolates consisted of 27 different species. Aquatic hyphomycetes were represented by six different species: Anguillospora longissima (1 isolate), Clavariopsis aquatica (2 isolates), Heliscus lugdunensis (3 isolates), Tetracladium marchalianum (7 isolates), Tricladium splendens (1 isolate) and Varicosporium elodeae (1 isolate). In 2001 the fungal isolation was restricted to suspected aquatic hyphomycetes using the conidial shape. Four sampling campaigns resulted in 14 fungal isolates in pure cultures. Eleven isolates could be morphologically determined to species level. Six different aquatic hyphomycete species were found. Three isolates were morphologically determined as Penicillium sp.. The aquatic hyphomycetes isolated within 2001 from the river Elbe were A. longissima (1 isolate), C. aquatica (1 isolate), H. lugdunensis (2 isolates), T. marchalianum (5 isolates), Tricladium angulatum (1 isolate) and Tricladium splendens (1 isolate). A list of all fungal isolations throughout the years 1999, 2000 and 2001 is given in Table 10.

35 Results A

Table 10: Isolation frequencies of fungi isolated from the river Elbe throughout the years 1999, 2000 and 2001, aquatic hyphomycetes are indicated in bold letters.

Species Isolation frequency 1999 2000 2001 Acremonium rutilum 2 2 Acremonium kiliense 0 2 Alternaria alternata 2 3 Anguillospora longissima 0 1 1 Arthrinium phaeospermum 2 0 Chalara sp. 1 2 0 Chalara sp. 2 2 0 Cladosporium cladosporioides 9 5 Clavariopsis aquatica 1 2 1 Emericellopsis sp. 1 0 Fusarium aquaeductum 3 2 Fusarium culmorum 2 2 Fusarium sambucinum 2 1 Fusarium sp. 2 2 Heliscus lugdunensis 3 3 2 Heterobasidiomycet 4 2 Hormonema sp. 2 0 Mucor hiemalis 0 2 Penicillium sp. 5 2 3 Pachnocybe albida 3 0 Pachnocybe ferruginea 0 2 Paecilomyces farinosus 0 1 Paecilomyces lilacinus 0 1 Penicillium chrysogenum 2 2 Penicillium restrictum 2 2 Phoma sp. 2 2 Plectosporium tabacinum 4 3 Ramichloridum sp. 1 0 Septofusidium elegans 1 0 Sesquicillium candelabrum 3 1 Tetracladium marchalianum 4 7 5 Trichoderma viride 1 1 Tricladium angulatum 0 0 1 Tricladium splendens 1 1 1 Verticillium lecanii (group) 0 2 Varicosporium elodeae 0 1 Verticillium nigrescens 1 1 mycelia sterilia 31 22 Total 100 80 14

1.1. Fungal isolates from different substrata During 16 sampling campaigns throughout the years 1999 and 2000, 127 fungi were isolated from leaves, foam, wood, alder roots, river snow and water samples. The distribution of fungi isolated from different substrata is shown in Figure 2. From a total of 127 isolates a fraction of 50.5 % (64) were

36 Results A obtained from leaves. Only three isolates (2.4 %) belonged to the group of aquatic hyphomycetes, represented by Clavariopsis aquatica. Foam samples yielded 48 (37.8 %) of all fungal isolates. Within these samples 16.5 % were aquatic hyphomycetes represented by various species (s. Table 10) except of C. aquatica. In contrast only 8.7 % of all fungal isolates were isolated from wood, 2.4 % from alder roots and 0.7 % from river snow. From river snow only one isolate of Alternaria alternata could be obtained. Using river water no fungi could be isolated. In 2001, all aquatic hyphomycetes with the exception of C. aquatica were obtained from foam samples. As in the years 1999 and 2000 C. aquatica was found on leaves only. Leaf and wood samples allowed to isolate 3 Penicillium sp.. Leaf material collected for fungal isolations comprised of Alnus glutinosa, Populus nigra, Salix alba, Fagus sylvatica, Juncus effusus, Polygonatum multiflorum and various undetermined leaves. Remarkably, all four isolates of C. aquatica were gained from Alnus glutinosa leaves. The majority of the 16 isolates belonging to the genus Fusarium were obtained from leaves. Only two isolates of Fusarium aquaeductum were isolated from foam.

60

50

40 s e t aquatic hyphomycetes a l

o other fungi

l is 30 a t o t f o

% 20

10

0 leaves foam wood alder root river snow river water substrata

Figure 2: Distribution of fungal isolates from different substrata given as percentage of total isolates during the years 1999 and 2000 in the river Elbe.

1.2. Seasonal patterns

Sampling campaigns within the river Elbe were performed every three months ensuring at least one sampling for each season. Throughout the years 1999 and 2000 almost half (49.5 %) of all fungal isolates were obtained in autumn, of which 16.5 % were aquatic hyphomycetes. 21.3 % of all fungal

37 Results A isolates could be isolated from samples taken in spring, of which 2.4 % were aquatic hyphomycetes represented by three isolates of C. aquatica. Almost equal numbers of fungal isolates, but no aquatic hyphomycetes could be obtained in summer (15 %) and winter (14.2 %). In 2001 the majority of aquatic hyphomycetes could be isolated from foam collected in autumn. Only one isolate of C. aquatica could be isolated from an alder leaf collected in summer. The seasonal distribution of fungal isolates is indicated in Figure 3.

60

50

40 aquatic hyphomycetes other fungi 30 total isolates total f o

% 20

10

0 Spring Summer autumn Winter Season

Figure 3: Seasonal distribution of fungal isolates given in percentage of total isolates during the years 1999 and 2000 in the river Elbe.

38 Results A

Decaying leaf material, wood, alder roots, river snow and water samples were available throughout the year. In contrast, foam was only available in spring and autumn. Regarding the isolation frequencies of aquatic hyphomycetes in relation to substrate and seasonal distribution within the years 1999, 2000 and 2001, it is noteworthy that the majority (31 of 35) of isolates could be obtained from foam collected in autumn (Figure 4). C. aquatica could be isolated from alder leaves only which have been collected in spring and summer.

35

30 31

25

20

15 ber of Ingoldian isolates

m 10 nu

5 1 3 0 foam/autumn leaves/spring leaves/summer substrate/season

Figure 4: Isolation frequencies of aquatic hyphomycetes within the years 1999, 2000 and 2001 in relation to substrate and seasonal distribution

1.3. Freshwater fungal conidia abundant in fixed foam samples Foam samples for fixation and subsequent examination by phase contrast microscopy for conidia of aquatic hyphomycetes were available in spring and autumn, only. At the sampling site km322 foam samples could be taken near the river bank adhered to small floating twigs. At the sampling site FERRY, foam samples were taken directly from foam accumulated at the wall of the ferry ship. At nine sampling campaigns foam was available. Six times at the sampling site FERRY. At each sampling event three to six foam samples were collected. From each foam sample 10 ml were examined for conidia. In foam samples taken in spring no conidia of aquatic hyphomycetes could be detected. Only foam samples taken in autumn showed small numbers of conidia. Within 12 foam samples 45 conidia could be identified and could be further grouped as eleven different species of aquatic hyphomycetes.

39 Results A

Most abundant were the conidia of Alatospora acuminata sensu stricto (Marvanová & Descals 1985) with eight conidia found in four foam samples. A significant difference within the abundance of conidia found in foam samples at the sampling sites km322 and FERRY could not be observed. A list of all conidia found in fixed foam samples from the river Elbe is given in Table 11.

Table 11: Conidia of aquatic hyphomycetes observed in fixed foam samples of the river Elbe throughout the years 1999, 2000 and 2001.

Species Number of conidia Alatospora acuminata 8 Flagellospora curvula 6 Tetracladium marchalianum 6 Tricladium angulatum 6 Tricladium splendens 5 Anguillospora longissima 4 Clavariopsis aquatica 2 Diplocladiella scalarioides 2 Heliscus lugdunensis 2 Tetrachaetum elegans 2 Varicosporium elodeae 2

2. Oberer Seebach During three weeks in autumn 2000 (26.9.-15.10.2000) and three weeks in summer 2001 (2.8.- 23.8.2001), sampling and isolation of aquatic hyphomycetes took place in the RITRODAT study area of the Oberer Seebach in Lunz / Austria. In autumn 2000 aquatic hyphomycetes were very abundant on all different substrata. Due to the limited time available during the three week stay, only qualitative analysis could be performed. Aquatic hyphomycetes from the Oberer Seebach were isolated if the same species or genus had been found in the river Elbe as this allows investigating the genetic variability of different strains and for evaluating different strain specific oligonucleotide probes. Occasionally, some additional species were isolated. In summer 2001 large numbers of conidia of aquatic hyphomycetes were found after heavy rain showers. Therefore the same criteria as in autumn 2000 were applied to isolate aquatic hyphomycetes.

2.1. Fungal isolates from different substrata Foam was available from the Oberer Seebach throughout the three weeks in 2000 and for seven days in summer 2001. Decaying leaf material collected from the stream body and banks, and exposed autoclaved leaf material comprised of Fraxinus excelsior, Acer pseudoplatanus, Fagus sylvatica,

40 Results A

Corylus avellana, Tilia chordata, Salix caprea and Picea abies. There was hardly any river snow in the water of the Oberer Seebach.

The small number of three river snow particles in 1l Oberer Seebach water comprised algae (diatoms) and detritus. From water samples various aquatic hyphomycetes could be isolated. No isolations could be made from exposed cellulose packages. Table 12 lists the fungi isolated from the various substrates.

Table 12: Distribution of fungal isolates with reference of isolation from foam or colonised substrata in autumn 2000 (26.9.-15.10.) and in summer 2001 (2.8.-23.8.). Elbe also = isolated from river Elbe, too. F= foam; W= water; Ac= Acer pseudoplatanus; Co= Corylus avellana; Fa= Fagus sylvatica; Fr= Fraxinus excelsior; Sa= Salix caprea; Ti= Tilia chordata.

Isolates 2000 Number Substrate Elbe also Alatospora acuminata 3 F Yes Tetracladium marchalianum 3 F, Fa, W Yes Clavariopsis aquatica 1 F Yes Anguillospora furtiva 1 F Genus Anguillospora longissima 1 F Yes Anguillospora crassa 1 Fa Genus Tetracladium setigerum 1 F No Articulospora tetracladia 1 F No L2 1 Sa No Heliscus lugdunensis 2 W, Ac Yes Tetracladium maxilliforme 1 F No Isolates 2001 Number Substrate Elbe also Flabellospora sp. 2 F, Ac No Tripospermum myrtii 1 W No Calcidospora gravida 1 F No Volucrispora graminea 1 F No Alatospora flagellata 1 F No Pleuropedium tricladioides 1 F No Actinospora megalosporum 1 F No Articulospora tetracladia 1 F No Anguillospora crassa 1 F Genus Anguillospora longissima 1 F Yes Tetracladium setigerum 1 Co No Tetracladium marchalianum 3 F, Fr, Ti Yes Camposporium pellucidum 1 F No Dactylaria aquatica 1 F No Heliscus lugdunensis 1 F Yes

41 Results A

2.2. Freshwater fungal conidia abundant in fixed foam samples Foam samples collected from the Oberer Seebach in autumn 2000 and summer 2001 were fixed immediately. Examination using a phase contrast microscope revealed a dense accumulation of conidia within these samples. Twelve samples (10 ml each) were scanned under the phase microscope and conidia were counted. Tables 13 and 14 show numbers and percentages of conidia in the foam samples from the Oberer Seebach of 2000 and 2001.

A total of 67837 conidia could be counted in 12 foam samples of autumn 2000: On average, 5653 conidia could be observed in each of the 12 samples, comprising 25 different types of conidia. Conidia of Alatospora acuminata (sensu stricto type, Marvanová & Descals 1985) were most abundant (37 % of total number), followed by cf. Flagellospora curvula (19 %), T. marchalianum (17 %), A. acuminata (10 %; resembling the sensu lato type with narrowed branch bases, (Marvanová & Descals 1985) and Anguillospora longissima (10 %). Two conidia (Unknown No. 13 and 8) could not be identified to species level, but were described previously by Marvanová & Gulis 2000 from the softwater stream Ysper, Austria.

With 31845 conidia, less than a half of the number of conidia in autumn 2000 samples could be counted in summer 2001: On average, 2653 conidia could be observed in each foam sample comprising 13 different types of conidia. Here conidia of T. marchalianum were most abundant (39 %) followed by A. acuminata, sensu stricto (33 %) and A. acuminata, sensu lato (14 %). Twelve species observed in the autumn 2000 samples could not be seen in the summer 2001 samples.

42 Results A

Table 13: Mean numbers, standard deviation and percentages of conidia in 10 ml foam samples from the Oberer Seebach taken between 26.9. and 14. 10. in autumn 2000. Lunz fixed foam 2000, species Numbers ± % of total Alatospora acuminata sensu lato 2083 615 36.85 cf. Flagellospora curvula 1066 86 18.87 Tetracladium marchalianum 933 107 16.51 Alatospora acuminata sensu stricto 583 74 10.32 Anguillospora longissima 566 83 10.02 Anguillospora filiformis 92 17 1.62 Articulospora tetracladia 92 17 1.62 Anguillospora furtiva 50 18 0.88 Tetrachaetum elegans 34 11 0.60 Volucrispora graminea 25 12 0.44 Unknown No.13 (Marvanová 2000) 25 13 0.44 Tetracladium setigerum 17 10 0.29 Fusarium sp. 17 10 0.29 Pleuropedium tricladioides 17 11 0.29 Heliscus lugdunensis 17 8 0.29 Clavariopsis aquatica 9 4 0.16 Tricladium splendens 8 4 0.15 Heliscina antennata 8 2 0.13 Triscelophorus monosporus 5 1 0.09 Lemnoniera cornuta 3 1 0.04 Erynia conica 2 1 0.03 Alatospora flagellata 1 1 0.01 Unknown No.8 (Marvanová 2000) 0.3 0.5 0.006 Petrakia sp. 0.2 0.4 0.003 Lemonniera centrosphaera 0.2 0.4 0.003 Total 5653,7 100

Table 14: Mean numbers, standard deviation and percentages of conidia in 10 ml foam samples from the Oberer Seebach taken between 2.8. and 22.8. in summer 2001. Lunz fixed foam 2001, species Numbers ± % of total Tetracladium marchalianum 1025 128 38.62 Alatospora acuminata sensu lato 875 70 32.97 Alatospora acuminata sensu stricto 367 50 13.82 Anguillospora filiformis 108 21 4.08 Anguillospora longissima 92 11 3.45 cf. Flagellospra curvula 71 9 2.67 Clavariopsis aquatica 25 10 0.94 Heliscus lugdunensis 25 8 0.94 Tetracladium setigerum 25 9 0.94 Anguillospora crassa 25 8 0.94 Tripospermum myrtii 8 5 0.31 Gyoerfyella rotula 4 2 0.16 Tetracladium palmatum 4 1 0.16 Total 2654 100.00

43 Results B

B Phylogenetic relationships of freshwater fungi

1. ARB modification

1.1. Databases of fungal sequences The 18S rDNA sequence database implemented in the ARB software package could be extended from 314 fungal sequences (dataset Dec 1998) to over 2000 sequences with more than 1000 nucleotides each. New databases were set up for 28S rDNA sequences and sequences of the internal transcribed spacer (ITS) region. The effort resulted in a new 28S rDNA database that includes 700 partial and complete sequences. The database for the ITS1, 5.8S, ITS2 region consists of 650 complete fungal sequences (May 2002). Changing the reference organism from E. coli to S. cerevisiae was successfully completed. The alignment function taking into account the newly adjusted secondary structure of the 18S and the 28S rRNA molecule could be established as well. The sequences of freshwater fungi obtained in this study were included into the databases to perform phylogenetic analysis and evaluation of possible target signatures for designing specific oligonucleotide probes.

2. Inference of phylogenetic relationships of freshwater fungi based on rDNA data In order to place several strains of the aquatic hyphomycetes Tetracladium marchalianum, Alatospora acuminata, Anguillospora crassa, Anguillospora longissima, Tricladium splendens, Tricladium angulatum, Lemonniera terrestris, Lemonniera aquatica and Heliscus lugdunensis within Ascomycetes orders, three regions of the rRNA gene cluster (18S, ITS spacer regions and partial 28S) were sequenced. The obtained sequences were aligned with respect to the sequences consistent in the respective databases. Phylogenetic analyses were performed with sequences of selected orders as shown in Table 8 (see chapter Material and Methods). Sequences of fungi, which were suspected to be closely related either by representing the same genera or species as the own strains, or by representing genera recorded as suspected teleomorph genera (Shearer, www: freshwater ascomycete database: http://fm5web.life.uiuc.edu:23523/ascomycete/default.html) were additionally included into the analyses. Representatives of the Hemiascomycetes and Basidiomycetes were used as the outgroup. The alignments are given in the appendix.

2.1. Tetracladium marchalianum

Tetracladium marchalianum DEWILD is the type species of the genus Tetracladium. The anamorph genus is relatively well defined (Roldán et al. 1989) with moderately slow growing whitish, yellowish or orange pigmented mycelium. Aerial mycelium is sparse. Conidiogenesis is holoblastic generating stauroform conidia composed of a stalk and globose, finger-like, subulate, acicular and filiform elements. Some elements may be absent in some species or present in double number. Often shapes are varying and intermediate forms can be observed inter- and intraspecifically. Tetracladium marchalianum is a well recognisable taxon frequently observed in foam samples. The conidia consist of an axis with a mostly globose distal cell and two acicular primary branches, one wedge-shaped with

44 Results B a globose cell at the apex. A secondary branch is acicular. The seven strains investigated in the present study did not show any differences within their conidium morphology. Sequencing of the SSU of Tetracladium marchalianum strains (F-26399; F-19399; F-26199, F-26299; ELBE50; ELBE90; L27) resulted in sequences of 1727-1753 nucleotides in length. Using BLAST searches the sequences were most closely related to SSU sequences of Tetracladium marchalianum species consistent in GenBank followed by Bulgaria inqinans (Helotiales) and Oidiodendron tenuissimum (Myxotrichaceae formerly Onygenales) sequences. The SSU sequence of T. marchalianum isolate F-312 was obtained from GenBank (Nikolcheva & Bärlocher 2002) and included into the analyses. Phylogenetic analysis with neighbour joining (NJ, Figure 5), parsimony (Figure 6) and maximum likelihood (ML see Figure A1 in appendix) algorithms consistently placed the seven strains within Tetracladium species of which sequences from GenBank were included into the analysis. No bootstrap support could be observed for the grouping of the Tetracladium species within three different clusters. The NJ analysis resulted in a non supported (51 % bootstrap value) placement of T. marchalianum species close to Loramyces juncicola, which is classified within the order Helotiales. Parsimony analysis was performed with 294 potentially phylogenetically informative positions. Figure 6 shows one of 67 most parsimonious trees of 1848 steps derived from the analysis. Here Tetracladium species are long branch placed as sistergroup to Oidiodendron tenuissimum and Myxotrichum deflexum but without any support by bootstrap analysis. ML and Bayesian (Figure 7) analysis placed Tetracladium species also within the family Myxotrichaceae represented by Oidiodendron tenuissimum and Myxotrichum deflexum. This placement of Tetracladium close to the Myxotrichaceae was strongly supported in Bayesian analysis through a re-arrangement value of 99 %.

45 Results B

Cudonia confusa Rhytismatales, Spathularia flavida 90 Cyttaria darwinii Helotiales & Hymenoscyphus fructigenus Leotiales Bulgaria inquinans Leotia lubrica Pseudogymnoascus roseus 61 Geomyces pannorum var. pannorum Myxotrichaceae Phyllactinia guttata 51 Blumeria graminis Erysiphales Hymenoscyphus virgultorum 55 Fabrella tsugae Helotiales 99 Myxotrichum deflexum 71 Oidiodendron tenuissimum Myxotrichaceae Tetracladium marchalianum 26399 Tetracladium marchalianum 26299 Tetracladium marchalianum ELBE90 Tetracladium marchalianum 26199 Tetracladium marchalianum 19399 Tetracladium furcatum Tetracladium maxilliforme Tetracladium marchalianum 312 Helotiales ? Tetracladium apiense Tetracladium marchalianum ELBE50 98 Tetracladium setigerum 51 Tetracladium marchalianum L27 78 Loramyces juncicola Ophiobolus herpotrichus Leptosphaeria doliolum 95 Cucurbitaria elongata 95 Pleospora betae Kirschsteiniothelia elaterascus Pleosporales Mycosphaerella mycopappi 72 Herpotrichia juniperi 81 Kirschsteiniothelia maritima Massarina bipolaris 92 Massarina australiensis Hypocrea lutea Hypomyces chrysospermus Gibberella pulicaris Hypocreales 99 Nectria cinnabarina 51 Geosmithia putterillii Pseudallescheria boydii Petriella setifera Microascus cirrosus Microascales 99 Halosarpheia retorquens 95 Graphium penicillioides Neurospora crassa 91 Sordaria firmicola 54 Chaetomium elatum Sordariales Kionochaeta ivoriensis Monascus purpureus 99 Paecilomyces variotii Eurotiales Onygena equina Onygenales 62 Kirschsteiniothelia aethiops Aureobasidium pullulans 99 Hortaea werneckii Dothideales & Chaetothyriales 76 Arthrobotrys superba Orbilia fimicola 67 Arthrobotrys oligospora Orbiliales 100 Monacrosporium doedycoides Orbilia delicatula Hydnum repandum Hemiascomycetes 99 Peniophora nuda Bulleromyces albus and Basidiomycetes Saccharomyces cerevisiae (outgroup)

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Figure 5: 18S rDNA sequence based phylogenetic tree derived using neighbour joining, showing the placement of seven Tetracladium marchalianum strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). The scale bar indicates one base change per 100 nucleotide positions. Bootstrap values were calculated from 1000 resampled data sets.

46 Results B

Tetracladium maxilliforme Tetracladium furcatum Tetracladium setigerum Tetracladium apiense Tetracladium marchalianum ELBE50 Tetracladium marchalianum L27 99 Tetracladium marchalianum 312 Tetracladium marchalianum 26299 Tetracladium marchalianum 19399 Tetracladium marchalianum 26399 Tetracladium marchalianum 26199 Tetracladium marchalianum ELBE90 77 Oidiodendron tenuissimum 70 Myxotrichum deflexum 86 Loramyces juncicola 60 Hymenoscyphus virgultorum Phyllactinia guttata 60 Blumeria graminis Cyttaria darwinii Pseudogymnoascus roseus 89 Geomyces pannorum var. pannorum Leotia lubrica Bulgaria inquinans 83 Hymenoscyphus fructigenus Cudonia confusa 93 Spathularia flavida 87 Fabrella tsugae 98 Halosarpheia retorquens 57 Microascus cirrosus Pseudallescheria boydii 99 Petriella setifera 91 Graphium penicillioides 56 Geosmithia putterillii 60 Nectria cinnabarina 57 Gibberella pulicaris Hypocrea lutea 100 Hypomyces chrysospermus 94 Neurospora crassa 94 Sordaria firmicola Chaetomium elatum 79 Kionochaeta ivoriensis 94 Aureobasidium pullulans 70 Hortaea werneckii 89 Kirschsteiniothelia aethiops 86 Ophiobolus herpotrichus 56 Leptosphaeria doliolum Cucurbitaria elongata 94 Pleospora betae 60 Kirschsteiniothelia elaterascus Mycosphaerella mycopappi 99 Herpotrichia juniperi 87 Massarina bipolaris 63 Massarina australiensis 89 Kirschsteiniothelia maritima Monascus purpureus 100 Paecilomyces variotii 99 Onygena equina Arthrobotrys superba Orbilia fimicola Arthrobotrys oligospora 97 Monacrosporium doedycoides Orbilia delicatula 91 Hydnum repandum Peniophora nuda Bulleromyces albus Saccharomyces cerevisiae

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Figure 6: Phylogenetic tree using maximum parsimony, showing the placement of seven Tetracladium marchalianum strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. One of 67 equally parsimonious trees based on 294 parsimony informative data of 18S rDNA sequences. Bootstrap values were derived from 1000 resampled data sets.

47 Results B

100 Monascus purpureus 100 Eurotiales & Paecilomyces variotii Onygenales Onygena equina 93 Mycosphaerella mycopappi Herpotrichia juniperi Dothideales 100 51 Pleospora betae Ophiobolus herpotrichus 73 100 Pleosporales 97 Cucurbitara elongata 84 Leptosphaeria doliolum Hortaea werneckii Chaetothyrialales. Aureobasidium pullulans Dothideales Monacrosporium doedycoides 55 65 100 Arthrobotrys superba Orbiliales 100 100 Orbilia fimicola Arthrobotrys oligospora Orbilia delicatula 100 Sclerotinia sclerotiorum Monilinia fructicola Helotiales 100 Blumeria graminis & Phyllactinia guttata Erysiphales Spathularia flavida Leotia lubrica 71 Alatospora acuminata 37194 100 Leotiales 91 Alatospora acuminata 13089 Alatospora acuminata L8 100 Geomyces pannorum Pseudogymnoascus roseus 98 Myxotrichum deflexum Oidiodendron tenuissimum Myxotrichaceae 94 99 Tetracladium marchalianum 26199 (Onygenales) Tetracladium marchalianum 19399 100 Tetracladium marchalianum E 50 Tetracladium marchalianum L27 Tetracladium marchalianum 26399 Bulgaria inquinans Helotiales Cyttaria darwinii Cyttariales 85 Microascus cirrosus 100 Petriella setifera Microascales 97 100 Halosarpheia retorquens Graphium penicillioides 100 100 Hypomyces chrysospermus 80 Hypocrea lutea 57 Gibberella pulicaris Hypocreales 61 74 Nectria cinnabarina 100 Geosmithia putterillii 100 Neurospora crassa 94 85 Sordaria firmicola 100 Chaetomium elatum Sordariales 56 100 Kinochaeta ivoriensis Ophiostoma ulmi Ophiostomatales. 96 Xylaria carpophila Xylariales Saccharomyces cerevisiae Saccharomycetales. Bulleromyces albus Hydnum repandum Basidiomycetes (outgroup) Peniophora nuda Auricularia auricula

Figure 7: Majority rule consensus tree of 19863 MCMC-sampled trees (average Ln likelihood = - 3397552949) derived from Bayesian analysis on the basis of 294 phylogenetically informative positions of 18S rDNA sequences showing the placement of five strains of T. marchalianum and three strains of Alatospora acuminata within the Ascomycota, names in red indicate DNA sequences newly determined in this study. The number above each branch corresponds to the posterior probability (%) of the node to which it points. Classification of species in orders after Eriksson et al. 2002.

48 Results B

Sequences of the ITS spacer regions including the 5.8S rDNA were obtained for the same T. marchalianum strains as in SSU analyses with the exception of the isolate ELBE90. In all strains examined the 5.8S rDNA sequences were identical and156 bp long. The ITS1 and ITS2 sequences were identical or very similar as shown in the similarity matrix in Table 15. The ITS sequences were 160 or 161 (ITS1) and 200, 201 and 202 bp (ITS2) in length. The isolates F- 19399, L27 from the Oberer Seebach and ELBE50 had identical ITS sequences. BLAST searches showed T. marchalianum strains and T. furcatum, T. maxilliforme and T. apiense followed by Dactylaria dimorphospora and an axenic ectomycorrhizal isolate (unclassified Helotiales) and two Helotiales sp. and two ericoid mycorrhizal species to have most similar sequences. The closest matches from BLAST searches were included into phylogenetic analyses. Because of the unclear placement of T. marchalianum (either close to Helotiales or to anamorphic Onygenales), ITS sequences of representatives of both orders were additionally included into the analyses. Unfortunately ITS sequences of Loramyces juncicola, which occurred to cluster with T. marchalianum in SSU analyses, were not available. The similarity matrix shows that T. furcatum, T. maxilliforme and T. apiense strains with similarity values of 95.6 to 97.4 % were closely related to the T. marchalianum strains. Sequences of T. marchalianum strains and of the axenic ectomycorrhizal isolate showed 93.4 to 94.4 % similarity. Slightly lower similarity values of 92.1 – 93 % were obtained for Dactylaria dimorphospora sequences and T. marchalianum. ITS sequences of the Helotiales sp. and of ectomycorrhizal isolate ARONR1165 showed 90.1 – 90.9 % similarity to T. marchalianum sequences. Ericoid mycorrhizal sp. Sd9 and Sm5 showed 82.5 – 86.9 % similarity to T. marchalianum sequences. Comparing the ITS sequence of Oidiodendron tenuissimum (accession No. AF062808) with the ITS sequences of T. marchalianum strains yielded similarity values of 84.4 to 85.7 %. This is relevant as O. tenuissimum occurred to be related with T. marchalianum strains in SSU parsimony, ML and Bayesian analyses.

49 Results B

Table 15: ITS rDNA (including 5.8S rDNA) sequence similarity matrix for Tetracladium strains, Hymenoscyphus species and Oidiodendron tenuissimum % rRNA sequence similarity

Tmarc Tmarc Tmarc Tmarc Tmarc Tmarc Taxon 19399 26199 26299 26399 ELBE50 L27 T. marchalianum CCM F-19399 T. marchalianum CCM F-26199 98,8 T. marchalianum CCM F-26299 99,4 99,8 T. marchalianum CCM F-26399 99,4 99,4 99,8 T. marchalianum ELBE50 100 98,8 99,4 99,4 T. marchalianum L27 100 98,8 99,4 99,4 100 T. marchalianum CCM F-312 acc. no. AF411023 a 100 98,8 99,4 99,4 100 100 T. furcatum acc. no. AF411026 a 96,6 96,8 97,4 97,4 96,6 96,6 T. maxilliforme acc. no. AF411027 a 96,4 96,4 97 97 96,4 96,4 T. apiense acc. no. AF411025 a 95,8 95,8 96,4 96,4 95,8 95,8 Axenic ectomycorrhizal isolate acc. no. AJ430405b 93,9 94,4 94,4 93,4 94,4 94,4 Dactylaria dimorphospora acc. no. U51980e 92,3 93 93 92,1 93 93 Helotiales sp. ARON3063.S acc. no. AJ292198.1b 90,1 90,9 90,1 90,9 90,9 90,9 Ectomycorrhizal isolate ARONR1165 acc. no. AJ430411 b 90,1 90,9 90,9 90,1 90,9 90,9 Ericoid mycorrhizal sp. Sd9 acc. no. AF269067.1 c 85,7 86,3 86,1 86,9 86,4 86,4 Ericoid mycorrhizal sp. Sm5 acc. no. AY0464c 82,5 83 83,3 83 84 84 Oidiodendron tenuissimum acc. no. AF062808 d 84,4 85,2 85,1 85,7 85,4 85,4 a (Nikolcheva & Bärlocher 2002) b (Vrålstad 2001, 2002) c (Bergero et al. 2000) d (Hambleton et al. 1998) e (Liou & Tzean 1997) Tmarc: Tetracladium marchalianum; CCM: Czech Collection of Microorganisms; acc. no.: GenBank accession number.

The tree shown in Figure 8 reflects the phylogenetic relationships of the seven T. marchalianum strains to their next known relatives, and O. tenuissimum and Helotiales spp. based on ITS rDNA sequences. All of the T. marchalianum strains, sequenced in this study, clustered with T. marchalianum strain F- 312 and showed close relation to T. furcatum, T. maxilliforme and T. apiense. The parsimony tree showed the ericoid mycorrhizal, ARON isolates and Oidiodendron tenuissimum to be less closely related to Tetracladium than the axenic ectomycorrhizal isolate and D. dimorphospora. This was supported by high bootstrap values. The NJ and ML analyses (Fig. A2 and A3 in appendix) resulted in the same tree topology as parsimony.

50 Results B

Tetracladium marchalianum 26199 Tetracladium marchalianum 19399 Tetracladium marchalianum ELBE50 Tetracladium marchalianum 26399 100 Tetracladium marchalianum 312 Tetracladium marchalianum L27 97 Tetracladium marchalianum 26399 93 Tetracladium apiense 91 Tetracladium maxilliforme 94 Tetracladium furcatum axenic ectomycorrhizal isolate 70 Dactylaria dimorphospora Oidiodendron tenuissimum ectomycorrhizal isolate ARONR1165 Helotiales sp. ARON3063.S ericoid mycorrhizal sp. Sd9 ericoid mycorrhizal sp. Sm5

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Figure 8: Phylogenetic tree using maximum parsimony, based on ITS rDNA (including 5.8S rDNA), showing relationships of ten Tetracladium strains and representatives of the Ascomycetes orders Helotiales and Oidiodendron tenuissimum. Names in red indicate DNA sequences newly determined in this study. Single obtained most parsimonious tree (358 steps). Bootstrap values were derived from 1000 resampled data sets.

From T. marchalianum strain F-26199 the first 625 nucleotides of the LSU (28S rDNA) were sequenced. BLAST searches indicated relative low similarity to sequences of an Antarctic yeast CBS 839.1 (GenBank accession number: AY040649, Thomas-Hall et al. 2001), a salal associated root fungus (AF300724, Allen et al. 2000) and several Chalara sp. with phylogenetic affinity to the order Helotiales (Paulin & Harrington 2000). NJ, parsimony and ML phylogeny reconstruction included representatives of several Ascomycetes orders. NJ analysis (Figure A4 in appendix) placed T. marchalianum close to Hymenoscyphus virgultorum without bootstrap support. Parsimony (Figure 9) and ML (Figure A5 in appendix) analyses placed T. marchalianum next to the Antarctic yeast isolate with a bootstrap support of 71 and 86 %, respectively. This clade clustered in both analyses with Chalara spp., Rhytisma acerinum and the salal root associated fungus. The 625 bp of the T. marchalianum sequence were aligned to the same range of the LSU sequences included in all analyses. For parsimony analysis 64 ambiguously aligned positions were excluded. Searches for the most parsimonious trees were performed based on 213 potentially phylogenetically informative data. Figure 9 shows one consensus tree of 105 most parsimonious trees requiring 2110 steps.

51 Results B

99 Sordaria fimicola 100 Chaetomium globosum Neurospora crassa Gibberella pulicaris Nectria cinnabarina 83 Geosmithia putterillii Hypomyces chrysospermus 100 Hypocrea lutea 82 Pseudallescheria boydii Petriella setifera 59 Microascus cirrosus 66 Halosarpheia retorquens Graphium penicillioides 83 Curvularia inaequalis 100 Curvularia lunata 74 Bipolaris spicifera Curvularia eragrostidis Pleospora herbarum var. herbar 50 Ophiobolus herpotrichus Leptosphaeria doliolum 97 Mycosphaerella mycopappi 99 Westerdykella cylindrica 63 Anguillospora longissima 00980 64 Aureobasidium pullulans Chalara longipes Chalara microchona Rhytisma acerinum salal root associated fungus UBC 61 Chalara constricta 71 Tetracladium marchalianum 26199 Antarctic yeast CBS 8931 Hymenoscyphus virgultorum Cudonia lutea Spathularia flavida Geomyces pannorum var. pannorum Pseudogymnoascus roseus Leotia viscosa 82 Oidiodendron tenuissimum Myxotrichum deflexum Blumeria graminis f. sp. bromi Phyllactinia moricola Peziza vesiculosa Bulleromyces albus

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Figure 9: Phylogenetic tree using maximum parsimony, showing the placement of Tetracladium marchalianum 26199 and Anguillospora longissima 00980 within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Shown is one of the three equally parsimonious trees based on 28S rDNA sequences. Bootstrap values were derived from 1000 resampled data sets.

52 Results B

2.2. Alatospora acuminata

Alatospora acuminata INGOLD is the type species of the genus Alatospora. The anamorphic genus is relatively homogenous. It forms slow-growing, glabrous dense colonies with an entire margin and elevated centre. Colonies are typically beige, but blackish sectors are also present in some species, at least in the primocultures (Marvanová 1977; Dyko 1978). Conidiogenous cells are phialidic. Conidia develop by forming one axis and two synchronous lateral branches arising side by side from near its middle. Conidial secession is schizolytic. Marvanová & Descals (1985) distinguished two morphotypes, based on conidial morphology: A. acuminata sensu stricto with unconstricted insertion of conidial branches (Fig. A6, appendix) and A. acuminata sensu lato with conidial branch insertion distinctly constricted (Fig. A7, appendix).

Sequencing of the SSU of four Alatospora acuminata strains (F-37194; F-13089; F-02383 and L8) resulted in sequences of 1763 – 1839 nucleotides in length. With the exception of strain L8, all other strains showed an intron sequence around position 1397 of 81 nucleotides in length. The intron sequences were eliminated before phylogenetic analyses. In BLAST searches all sequences were nearest affiliated to SSU sequences of Bulgaria inquinans (Helotiales). In NJ, parsimony, ML and Bayesian analysis all strains of A. acuminata were placed in one cluster. All four phylogenetic methods resulted in placement of A. acuminata strains close to Leotia lubrica, which is a representative of the family Leotiaceae (Helotiales). Highest bootstrap support for this placement was displayed using the parsimony method (92 %, Figure 10) whereas the lowest support was shown employing Bayesian analysis (71 %, Figure 7). In parsimony and Bayesian analyses 294 phylogenetically informative positions were included into tree inference. The trees inferred with NJ and ML methods are given in the appendix (Figures A8 and A9).

53 Results B

Spathularia flavida 60 Cudonia confusa 86 Loramyces juncicola 56 Hymenoscyphus virgultorum Geomyces pannorum var. pannorum 60 Pseudogymnoascus roseus Bulgaria inquinans Alatospora acuminata 37194 Alatospora acuminata L8 99 Alatospora acuminata 13089 92 Alatospora acuminata 02383 Leotia lubrica 56 Hymenoscyphus fructigenus Phyllactinia guttata 87 Blumeria graminis Myxotrichum deflexum 93 Oidiodendron tenuissimum 95 Cyttaria darwinii 57 Pseudallescheria boydii 94 Petriella setifera 68 Microascus cirrosus Halosarpheia retorquens 57 Graphium penicillioides Gibberella pulicaris 56 Nectria cinnabarina 86 Geosmithia putterillii Hypomyces chrysospermus 99 Hypocrea lutea 94 Sordaria firmicola 98 Neurospora crassa 99 Chaetomium elatum 73 Kionochaeta ivoriensis 86 Fabrella tsugae 94 Hortaea werneckii 83 Aureobasidium pullulans 79 Kirschsteiniothelia aethiops 56 Cucurbitaria elongata Pleospora betae 86 Ophiobolus herpotrichus 94 Leptosphaeria doliolum 60 Kirschsteiniothelia elaterascus Mycosphaerella mycopappi Herpotrichia juniperi 87 Massarina australiensis 63 Massarina bipolaris 91 Kirschsteiniothelia maritima Paecilomyces variotii Monascus purpureus 99 Onygena equina Arthrobotrys superba Orbilia fimicola Arthrobotrys oligospora Monacrosporium doedycoides Orbilia delicatula 91 Hydnum repandum Peniophora nuda Bulleromyces albus Saccharomyces cerevisiae

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Figure 10: Phylogenetic tree using maximum parsimony, showing the placement of four Alatospora acuminata strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. One of 43 equally parsimonious trees is shown, based on 294 parsimony informative data of 18S rDNA sequences. Bootstrap values were derived from 1000 resampled data sets.

54 Results B

Sequences of the ITS spacer regions including the 5.8S rDNA were obtained for the same A. acuminata strains as mentioned above. Additionally, strain F-12186 of the CCM was included in the analysis. In all strains examined the 5.8S rDNA sequences were identical and156 bp long. The ITS1 and ITS2 sequences were of almost equal lengths of 156 to 158 and 199 to 200 bp, respectively. However, they did not have identical sequences, as shown in the similarity matrix in Table 16. BLAST searches were used to find the closest ITS sequences. The closest sequences were found to be the ones from Guignardia philoprina (Dothideomycetes et Chaetothyriomycetes incertae sedis; Botryosphaeriaceae), a Phoma-like Coelomycete, and a Leotiales species isolate Bjelland 61 (unclassified Helotiales). No ITS sequences were available for Leotia lubrica, which was related to the A. acuminata strains in analyses based on 18S rDNA sequence data. The closest matches of the BLAST searches were included in phylogenetic analyses along with Cudonia lutea (Rhytismatales) serving as outgroup taxon.

Table 16: ITS rDNA (including 5.8S rDNA) sequence similarity matrix for Alatospora acuminata strains, their most similar known sequences and Cudonia lutea

% rRNA sequence similarity

Alacumi Alacumi Alacumi Alacumi Alacumi Taxon 02383 L8 37194 12186 13089 A. acuminata CCM F-02383 A. acuminata L8 95.9 A. acuminata CCM F-37194 95.7 98.5 A. acuminata CCM F-12186 95.9 99.2 98.1 A. acuminata CCM F-13089 97.6 96.5 96.3 94.4 Leotiales species Bjelland 61 acc. no: AY011014a 87.1 86.4 85.6 86.7 87.7 Guignardia philoprina acc. no. AB041243b 87.7 87.1 87.7 87.8 85.0 Phoma-like coelomycete 1L35 acc. no. AJ310558c 86.7 86.5 86.9 87.0 87.2 Cudonia lutea acc. no. AF433151d 80.9 80.0 80.2 81.0 80.2 a (Ekman & Bjelland 2000) b (Okane et al. 2001) c (Schroeder 2001) d (Wang et al. 2002) Alacumi: Alatospora acuminata; CCM: Czech Collection of Microorganisms; acc. no.: GenBank accession number.

The ITS rDNA sequence similarity of strains of A. acuminata ranged between 94.4 % between strains F-13089 and F-12186 and 99.2 % between strains F-12186 and L8. The differences in the ITS sequences occurred in both the ITS1 and ITS2 regions. About the same amount of differences were observed in both regions (see Fig. A39, appendix). The ITS sequence similarity of A. acuminata strains and the closest BLAST hits Leotiales sp., G. philoprina, and the Phoma-like Coelomycete ranged between 85.0 % between G. philoprina and strain F-13089 and 87.8 % between G. philoprina and strain F-12186. The similarity of ITS sequences of A. acuminata strains and Cudonia lutea ranged between 80 and 81 %. Figure 11 shows a phylogenetic tree based on ITS rDNA sequences. It indicates a possible relationship of A. acuminata and those fungal species that are displaying similar sequences. For parsimony analysis the data set included 511 characters, from which 56 were parsimony informative.

55 Results B

The estimated phylogenetic tree is one of 6 most parsimonious trees with a length of 87 steps. In the parsimony tree two distinct clusters of A. acuminata strains are visible. One cluster (“sensu lato- cluster”) was formed by strains F-12186, F-37194 and the strain L8 isolated from the Oberer Seebach. The other cluster (“sensu stricto-cluster”) was formed by the two A. acuminata strains F-13089 and F- 02383. This branching order was proven by NJ (Fig. A10, appendix) and ML (Figure 12) analyses with bootstrap supports of 93 and 91 % for the “ sensu lato -cluster”, and lower bootstrap values of 63 and 70 % for the “sensu stricto -cluster”, respectively. All analyses based on ITS sequence data placed the Leotiales sp. Bjelland61 next to the A. acuminata clade with a bootstrap value of 82 % in NJ analysis and 79 % in ML analysis. G. philoprina and the Phoma-like Coelomycete were always placed as a sistergroup of the A. acuminata – Leotiales sp. group.

Phoma - like Coelomycete Guignardia philoprina Cudonia lutea

Leotiales sp.Bjelland 61

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Alacumi 13089 sensu stricto cluster sensu lato Alacumi 12186 Alacumi 02383 cluster Alacumi L8 Alacumi 37194

Figure 11: Phylogenetic tree reconstructed by maximum parsimony analysis, showing the possible relationships of five Alatospora acuminata strains and fungal species with the highest ITS rDNA sequence similarity obtained in BLAST searches. Cudonia lutea was used as outgroup. Names in red indicate DNA sequences newly determined in this study. One of 6 equally parsimonious trees based on 329 bp ITS1, 5.8S, ITS2 rDNA data.

56 Results B

Alatospora acuminata 12186 91 Alatospora acuminata L8 81 Alatospora acuminata 37194 63 Alatospora acuminata 02383 79 Alatospora acuminata 13089 Leotiales sp. Bjelland 61 98 Guignardia philoprina Phoma-like coelomycete 1-L-3-5 Cudonia lutea

0.01 substitutions / site

Figure 12: Phylogenetic tree reconstructed by maximum likelihood analysis, consensus of 16 equally likely trees (Ln likelihood = -2337,37721), based on the same data as Figure 11, but including parsimony uninformative data. Bootstrap values were derived from 1000 resampled data sets. The tree shows the possible relationships of five Alatospora acuminata strains (indicated in red) and fungal species with the most known similar ITS rDNA sequences. Cudonia lutea was used as outgroup.

2.3. Tricladium splendens and Tricladium angulatum

Tricladium splendens INGOLD is the type species of a large and heterogeneous genus. On malt agar, T. splendens grows as a dark grey colony with abundant aerial mycelium and blackish reverse. Conidiophores are simple or sparsely branched. Conidiogenous cells are integrated, proliferation is percurrent. The conidia consist of an axis and typically two lateral branches. The axis is gently curved, branches are dorsal, diverging, with distinctly constricted insertion (Fig. A11, appendix).

The species T. angulatum INGOLD has four morphological characters departing from the generic concept erected from the characters of the type species: Colonies growing on malt agar are hyaline and the conidiogenous cells proliferate sympodially. Additionally the conidial axis is bent at branch insertion and the branch bases join the axis by full width without any constriction (Fig. A11, appendix). The SSU sequences obtained from four strains of Tricladium splendens (F- 11989; F- 12386; F-

19087 and F- 16599), and four strains of T. angulatum INGOLD (F- 01380; F- 139; F- 10200 and F- 14186) ranged from 1716 to 1778 nucleotides in length. Employing BLAST searches, the nearest SSU sequence for all strains was that of Blumeria graminis, which is located in the Ascomycete order Erysiphales. In NJ, parsimony and ML analyses all strains of T. splendens and T. angulatum clustered together, respectively. Phylogenetic analysis with NJ placed T. splendens strains close to Hymenoscyphus fructigenus within the order Helotiales. This was supported by a bootstrap value of 95 % (Figure 13). In contrast to this, the parsimony analysis placement of T. splendens is far less clearly. It is displaying a very long branch next to H. fructigenus, there is no bootstrap support. Within the ML analysis T. splendens strains clustered together with Blumeria graminis and Phyllactinia guttata (Erysiphales). The bootstrap value was only 55 % (Figure 14). For the strains of T. angulatum, only ML analysis showed a placement close to Hymenoscyphus virgultorum within the order Helotiales displaying a bootstrap support of 92 % (Figure 14). In NJ and parsimony analyses, T. angulatum was placed with very long branches either next to Fabrella tsugae (Helotiales) and H. virgultorum (NJ, Figure 13) or as a sister clade beside T. splendens (bootstrap value 56 %) (parsimony, Fig. A12 in appendix).

57 Results B

Tricladium angulatum 10200 Tricladium angulatum139 100 Tricladium angulatum14186 89 Tricladium angulatum 01380 95 Fabrella tsugae 81 Hymenoscyphus virgultorum Blumeria graminis 55 Phyllactinia guttata Myxotrichum deflexum 87 Oidiodendron tenuissimum Cudonia confusa Spathularia flavida Loramyces juncicola Tricladium splendens 11989 100 Tricladium splendens 12386 Tricladium splendens 19087 95 Tricladium splendens 16599 Hymenoscyphus fructigenus Bulgaria inquinans Leotia lubrica Geomyces pannorum var. pannorum 51 Pseudogymnoascus roseus Cyttaria darwinii Aureobasidium pullulans 93 Hortaea werneckii Leptosphaeria doliolum Ophiobolus herpotrichus Cucurbitaria elongata Pleospora betae Kirschsteiniothelia elaterascus Mycosphaerella mycopappi Herpotrichia juniperi 100 Kirschsteiniothelia maritima Massarina bipolaris 82 Massarina australiensis 95 Kirschsteiniothelia aethiops Pseudallescheria boydii Petriella setifera Microascus cirrosus 99 Halosarpheia retorquens 98 Graphium penicillioides Hypocrea lutea Hypomyces chrysospermus Gibberella pulicaris 99 Nectria cinnabarina 100 Geosmithia putterillii Neurospora crassa Sordaria firmicola 100 Chaetomium elatum Kionochaeta ivoriensis Monascus purpureus 99 Paecilomyces variotii 100 Onygena equina Arthrobotrys superba Orbilia fimicola Arthrobotrys oligospora 100 Monacrosporium doedycoides Orbilia delicatula Hydnum repandum 99 Peniophora nuda 64 Bulleromyces albus Saccharomyces cerevisiae

0.01 changes

Figure 13: 18S rDNA sequence-based phylogenetic tree derived using neighbour joining analysis, showing the placement of four Tricladium angulatum strains and four Tricladium splendens strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). The scale bar indicates one base change per 100 nucleotide positions. Bootstrap values were calculated from 1000 resampled data sets.

58 Results B

Tricladium angulatum 01380 Tricladium angulatum 139 100 Tricladium angulatum 14186 Tricladium angulatum 10200 92 Hymenoscyphus virgultorum Loramyces juncicola Myxotrichum deflexum Oidiodendron tenuissimum 64 Cyttaria darwinii Tricladium splendens16599 Tricladium splendens19087 100 Tricladium splendens12386 55 Tricladium splendens11989 Blumeria graminis 71 Phyllactinia guttata Cudonia confusa Spathularia flavida 85 Fabrella tsugae Leotia lubrica Geomyces pannorum var. pannorum Pseudogymnoascus roseus Bulgaria inquinans 86 Hymenoscyphus fructigenus Microascus cirrosus Petriella setifera Halosarpheia retorquens 99 Pseudallescheria boydii Graphium penicillioides Hypocrea lutea Hypomyces chrysospermus Gibberella pulicaris 82 Nectria cinnabarina 100 Geosmithia putterillii Neurospora crassa Sordaria firmicola 100 Chaetomium elatum 77 Kionochaeta ivoriensis Aureobasidium pullulans Hortaea werneckii 95 Kirschsteiniothelia aethiops Leptosphaeria doliolum Ophiobolus herpotrichus Pleospora betae 99 Cucurbitaria elongata Kirschsteiniothelia elaterascus Mycosphaerella mycopappi 90 Herpotrichia juniperi Massarina bipolaris Massarina australiensis 87 Kirschsteiniothelia maritima Monascus purpureus 98 Paecilomyces variotii 100 Onygena equina Orbilia fimicola Arthrobotrys superba Arthrobotrys oligospora 99 Monacrosporium doedycoides Orbilia delicatula Hydnum repandum 86 Peniophora nuda Bulleromyces albus Saccharomyces cerevisiae

0.01 substitutions / site

Figure 14: Single phylogenetic tree obtained by maximum likelihood analysis (Ln likelihood = –13259,8646), based on 18S rDNA sequence data. Bootstrap values were derived from 1000 resampled data sets. The tree shows the possible placement of four Tricladium angulatum strains and four Tricladium splendens strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study.

59 Results B

Sequences of the ITS spacer regions including the 5.8S rDNA were obtained for the same strains of T. angulatum and T. splendens as for the SSU analyses. The ITS sequences of T. angulatum were 505 – 513 bp long. The ITS1 region comprised 153 nucleotides, the 5.8S rDNA 156 bp, and the ITS2 region 193 – 201 nucleotides in all four T. angulatum strains. The 5.8S rDNA of all four strains was identical. The similarity of the ITS sequences of T. angulatum strains ranged between 99.8 and 100 %. The ITS sequences of the strains F-10200, F-139 and F-14186 were identical. In BLAST searches the nearest sequences were the ITS sequences of an ericoid mycorrhizal sp. Sm5, an ericoid endophyte sp. C37, Cistella grevillei (Helotiales) and various Hymenoscyphus spp. (Helotiales). The ITS sequences of the T. splendens strains were 513 – 524 nucleotides in length. The ITS1 region comprised 164 bp, the 5.8S rDNA 156 bp, and the ITS2 region 191 – 201 bp in all four T. splendens strains. The 5.8S rDNA of all four strains was identical. The similarity values of the ITS sequences of T. splendens strains ranged between 99.6 and 100 %, and the sequences were identical for the strains F-16599 and F-19087. In BLAST searches the nearest sequences were the ITS sequences of Zalerion varium, Lachnum bicolor (Helotiales) and various Hymenoscyphus spp. As shown in the similarity matrix in Table 17, the ITS sequence similarity values between T. angulatum and T. splendens ranged between 86.3 % for the T. angulatum strains F-10200, F-139, F-14186 and T. splendens strain F-11989 and 87.7 % for T. angulatum F-01380 and T. splendens F-19087 and F- 16599.

Table 17: ITS rDNA (including 5.8S rDNA) sequence similarity matrix for Tricladium angulatum and Tricladium splendens % rRNA sequence similarity T.angu. T.angu. T.angu. T.angu. T.splen. T.splen. T.splen. Taxon 01380 10200 139 14186 11989 12386 16599 T. angu. F-01380 T. angu. F-10200 99.8 T. angu. F-139 99.8 100 T. angu. F-14186 99.8 100 100 T. splen. F-11989 86.7 86.3 86.3 86.3 T. splen. F-12386 86.9 86.4 86.4 86.4 99.8 T. splen. F-16599 87.5 86.9 86.9 86.9 99.6 99.8 T. splen. F-19087 87.5 86.9 86.9 86.9 99.6 99.8 100 T. angu. = Tricladium angulatum; T. splen. = Tricladium splendens

Because of the relative low similarity values of T. angulatum and T. splendens sequences, phylogenetic analyses of the ITS1, 5.8S, ITS2 sequence data were evaluated for each species separately. In order to reconstruct phylogenetic relationships of T. angulatum on the basis of the spacer regions, the sequences of the closest BLAST hits, various Hymenoscyphus species and Fabrella tsugae, which was placed close to T. angulatum in SSU maximum likelihood analysis, were included into NJ, parsimony and ML analyses. However, the ITS sequences of Fabrella tsugae and the ericoid

60 Results B endophyte sp. C37 could not be unambiguously aligned, and were therefore excluded from the further analyses. Table 18 shows the similarities of ITS sequences of T. angulatum strains and their closest BLAST hits and Hymenoscyphus spp..

Table 18: ITS rDNA (including 5.8S rDNA) sequence similarity matrix for Tricladium angulatum strains, their most similar known sequences and Hymenoscyphus spp. % rRNA sequence similarity Triang Triang Triang Triang Taxon 01380 10200 139 14186 Ericoid mycorrhizal sp. Sm5 acc. no. AY046400 a 91.9 91.7 91.7 91.7 Cistella grevillei acc. no. U57089 b 89.7 89.5 89.5 89.5 Hymenoscyphus ericae 6 acc. no. AF252851 c 87 87.3 87.3 87.3 Hymenoscyphus ericae 7 acc. no. AF252835 c 83.5 83.2 83.2 83.2 Hymenoscyphus sp. GU30 acc. no. AF252836 c 85 84.5 84.5 84.5 Hymenoscyphus fructigenus acc. no. AJ430396 d 84.1 83.8 83.8 83.8 a(Bergero et al. 2001); b(Cantrell & Hanlin 1997); c(Sharples et al. 2000); d(Vralstad 2001); Triang = Tricladium angulatum; acc. no.: GenBank accession number

The highest similarity values of 91.7 to 91.9 % of sequences of T. angulatum strains were found within the sequence of the ericoid mycorrhizal sp. Sm5. It is noteworthy to say, that the ITS sequence similarities of T. angulatum with the ericoid mycorrhizal sp. Sm5 were higher than that of T. angulatum sequences with ITS sequences of T. splendens (Table 17). Sequence similarities with Cistella grevellei and T. angulatum sequences ranged between 89.5 and 89.7 %. The sequence similarities of T. angulatum strains to Hymenoscyphus sp. GU30, two representatives of the Hymenoscyphus ericae complex (Sharples et al. 2000) and H. fructigenus ranged between 83.2 and 87.3 %. In NJ, parsimony and ML analyses the T. angulatum strains were always placed within one cluster. All three tree estimating methods resulted in equal tree topologies, forming two groups consisting of a T. angulatum - Cistella grevillei - ericoid mycorrhizal sp. Sm5 - clade and a Hymenoscyphus species - clade. A statistically highly supported subclade placed T. angulatum together with Cistella grevillei (NJ, Figure 15; parsimony, Fig. A13 and ML, Fig. A14 in appendix).

61 Results B

Tricladium angulatum 10200 Tricladium angulatum 14186 99 Tricladium angulatum 139 96 Tricladium angulatum 01380 98 Cistella grevillei Ericoid mycorrhizal sp. Sm5 99 Hymenoscyphus ericae 7 Hymenoscyphus sp.GU30 Hymenoscyphus ericae 6 Hymenoscyphus fructigenus

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Figure 15: ITS rDNA (including 5.8S rDNA) sequence-based phylogenetic tree derived using neighbour joining, showing relationships of four Tricladium angulatum strains and closest BLAST hits of the Ascomycetes order Helotiales. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). The scale bar indicates one base change per 100 nucleotide positions.

Phylogenetic analyses of the ITS sequences of T. splendens strains were performed with the closest BLAST hits and Hymenoscyphus spp. In some analyses the ITS sequence of Blumeria graminis f. sp. bromi was used as an outgroup, because of its placement as sister clade to T. splendens in ML SSU analyses. Table 19 shows a similarity matrix of ITS sequences of T. splendens and the closest BLAST hits.

Table 19: ITS rDNA (including 5.8S rDNA) sequence similarity matrix for Tricladium splendens strains, their most similar known sequences and Hymenoscyphus spp % rRNA sequence similarity Taxon Trisplen Trisplen Trisplen Trisplen 11989 12386 16599 19087 Zalerion varium ATCC28878 acc. no. AF169303a 94.4 94.4 94.6 94.6 Lachnum bicolor acc. no. AJ430394b 81.6 81.4 82.2 82.2 Hymenoscyphus ericae 6 acc. no. AF252851c 84.4 83.9 83.9 83.9 Hymenoscyphus ericae 7 acc. no. AF252835c 81.5 80.9 81.2 81.2 Hymenoscyphus sp. GU30 acc. no. AF252836c 82.8 82.6 82.8 82.8 Hymenoscyphus fructigenus acc. no. AJ430396b 79.0 78.7 78.7 78.7 a(Bills et al. 1999); b(Vrålstad 2001); c(Sharples et al. 2000); Trisplen = Tricladium splendens; acc. no.: GenBank accession number

The highest sequence similarities of 94. 4 and 94. 6 % were observed between T. splendens strains and the sequence of Zalerion varium. The similarity value is larger than between T. splendens and T. angulatum sequences. The sequence similarity between T. splendens strains and Lachnum bicolor

62 Results B was 83.9 and 84.4 %. Sequence similarity values between T. splendens strains and Hymenoscyphus spp. ranged between 78. 7 – 79.0 for H. fructigenus and 80.9 – 84.4 % for the H. ericae spp. and Hymenoscyphus. sp. GU30. Phylogenetic trees obtained performing NJ, parsimony and ML methods always showed a placement of T. splendens strains close to Zalerion varium, statistically supported by bootstrap values of 97 % in parsimony analysis and 95 % in ML analysis, but without support in NJ analysis. All analysis methods placed Lachnum bicolor as sister clade to the T. splendens. But the placement shows a long branch. Figure 16 shows a phylogenetic tree obtained by NJ indicating the close placement of T. splendens near Zalerion varium of which phylogenetic classification is still unclear. Trees obtained by parsimony and ML analyses are given in the appendix (Fig. A16 and A17).

H. fructigenus

H. sp. GU 30

H. ericae 7 H. ericae 6

Lachnum bicolor

0.01 changes

Trispl 19087 Zalerion varium Trispl 11989 Trispl 16599 Trispl 12386

Figure 16: ITS rDNA (including 5.8S rDNA) sequence-based phylogenetic tree derived using neighbour joining analysis, showing relationships of four Tricladium splendens strains and closest BLAST hits. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). The scale bar indicates one base change per 100 nucleotide positions. H. = Hymenoscyphus; Trispl = Tricladium splendens.

63 Results B

2.4. Heliscus lugdunensis

Heliscus lugdunensis SACC.&THERRY is the type species of the genus Heliscus. On malt agar H. lugdunensis produces beige colonies with woolly, often funiculose aerial mycelium. The conidiophores are simple or sparsely branched. Conidiogenous cells are phialidic and arranged in penicillate groups. Conidia from nature are clove-shaped, typically with three longer apical outgrowths. Conidia from culture are mostly clavate or wedge-shaped and have mostly two or three very short apical outgrowths. The conidial secession is schizolytic. The teleomorph is Nectria lugdunensis. This connection was established by Webster (1959b). He obtained nectrioid perithecia in pure culture from conidia of the H. lugdunensis state. Unfortunately, a culture of N. lugdunensis was not available for the present study. SSU sequences from Heliscus lugdunensis were obtained from three strains isolated from the river Elbe (ELBE 98), the Oberer Seebach (L5) and taken from the CCM (F- 245). Partial SSU sequences of 1004 – 1761 nucleotides in length were used for phylogenetic analyses by NJ, parsimony and ML. Within BLAST searches the closest SSU sequences were that from Nectria cinnabarina and Gibberella pulicaris, both are representatives of the order Hypocreales. In phylogenetic analyses all three strains formed a distinct cluster within the order Hypocreales with placement next to G. pulicaris. Figure 17 shows one of 42 most parsimonious trees. The placement of H. lugdunensis is supported by a bootstrap value of 90 %. Phylogenetic trees obtained with NJ and ML (appendix, Fig. A18 and A19) showed the same placement as the parsimony tree, but with even lower bootstrap values (73 and 80 %).

64 Results B

61 Microascus cirrosus 98 Halosarpheia retorquens 52 Petriella setifera Pseudallescheria boydii 60 Graphium penicillioides 98 Heliscus lugdunensis ELBE98 Heliscus lugdunensis L5 90 Heliscus lugdunensis 245 69 Gibberella pulicaris Geosmithia putterillii Nectria cinnabarina Hypomyces chrysospermus 100 Hypocrea lutea 90 Sordaria firmicola 97 Neurospora crassa Chaetomium elatum 71 Kionochaeta ivoriensis 96 Fabrella tsugae Geomyces pannorum var. pannorum 89 Pseudogymnoascus roseum 54 Leotia lubrica 77 Bulgaria inquinans Spathularia flavida 52 Cudonia confusa 74 Loramyces junicola 74 Hymenoscyphus virgultorum Blumeria graminis 73 Phyllactinia guttata Oidiodendron tenuissimum 51 Myxotrichum deflexum Cyttaria darwinii 85 Hymenoscyphus fructigenus 95 Aureobasidium pullulans 77 Hortaea werneckii 92 Kirschsteiniothelia aethiops 93 Ophiobolus herpotrichus 53 Leptosphaeria doliolum 88 Cucurbitaria elongata 93 Pleospora betae 52 Kirschsteiniothelia elaterascus Herpotrichia juniperi Mycosphaerella mycopappi 86 Massarina australiensis 93 Massarina bipolaris 94 Kirschsteiniothelia maritima Paecilomyces variotii 99 Monascus purpureus 100 Onygena equina Orbilia fimicola Arthrobotrys superba Arthrobotrys oligospora Monacrosporium doedycoides Orbilia delicatula 89 Peniophora nuda Hydnum repandum 94 Bulleromyces albus Saccharomyces cerevisiae

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Figure 17: Phylogenetic tree reconstructed by maximum parsimony analysis, showing the possible placement of three Heliscus lugdunensis strains within the Ascomycota. One of 42 equally parsimonious trees based on 659 bp 18S rDNA data. Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets.

65 Results B

2.5. Anguillospora crassa and Anguillospora longissima The genus Anguillospora is heterogeneous. It encompasses anamorphs of 7 teleomorphs belonging to 6 ascomycete orders and 3 anamorphs with unknown phylogenetic affinities. The characters common to all species are blastic conidiogenesis, integrated conidial initiation, percurrent proliferation of conidiogenous cells and scolecoform septate conidia.

Anguillospora longissima (SACC.&P.SYD.) INGOLD is the type species of the genus Anguillospora. It has dark grey colonies with radiating woolly mycelium. The conidiophores are simple. The conidia are scolecoform, sigmoid or curved or rarely straight (Fig. A20, appendix). Conidial secession is rhexolytic and takes place by break down of a separating cell connecting the conidium with the conidiogenous cell. The remnants of the separating cell are clearly visible at the conidial base short after secession. Later the remaining collarette becomes masked by percurrent basal extension which makes distinguishing from other Anguillospora spp. difficult. A. longissima has a Massarina sp. (Pleosporales, bitunicate loculoascomycetes) teleomorph which was described by Willoughby & Archer (1973) and later confirmed by Webster & Descals (1979). Anguillospora crassa was described as a second species of the genus. Colonies on malt agar are grey, with woolly aerial mycelium. Conidiophores are simple or branched, conidiogenous cells proliferate percurrently but in culture rarely also sympodially. Conidial secession is schizolytic. Conidia are scolecoform, sigmoid or curved, cells sometimes inflated (Fig. A21 and A22, appendix). The holomorph, according to Webster (1961) is Mollisia uda (Helotiales, inoperculate discomycetes). The isolates sequenced in this study were derived from either stalked (F-07082), sessile Mollisia (not M. uda, F-15583) or sessile apothecia (F-05584; F-13483; F-15283).

Sequencing of the SSU of four strains of Anguillospora crassa INGOLD (F- 15583; F- 13483; F- 05584 and F- 07082) and three strains of Anguillospora longissima (F- 11891; F- 00980 and L22) resulted in sequences of 1703 – 2166 nucleotides in length. Within the strains of A. crassa the sequence of isolate F- 05584 showed an intron of 77 nucleotides inserted at position 1362. Regarding the sequences of A. longissima strains, isolate F- 00980 showed an intron of 508 nucleotides inserted at position 1551. Nucleotides belonging to intron insertions were eliminated before phylogenetic analyses. Using BLAST searches the nearest SSU sequences for A. crassa strains were Phialophora sp. (GenBank accession number AJ278753) and Blumeria graminis (Erysiphales). In contrast BLAST searches with sequences of A. longissima resulted in closest hits with the SSU sequence of Kirschsteiniothelia maritima, which is a representative of the order Pleosporales. Phylogeny reconstruction with NJ, parsimony and ML methods gave no clear results for the placement of A. crassa within the order Erysiphales whereas A. longissima was clearly placed within the order Pleosporales. Figure 18 shows a phylogenetic tree derived from ML analysis indicating A. crassa strains as sister group to B. graminis and Phyllactinia guttata without bootstrap support, but a distinct (98 % bootstrap support) clustering of A. longissima strains with K. maritima and Massarina spp. This is in good accordance with the assumption of Willoughby and Archer (1973) who described a none- named species of the genus Massarina as the perfect state of A. longissima. NJ analysis (Figure 19) showed A. longissima isolates F- 00980 and K. maritima forming a strongly supported (98 %) additional clade within the cluster. Here A. crassa is also placed as sister clade to Erysiphales with the

66 Results B rather low bootstrap value of 74 %. Parsimony analyses resulted in a very similar tree topology as NJ analysis, but without any bootstrap support for the placement of A. crassa (Fig. A23, appendix).

Anguillospora crassa 07082 Anguillospora crassa 15583 100 Anguillospora crassa 13483 Anguillospora crassa 05584 96 Blumeria graminis Phyllactinia guttata Hymenoscyphus fructigenus Loramyces juncicola 87 Hymenoscyphus virgultorum 87 Oidiodendron tenuissimum Myxotrichum deflexum 76 Cyttaria darwinii Geomyces pannorum var. pannorum Pseudogymnoascus roseus Leotia lubrica 52 Bulgaria inquinans Spathularia flavida 82 Cudonia confusa 84 Fabrella tsugae Hypomyces chrysospermus Hypocrea lutea Gibberella pulicaris Nectria cinnabarina 87 Geosmithia putterillii Microascus cirrosus Halosarpheia retorquens Pseudallescheria boydii 96 Petriella setifera 100 Graphium penicillioides Sordaria firmicola Neurospora crassa 99 Chaetomium elatum Kionochaeta ivoriensis Hortaea werneckii 61 Aureobasidium pullulans 83 Kirschsteiniothelia aethiops Leptosphaeria doliolum Ophiobolus herpotrichus Cucurbitaria elongata Pleospora betae 84 Kirschsteiniothelia elaterascus Herpotrichia juniperi 100 Mycosphaerella mycopappi Anguillospora longissima 11891 Anguillospora longissima L22 98 Anguillospora longissima 00980 98 Kirschsteiniothelia maritima Massarina australiensis 97 Massarina bipolaris Paecilomyces variotii 99 Monascus purpureus 99 Onygena equina Arthrobotrys superba Orbilia fimicola Arthrobotrys oligospora 100 Monacrosporium doedycoides Orbilia delicatula Peniophora nuda 98 Hydnum repandum Bulleromyces albus Saccharomyces cerevisiae

0.01substitutions / site

Figure 18: One phylogenetic tree of 346 obtained by maximum likelihood analysis (Ln likelihood = – 13639.55185), using 18S rDNA sequence data. Bootstrap values were derived from 1000 resampled data sets. The tree shows the possible placement of four Anguillospora crassa strains and three Anguillospora longissima strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study.

67 Results B

Anguillospora crassa 15583 Anguillospora crassa 13483 96 Anguillospora crassa 05584 74 Anguillospora crassa 07082 98 Blumeria graminis Phyllactinia guttata 74 Loramyces juncicola 80 Hymenoscyphus virgultorum 73 Oidiodendron tenuissimum Myxotrichum deflexum Cyttaria darwinii 71 Hymenoscyphus fructigenus 96 Geomyces pannorum var. pannorum 55 Pseudogymnoascus roseus Leotia lubrica 82 Bulgaria inquinans Spathularia flavida 86 Cudonia confusa 86 Fabrella tsugae Hypomyces chrysospermus 74 Hypocrea lutea 60 Gibberella pulicaris Nectria cinnabarina 88 Geosmithia putterillii Petriella setifera Microascus cirrosus 91 Halosarpheia retorquens 98 Pseudallescheria boydii 100 Graphium penicillioides 81 Sordaria firmicola 92 Neurospora crassa Chaetomium elatum 65 Kionochaeta ivoriensis 86 Hortaea werneckii 64 Aureobasidium pullulans 86 Kirschsteiniothelia aethiops Anguillospora longissima 11891 96 Anguillospora longissima L22 98 Anguillospora longissima 00980 Kirschsteiniothelia maritima 90 Massarina australiensis Massarina bipolaris Herpotrichia juniperi Mycosphaerella mycopappi Leptosphaeria doliolum Ophiobolus herpotrichus 82 Cucurbitaria elongata 60 Pleospora betae 93 Kirschsteiniothelia elaterascus Paecilomyces variotii Monascus purpureus 99 Onygena equina Arthrobotrys superba Orbilia fimicola 87 Arthrobotrys oligospora Monacrosporium doedycoides Orbilia delicatula 96 Hydnum repandum Peniophora nuda Bulleromyces albus Saccharomyces cerevisiae

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Figure 19: 18S rDNA sequence-based phylogenetic tree derived using neighbour joining, showing the placement of four Anguillospora crassa strains and three Anguillospora longissima strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). The scale bar indicates one base change per 100 nucleotide positions. Bootstrap values were calculated from 1000 resampled data sets.

68 Results B

Sequences of the ITS region of Anguillospora crassa could be obtained for the same strains as for SSU analysis with the exception of strain CCM F-13483, which was replaced by strain CCM F-15283. Sequences of the ITS region of Anguillospora longissima were obtained for the same strains as for SSU analysis and additionally the strains CCM F-10691 and F-11791 were included in the analysis. The length of ITS sequences from A. crassa strains ranged from 436 – 471 nucleotides. The ITS1 regions were 161 – 168 bp long, the 5.8S region was 152 bp and the ITS2 region 148 – 149 bp long. All four strains of A. crassa showed identical sequences within the spacer regions. The closest BLAST hits for A. crassa strains were the ITS sequences of Zalerion varium and, with smaller similarity that of Fabrella tsugae and Phacidium infestans (Helotiales). The ITS sequences obtained for the five strains of A. longissima were 446 – 449 bp long. The length of the ITS1 region ranged from 136 to 138 bp. The 5.8S rDNA was 152 bp long and identical in all strains. The sequences of the ITS2 region were 152 bp long in all strains. In BLAST searches the nearest sequences were that of Leptosphaeria contecta, L. doliolum and Curvularia eragrostidis (all Pleosporales). The ITS sequences of A. longissima strains F-00980, F-10691 and F-11791 were identical and showed 99.2 and 98.2 % similarity to the ITS sequences of the strains L22 and F-11891. Strains L22 and F-11891 showed a sequence similarity of 99.1 % (Table 20).

Table 20: ITS rDNA (including 5.8S rDNA) sequence similarity matrix for Anguillospora crassa and Anguillospora longissima % rRNA sequence similarity A. crassa Alongi Alongi Alongi Alongi Taxon all strains 00980 L22 11791 11891 A. crassa (all strains identical) A. longissima 00980 71.1 A. longissima L22 71.4 99.2 A. longissima 11791 71.1 100 99.2 A. longissima 11891 67.6 98.2 99.1 98.2 A. longissima 10691 71.1 100 99.2 100 98.2 Alongi = Anguillospora longissima

As already indicated from phylogenetic analyses of the SSU rDNA sequences, A. crassa and A. longissima are not closely related. The low ITS sequence similarities of A. crassa to A. longissima of 67 to 71.1 % (Table 20) clearly support the result from the SSU rDNA analyses. Therefore phylogenetic analyses of the ITS1, 5.8S, ITS2 sequence data were further evaluated for each species separately.

Phylogenetic analyses of the ITS sequences of A. crassa strains were performed with the closest BLAST hits and other representatives of the order Helotiales. Blumeria graminis f. sp. bromi was used as an outgroup, because of its placement as sister clade to A. crassa in SSU analyses. ITS sequences of Mollisia cinera and Mollisia minutella were included into the analyses because of a suspected teleomorph connection of A. crassa to this genus (Webster 1961). Table 21 shows a similarity matrix of ITS sequences of A. crassa, the closest BLAST hits and Mollisia spp..

69 Results B

Table 21: ITS rDNA (including 5.8S rDNA) sequence similarity matrix for Anguillospora crassa, closest BLAST hits and Mollisia spp. % rRNA sequence similarity Taxon A. crassa (all strains) Zalerion varium 96.2 Phacidium infestans acc. no.: U92305a 86.4 Fabrella tsugae acc. no.: U92304a 82.8 Mollisia cinera acc. no.: AJ430222b 82.9 Mollisia minutella acc. no.: AJ430223b 82.2 a (Gernandt et al. 1997); b (Vrålstad 2001); acc. no.: GenBank accession number

The highest sequence similarities of 96.2 % could be observed between the ITS sequences of Zalerion varium and the A. crassa strains. This is an even higher sequence similarity as it was observed between Zalerion varium and Tricladium splendens (94.4 – 94.6 % ITS sequence similarity). The ITS sequence similarity between A. crassa strains and T. splendens strains ranges from 88.8 to 89.9 % (data not shown in Table).The comparison of the ITS sequences of Phacidium infestans and A. crassa showed 86.4 % similarity whereas the similarities to Fabrella tsugae and the Mollisia spp. ranged between 82.2 and 82.9 %. Phylogenetic trees obtained with NJ, parsimony and ML methods always showed a placement of A. crassa strains close to Zalerion varium, rather weakly supported by bootstrap values of 87 % in NJ analysis and 73 % in parsimony analysis. ML analysis showed the same tree topology, but with a slightly stronger bootstrap support of 95 %. In ML and parsimony analyses Phacidium infestans and Fabrella tsugae were placed in a sister clade to A. crassa and Z. varium. Figure 20 shows a phylogenetic tree obtained by ML indicating the close placement of A. crassa near Zalerion varium Trees obtained by parsimony and NJ analyses are given as Figures A24 and A25 in the appendix.

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Anguillospora crassa 15283 Anguillospora crassa 07082 74 Anguillospora crassa 15583 95 Anguillospora crassa 05584 Zalerion varium 71 Fabrella tsugae 90 Phacidium infestans 99 Mollisia minutella Mollisia cinerea 96 Hymenoscyphus ericae 7 Hymenoscyphus sp. GU30 Leotiales sp. Bjelland 61 Cudonia lutea Spathularia flavida Blumeria graminis f. sp. bromi

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Figure 20: Single phylogenetic tree obtained by maximum likelihood analysis (Ln likelihood = – 3478,26204), based on ITS rDNA sequence data. Bootstrap values were derived from 1000 resampled data sets. The tree shows the most possible placement of four Anguillospora crassa strains, closest BLAST hits and representatives of the orders Helotiales and Erysiphales. Names in red indicate DNA sequences newly determined in this study.

Phylogenetic analyses of the ITS sequences of A. longissima were performed with the closest BLAST hits and other representatives of the order Pleosporales. Eight ITS sequences of Massarina were included into the analyses because of an assumed teleomorph connection of A. longissima to this genus (Willoughby & Archer 1973). As indicated in Table 22, the closest BLAST hit sequence of Leptosphaeria contecta showed the highest similarity to A. longissima sequences of 89.8 % for the strains F-00980; F-10691 and F-11791. A. longissima strains L22 and 11891 showed 89.1 and 89.3 % sequence similarity with L. contecta. Lower sequence similarities of 82.5 to 83.2 % were observed for A. longissima strains and Curvularia eragrostidis. With sequences of the genera Massarina, Lophiostoma and Pleospora, A. longissima strains showed rather low sequence similarities of 67.4- 78.9 %. The sequences of A. longissima and Massarina spp. were difficult to align (Figure A40, appendix). Many ambiguously aligned regions were excluded before parsimony analyses. Parsimony analysis performed with 89 parsimony informative positions resulted in a 91 % bootstrap supported placement of A. longissima strains close to L. contecta (Figure 21). The nearest sistergroup consists of several Massarina spp. associated with Lophiostoma. NJ analysis resulted in a single A. longissima cluster and a low supported sistergroup including the same species as in parsimony analyses. The branching order dividing the A. longissima strains into two groups was not supported by bootstrapping. ML analyses resulted in similar tree topology as NJ with 99 % bootstrap support for the placement of A. longissima as sistergroup of a Massarina-Lophiostoma- Leptosphaeria doliolum cluster (Fig. A26 and A27, appendix).

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Table 22: ITS rDNA (including 5.8S rDNA) sequence similarity matrix for Anguillospora longissima, closest BLAST hits and Massarina spp. % rRNA sequence similarity A. longissima A. longissima A. longissima F-00980 L22 11891 Taxon F-10691 F-11791 Leptosphaeria contecta acc. no: AF181702a 89.8 89.1 89.3 Leptosphaeria doliolum 83 82.7 82.7 Curvularia eragrostidis acc. no: AF163077b 83.2 82.9 82.5 Pleospora papaveracea 78.9 78.5 78 Lophiostoma vagabundum acc. no.: AF383954c 77.5 77.4 77.9 Massarina corticola acc. no.: AF383957c 77.4 77.1 77.4 Massarina eburnea acc. no: AF383959c 78 77.4 76.7 Massarina walkeri acc. no.: AF383965c 71.7 71.7 71.2 Massarina rubi acc. no.: AF383964 69.9 68.7 67.4 a (Goodwin & Zinsmann 2001); b(Goh & Hyde 1999); c(Liew et al. 2002) acc. no: GenBank accession number

75 Massarina armatispora 77 Lophiostoma vagabundum 87 Lophiostoma caulium 91 Massarina bipolaris 77 Massarina frondisubmersa 72 Massarina rubi 93 Massarina corticola Anguillospora longissima 11791 Anguillospora longissima 00980 Anguillospora longissima 10691 Anguillospora longissima L22 91 Anguillospora longissima 11891 65 Leptosphaeria contecta Massarina walkeri 62 Massarina eburnea Curvularia eragrostidis 72 Pleospora papaveracea Leptosphaeria doliolum Massarina ramunculicola

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Figure 21: Phylogenetic tree estimated by maximum parsimony, showing the possible relationships of five Anguillospora longissima strains and fungal species with the highest ITS rDNA sequence similarity obtained in BLAST searches and Massarina spp.. Names in red indicate DNA sequences newly determined in this study. One of 3 equally parsimonious trees based on 89 bp ITS1, 5.8S, ITS2 rDNA data. A. longi = Anguillospora longissima

From A. longissima strain F-00980 the first 625 nucleotides of the LSU (28S rDNA) were sequenced. BLAST searches indicated the highest similarity to sequences of Pleosporales (Westerdykella, Curvularia and Leptosphaeria species). NJ, parsimony and ML phylogeny reconstruction included representatives of several Ascomycetes orders. NJ analysis (Fig. A4, appendix) placed A. longissima next to Leptosphaeria doliolum with a bootstrap value of 90 %. Parsimony (Figure 9) and ML (Fig. A5, appendix) analyses placed A. longissima within (99 % bootstrap support) or basal to a cluster

72 Results B comprising members of the order Pleosporales. The 625 bp of the A. longissima sequence were aligned to the same range of the LSU sequences included in all analyses. For parsimony analysis 64 ambiguously aligned positions were excluded. Searches for the most parsimonious trees were performed with 213 potentially phylogenetically informative data. Figure 9 shows a consensus tree of 105 most parsimonious trees requiring 2110 steps.

2.6. Lemonniera aquatica and Lemonniera terrestris Lemonniera is an anamorphic genus of which conidial and colony characters seem to be very homogeneous. On malt agar the colonies are fast growing with aerial mycelium. A very characteristic feature are the small blackish sclerotia appearing in concentric rings in agar cultures. Conidiophores are long stiped with apical branching in submerged standing cultures. Conidiogenous cells are phialidic bearing tetraradiate conidia. Conidial branches are diverging from a globose or tetrahedral conidial primordium at the phialide apex. Lemonniera aquatica DEWILD is the type species of the genus. It has conidia with four typically cylindrically arms, diverging from a common point (Fig. A28, appendix). Sometimes a small more or less distinct globose body in the middle of the conidium can be observed.

Lemonniera terrestris TUBAKI has a smaller conidial span than L. aquatica and the conidial arms are broader at the point of insertion, distinctly tapering towards the ends (Fig. A28, appendix). From Lemonniera aquatica strain CCM F-04480 and Lemonniera terrestris strains CCM F-125 and CCM F-11486 SSU sequences were obtained, yielding 1746-1765 nucleotides in length. Within BLAST searches the closest SSU sequences were that from Phialophora sp. Elec-N-14.PN (uncertain classification after Eriksson et al. 2002 in Sordariomycetes incertae sedis, Magnaporthaceae) for both L. aquatica and L. terrestris. In NJ, parsimony and ML phylogenetic analyses both species formed a distinct cluster within the order Helotiales with placement next to Loramyces juncicola. As indicated in Figure 22 the placement of both species in one cluster is weakly supported (82 % bootstrap) in NJ analysis. Parsimony and ML analyses (see appendix Fig. A29 and A30) placed both species in one cluster without any bootstrap support. L. juncicola was placed as a sister clade to Lemonniera in all three analyses with bootstrap values of 86 % in NJ, and 87 % in parsimony and ML.

2.7. Varicosporium elodeae and Anguillospora furtiva At the very last end of the practical work in this study two partial SSU sequences of Varicosporium elodeae CCM F-11783 (700 bp) and Anguillospora furtiva L 16 (1743 bp) were obtained. In analyses using NJ, parsimony and ML methods V. elodeae was placed as sister taxon to Loramyces juncicola within the order Helotiales. A. furtiva was placed in a monophylum with Anguillospora crassa (Fig. A31-33, appendix).

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88 Geomyces pannorum var. pannorum Pseudogymnoascus roseus Leotia lubrica Bulgaria inquinans Spathularia flavida Cudonia confusa 83 Oidiodendron tenuissimum Myxotrichum deflexum Cyttaria darwinii 56 Lemonniera terrestris 11486 82 Lemonniera terrestris 125 86 Lemonniera aquatica 04480 Loramyces juncicola Phialophora sp. Elec-N-1 4.PN Hymenoscyphus fructigenus 84 Phyllactinia guttata 61 Blumeria graminis Hymenoscyphus virgultorum Fabrella tsugae 95 Hortaea werneckii Aureobasidium pullulans Cucurbitaria elongata Ophiobolus herpotrichus 83 Leptosphaeria doliolum 57 Pleospora betae 59 Kirschsteiniothelia elaterascus 99 Herpotrichia juniperi Mycosphaerella mycopappi 99 Kirschsteiniothelia maritima 77 Massarina australiensis 98 Massarina bipolaris 86 Kirschsteiniothelia aethiops 53 Gibberella pulicaris 54 Nectria cinnabarina 87 Hypocrea lutea 91 Hypomyces chrysospermus 92 Geosmithia putterillii 96 Petriella setifera 60 Pseudallescheria boydii 79 Microascus cirrosus 97 Halosarpheia retorquens Graphium penicillioides 76 Neurospora crassa 98 Sordaria firmicola 99 Chaetomium elatum 91 Kionochaeta ivoriensis 95 Paecilomyces variotii Monascus purpureus 98 Onygena equina 74 Arthrobotrys superba 97 Orbilia fimicola 85 Arthrobotrys oligospora 99 Monacrosporium doedycoides Orbilia delicatula 76 Hydnum repandum Peniophora nuda 98 Bulleromyces albus Saccharomyces cerevisiae

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Figure 22:18S rDNA sequence-based phylogenetic tree derived using neighbour joining, showing the possible placement of two Lemonniera terrestris strains and one Lemonniera aquatica strain within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). The scale bar indicates one base change per 100 nucleotide positions. Bootstrap values were calculated from 1000 resampled data sets.

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C Fluorescence in situ hybridisation (FISH) of freshwater fungi

1. Development of rRNA targeted oligonucleotide probes In order to develop rRNA targeted oligonucleotide probes for the in situ detection of metabolically active freshwater fungi, complete and partial 18S and 28S rDNA sequences were scanned for specific signatures. The signatures are unique for different phylogenetic levels. Based on comparative sequence analysis 314 (database 1999) to 2000 (database 2002) aligned 18S rRNA and 700 28S rRNA sequence data were screened and evaluated. This was done using the Probe_Design tool of the ARB software package. If the probe design feature proposed several probes, the following criteria guided in the selection of probes (Manz 1999). The criteria were chosen due to their positive effect on the in situ accessibility.

i. 15 – 22 nucleotides probe length ii. a balanced G/C to A/T ratio with resulting melting points between 50 ° and 60 ° C iii. no self-complementary of the probe (target sites located at both sites of helical structures not longer than six nucleotides) iv. G/C base pairs at the ends to minimize cutting loose of terminal A/T basepairs

The 11 newly developed probes were tested using the ARB databases (Probe_Match tool) and GenBank for other organisms that may coincidentally contain identical nucleotide sequences. The results revealed at least one centrally located mismatch to all accessible 18S and 28S rRNA sequences for every probe. The last sequence data analysis was performed at September 10th 2002. All following sequence analysis data presented were updated on September 10th 2002. Probe binding sites of the newly designed probes within the 18S rRNA and 28 rRNA are shown in the secondary structure models of the rRNA molecules in Figures 25 and 26. Sequences of new oligonucleotide probes, target regions and formamide concentrations within the hybridisation buffer are summarized in Table 31.

1.1. Design and evaluation of oligonucleotide probes specific for the division Eumycota Two probes were designed on a phylogenetic level for detecting a broad range of Eumycota. These probes were used as positive fungal controls besides the Eucarya specific probe EUK516.

1.1.1. Fungal probe FUN1429 The probe FUN1429 was evaluated in 1999 against 314 partial and complete 18S rRNA sequences that were included in the ARB database as of December 1998. Designing one universal probe comprising all 314 fungal strains in the database was not possible. After elimination of some phylogenetic groups of fungi, e.g. the Dactylella – Monacrosporium complex (Rubner 1996), most Zygomycota and most lichenized Ascomycetes, probe FUN1429 could be designed for matching 146 Eumycota. Within the enlarged database from April 2002 the probe FUN1429 matches 908 fungal

75 Results C sequences. The probe consists of 18 nucleotides and binds within the region of helix 36 in the 18S rRNA secondary structure model (Figure 25). In order to check for sequence similarities within non – target organisms, all sequences of the ARB databases and of GenBank were tested against probe FUN1429. This showed no binding to other than fungal 18S rDNA sequences. Under in situ conditions probe FUN1429 showed no non-specific binding to the non – target organisms Aquabacterium commune, Methanosarcina barkeri, Scenedesmus quadricauda and Aspergillus nidulans. The species A. nidulans displays one mismatch within the probe target region. By adjusting the in situ hybridisation stringency by the addition of 35 % formamide to the hybridisation buffer, A. nidulans, could be unequivocally separated from target fungi tested. FISH of freshwater fungi using Cy3-or Oregon Green-labeled probe FUN1429 resulted in clear fluorescence hybridisation signals (Figure 23). Clear FISH signals were also obtained for Penicillium chrysogenum, Paecilomyces variotii and Aspergillus fumigatus. Fluorescence results for in situ hybridisations of fungal, algal and bacterial species with rRNA targeted probe FUN1429 are shown in Table 23.

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Table 23: Results of fluorescence in situ hybridisations of fungal, algal and bacterial species with rRNA targeted oligonucleotide probes FUN1429 and MY1574. Mismatches are underlined.

(-- = no probe match; — = no probe signal)

Probe sequence and target region Species FUN1429 MY1574 Probe signal GTGATGTACTCGCTGGCC TCCTCGTTGAAGAGC 1434-52 1560-1574 FUN1429 Penicillium chrysogenum GGCCAGCGAGUACAUCAC GCUCUUCAACGAGGA MY1574 1392-1409 1477-1491 FUN1429 Paecilomyces variotii GGCCAGCGAGUACAUCAC GCUCUUCAACGAGGA MY1574 1476-1494 1561-1574 FUN1429 Aspergillus fumigatus GGCCAGCGAGUACAUCAC GCUCUUCAACGAGGA MY1574 1431-1448 1500-1515 Aspergillus nidulans MY1574 GGGCAGCGAGUACAUCAC GCUCUUCAACGAGGA -- 1559- Verticillium lecanii — GCUCUUCAAGGAGGA Syncephalastrum -- 1522- — racemosum GCUCUUCAACUAGGA -- 1560-74 Alatospora acuminata MY1574 GCUCUUCAACGAGGA -- 1516-30 Anguillospora crassa MY1574 GCUCUUCAACGAGGA -- 1460-74 Anguillospora longissima MY1574 GCUCUUCAACGAGGA FUN1429 Helicodendron giganteum Unknown Unknown MY1574 FUN1429 Helicomyces roseus Unknown Unknown MY1574 FUN1429 Helicosporium phragmites Unknown Unknown MY1574 -- 1530-44 Heliscus lugdunensis MY1574 GCUCUUCAACGAGGA -- 1533-47 Lemonniera aquatica MY1574 GCUCUUCAACGAGGA -- 1533-47 Lemonniera terrestris MY1574 GCUCUUCAACGAGGA 1458-73 1542-56 FUN1429 Tetracladium marchalianum GGCCAGCGAGUACAUCAC GCUCUUCAACGAGGA MY1574 -- 1553-67 Tetracladium setigerum MY1574 GCUCUUCAACGAGGA -- 1530-44 Tricladium angulatum MY1574 GCUCUUCAACGAGGA -- 1543-57 Tricladium splendens MY1574 GCUCUUCAACGAGGA Varicosporium elodeae -- unknown MY1574 Scenedesmus quadricauda -- -- — Aquabacterium commune -- -- — Methanosarcina barkeri -- -- —

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F

10 µm Figure 23 A-F

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Figure 23: In situ hybridisations performed with probes FUN1429 and MY1574. A Phase contrast photomicrograph of Penicillium chrysogenum and B Corresponding epifluorescence photomicrograph of P. chrysogenum hybridised with Oregon Green-labeled probe FUN1429. C CLSM photomicrograph of Tetracladium marchalianum and Eubacteria after simultaneous in situ hybridisation with Cy3 labeled probe FUN1429 (red) and Oregon Green labeled probe EUB338. D CLSM photomicrograph of Tetracladium marchalianum in pure culture after hybridisation with Cy3- labelled probe MY1574 E CLSM photomicrograph of Varicosporium elodeae in pure culture after hybridisation with Cy3- labelled probe MY1574 F CLSM photomicrograph of Penicillium chrysogenum after hybridisation with Cy3-labelled probe MY1574.

1.1.2. Fungal probe MY1574 As another specific oligonucleotide, probe MY1574 was designed. It targets an even wider range of Eumycota than probe FUN1429. The 15 mer probe binds to a region close to helix 47 in the 18S rRNA secondary structure model (Figure 25). Within the enlarged ARB database as of April 2002, MY1574 matches 1385 fungal sequences without mismatches including mainly Ascomycetes, several Basidiomycetes, and some lichenized Ascomycetes. Testing of MY1574 against sequences of GenBank and ARB resulted in no other matches than fungal 18S rDNA sequences. Under in situ conditions in pure and mixed cultures probe MY1574 showed no unspecific binding to the non – target organisms Aquabacterium commune, Methanosarcina barkeri, Scenedesmus quadricauda and Verticillium lecanii and Syncephalastrum racemosum. V. lecanii and S. racemosum display one centrally located mismatch within the probe target region By adjusting the in situ hybridisation stringency by the addition of 20 % formamide to the hybridisation buffer V. lecanii and S. racemosum could unequivocally be separated from target fungi tested. Figure 23 shows successful in situ hybridisations of target fungi using MY1574. Table 23 shows the results of FISH of fungal, algal and bacterial target and non-target organisms.

1.2. Design and evaluation of FISH probes for freshwater fungi specific on genus and species level For Tetracladium, Alatospora acuminata, Anguillospora longissima, Heliscus lugdunensis and Tricladium angulatum genus or species-specific FISH probes could be designed and evaluated. They target either the 18S or the 28S rRNA region. No specific probes for Anguillospora crassa and Tricladium splendens, based on 18S rRNA, could be designed. Due to the high 18S rRNA sequence similarity of 99.8 % of both species no specific signatures for the probe design could be found (the complete SSU sequence similarity matrix is given in the appendix, Fig. A35).

1.2.1. Probe specific for the genus Tetracladium Freshwater fungi of the genus Tetracladium De Wild are ubiquitously distributed and regularly isolated from aquatic and terrestrial locations (Roldán et al. 1989). One specific 18S rRNA targeted probe was designed for already known sequences of fungi belonging to the genus Tetracladium. Sequences of the strains Tetracladium marchalianum CCM F-19399, CCM F-26199, CCM F-26299, CCM F-26399, ELBE 50, ELBE 90 and L27 were obtained in the present study whereas sequences of T. marchalianum strain CCM F-11391, T. maxilliforme strain CCM F-14286, T. setigerum CCM F-20987,

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T. furcatum CCM F-11883 and T. apiense CCM F-23199 were obtained from GenBank where they had been deposited by Nikolcheva and Bärlocher. Strain T. marchalianum CCM F-312 was sequenced both, in the present study and by Nikolcheva and Bärlocher, resulting in identical 18S rDNA sequences. Pure cultures of all Tetracladium strains mentioned above, and in addition T. marchalianum strain CBS 979.87 were available for evaluating the genus specific probe TCLAD1395. The 18 mer probe binds to a region at helix 44 in the 18S rRNA secondary structure model (Figure 25). Computer aided sequence comparison revealed that probe TCLAD1395 showed at least one mismatch to all other accessible 18S rRNA sequences. In situ hybridisations done in pure and mixed cultures of Tetracladium strains using probe TCLAD 1395 showed clear FISH signals. The non - target species Epicoccum nigrum displays two mismatches within the target region of TCLAD1395. It could be clearly separated by adjusting the hybridisation stringency to 35 %. Table 24 shows the probe sequence and the target sites of the 18S rRNA oligonucleotide probe specific for fungal strains of the genus Tetracladium.

Table 24: Sequence and target sites of 18S rRNA FISH probe TCLAD1395 specific for aquatic hyphomycetes of the genus Tetracladium. Probe TCLAD1395 (5’-3’) Species / strain CGCACTTCCATTTGCTTG target site T. marchalianum ELBE 50 1378-1396 T. marchalianum ELBE 90 1377-1395 T. marchalianum L 27 1377-1395 T. marchalianum CBS 979.87 Unknown T. marchalianum CCM F-312 1385-1403 T. marchalianum CCM F-11391 1387-1405 T. marchalianum CCM F-19399 1377-1395 T. marchalianum CCM F-26199 1387-1405 T. marchalianum CCM F-26299 1378-1396 T. marchalianum CCM F-26399 1377-1395 T. maxilliforme CCM F-14286 1387-1405 T. setigerum CCM F-20987 1384-1402 T. apiense CCM F-23199 1384-1402 T. furcatum CCM F-11883 1385-1403

1.2.1.1. Probes specific for Tetracladium marchalianum For detecting the metabolically active stages of one of the most often reported species of the genus Tetracladium, a species specific oligonucleotide probe for Tetracladium marchalianum was designed. In a first attempt a FISH probe Tmarch1096 targeting the 18S rRNA was designed and evaluated. As further sequences and cultures of other Tetracladium species became available, retesting of tmarch1096 revealed matches to other Tetracladium species. For that reason tmarch1096 can only be considered as a genus specific probe. Using the available 18S rRNA sequence data it was not possible to construct a probe on species level. Within the Tetracladium species the similarities of the

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18S rRNA sequences are too high. Therefore species specific probes for T. marchalianum needed to be designed for the larger 28S subunit of the ribosomal gene. Three different probes TmarchB10, TmarchC1_1 and TmarchC1_2 were evaluated in comparative sequence analyses within the ARB program and GenBank showing no non-specific matches with bacterial and fungal sequences. The closest non - target sequences showed at least three mismatches to the probes. In situ testing of the three probes performed against T. setigerum, T. furcatum, T. apiense and T. maxilliforme revealed no unspecific binding when the hybridisation stringency was adjusted to 20 % for probes TmarchC1_1 and TmarchC1_2 and to 40 % for probe TmarchB10. FISH of T. marchalianum with either 28S- targeted probe TmarchB10, TmarchC1_1 or TmarchC1_2 resulted in clear hybridisation signals in pure and mixed cultures (Figure 24) and environmental samples. Table 25 shows the sequences and target sites of the LSU targeted probes. Binding sites of the T. marchalianum specific probes are indicated in the model of the 28S rRNA secondary structure in Figure 26.

Table 25: Sequences and target sites of 28S rRNA oligonucleotide probes specific for Tetracladium marchalianum. Probe Sequence (5’-3’) Target site a Hybridisation stringency (%)b TmarchB10 GCTTAAGGTCAGGGGTAT 182-200 40 % TmarchC1_1 TCACCGGATGATCAACTG 605-623 20 % TmarchC1_2 GCCCATTCCCAGGCCTTT 679-697 20 % a S. cerevisiae numbering; b in percent formamide in the hybridisation buffer

1.2.2. Probes specific for Alatospora acuminata For the development of species specific oligonucleotide probes for Alatospora acuminata the intron positions within the 18S rRNA sequences (see results chapter part B) were eliminated. The intron positions were not tested because strain L8 did not show any intron. The first species specific FISH oligonucleotide for A. acuminata, probe Alacumi1491, designed and evaluated in May 2001 showed unacceptable secondary matches with bacteria when tested against ARB database as of December 2001. Therefore a new specific probe had to be designed. The new 18 mer probe ALacumi1698 binds to a region at helix 49 in the 18S rRNA secondary structure model (Figure 25) and showed at least two mismatches to other bacterial, fungal or algal sequences as tested against all sequences accessible in ARB databases and GenBank. Probe ALacumi1698 and probe Hlug1698, specific for Heliscus lugdunensis (see Results part C 1.2.5.) bind to the same target regions of the 18SrRNA molecule. Therefore probe ALacumi1698 was tested against Heliscus lugdunensis. The target regions of the probes ALacumi1698 and Hlug1698 had only five nucleotide positions in common. No unspecific binding of probe ALacumi1698 to H. lugdunensis was observed. Four fungi of the order Lecanorales (GenBank accession numbers U86695; U86696; U86697 and AF241543) and two fungi of the order Pertusariales (GenBank accession numbers AF274110 and AF274114) displayed two mismatches at positions 14 and 17 relative to the target region of probe ALacumi1698. None of the reference organisms, displaying two mismatches in the probe target region, were available for in situ testing. Therefore the hybridisation stringency was adjusted to 40 %. A. acuminata strains F-2380; F-13089;

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F-37194, F-12186; F-18799 and L8 showed clear FISH signals when hybridised with probe ALacumi1698 in pure cultures and environmental samples (Figure 24). Table 26 shows the probe sequence and target sites of probe ALacumi1698 for the specific detection of A. acuminata strains.

Table 26: Sequences and target sites of 18S rRNA oligonucleotide probe ALacumi1698 specific for Alatospora acuminata Probe ALacumi1698 (5’ – 3’) Alatospora acuminata strain CCGCGCTAGGTGGTCGTT target site CCM F-02383 1679-1697 CCM F-12186 1449-1467 CCM F-13089 1683-1701 CCM F-18799 1685-1703 CCM F-37194 1668-1686 L8 1681-1699

1.2.3. Probe specific for Tricladium angulatum For the Tricladium angulatum strains F-01380; F-139; F-10200 and F-14186 one specific oligonucleotide probe was designed. The probe TRIang322 targets the 18S rRNA region at helix 12 (Figure 25) and matches all four strains without mismatches. The closest 18S rRNA sequence of a non-target organism was that of the lichen Pertusaria amara (GenBank accession no: AF356682) showing two centrally located mismatches. All other currently known (September 10th 2002) sequences showed at least three mismatches within the probe target sequence. FISH signals exclusively of T. angulatum strains were obtained at a hybridisation stringency level of 35 % during in situ hybridisations testing T. angulatum strains, T. splendens strains and Tetracladium marchalianum strains (Figure 24). Table 27 shows the probe TRIang322 sequence and target sites of the sequences of the Tricladium strains. As no close relation between T. angulatum and T. splendens is visible (see results chapter B) it was not possible to design a genus specific oligonucleotide probe covering both Tricladium species. The construction of a probe specific for the genus Tricladium might only be possible if more sequences of Tricladium species are known. Even the attempt of designing a multiprobe array specific to T. splendens failed due to the high sequence similarities to Anguillospora crassa.

Table 27: Sequences and target sites of 18S rRNA oligonucleotide probe TRIang322 specific for Tricladium angulatum Probe TRIang322 (5’ – 3’) Tricladium angulatum strain GCCCACTACGCTACAATC target site CCM F-10200 297-315 CCM F-01380 304-322 CCM F-14186 291-309 CCM F-139 236-254

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1.2.4. Probe specific for Heliscus lugdunensis For Heliscus lugdunensis, another abundant species frequently occurring on leaves and in foam samples (Ingold 1975), an 18S rRNA targeted probe could be designed. The 18 mer probe Hlug1698 binds to a location close to helix 49 in the 18S rRNA secondary structure model (Figure 25). Computer assisted sequence comparisons showed that probe Hlug1698 showed one C/U mismatch to the 18S rRNA sequence of Trichoderma viride. In hybridisation experiments the formamide concentration had to be adjusted to 50 % for clearly separating T. viride from the H. lugdunensis target strains. Positive FISH signals with probe Hlug1698 were observed for all eight H. lugdunensis strains (F-245; F-13783; F-05185; F-10699; F-12486; L5; ELBE98; Bärlocher). This was done in cultures (Figure 24). Also in environmental samples FISH could unequivocally detect H. lugdunensis with the specific probe Hlug1698. Table 28 shows the probe sequence and target sites of sequenced H. lugdunensis strains and the non – target organism T. viride.

Table 28: Sequence and target sites of probe Hlug1698 specific for Heliscus lugdunensis Target region of probe Hlug1698 GCAACCACCACUCAGGGC Species / strain mismatches are underlined H. lugdunensis CCM F-13783 1667-1684 GCAACCACCACUCAGGGC H. lugdunensis CCM F-245 1667-1684 H. lugdunensis L5 1667-1684 Trichoderma viride 1694-1711 GCAACCACCACUUAGGGC

1.2.5. Probes specific for Anguillospora longissima Among the unbranched elongate conidia, the typically sigmoid or arcurate conidia of A. crassa and A. longissima are frequently reported by several authors (Ingold 1975; Marvanová 1984; Gönczöl et al. 1999a). No specific FISH probe for A. crassa on the basis of 18S rDNA sequence data could be designed. The sequence similarities of 18S rDNA sequence of Tricladium splendens and A. crassa are too high. The 28S rDNA sequence data of A. crassa could not be obtained. For the specific detection of A. longissima strains, probes on the basis of 18S and 28S rDNA sequence data could be designed. The 18S rRNA targeted probe Alongi340 binds to a region close to helix 12 in the 18S rRNA secondary structure model (Figure 25). The probe Alongi340 showed at least three 3’ end mismatches to the target regions of other organisms than A. longissima strains F-11891, F-00980 and L22. In situ testing of the probe Alongi340 was performed against Leptosphaeria bicolor which is frequently isolated from twigs and also a representative of the order Pleosporales. At 40 % formamide A. longissima could be clearly distinguished. Table 29 shows probe Alongi340 sequence and target regions of A. longissima strains. Performing FISH with the probe Alongi340, strains of A. longissima could be unambiguously detected (Figure 24).

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Table 29: Sequence and target sites of 18S rRNA targeted probe Alongi340 specific for Anguillospora longissima Probe Alongi340 (5’ – 3’) Anguillospora longissima strain CCCGTTGAAACCATAGGT target site CCM F-00980 292-309 CCM F-11891 310-328 L22 310-328

As another probe for the specific in situ detection of A. longissima, the 28S rRNA targeted probe AlongiB16 could be designed and successfully evaluated. The new probe AlongiB16 binds to a region of helix B16 in the LSU secondary structure model as shown in Figure 26. The probe AlongiB16 showed at least two mismatches in its target region of all other sequences available in databases. In situ testing of probe AlongiB16 against A. longissima strains F-00980, F-11891, F-10691, F-11791, L22 and the non – target organisms Leptosphaeria bicolor and Clavariopsis aquatica showed exclusively staining of the target organism when the stringency was adjusted to 35 % (Figure 24). Table 30 shows the AlongiB16 probe sequence and target site.

Table 30: Sequence and target site of the 28S rRNA oligonucleotide probe specific for Anguillospora longissima Probe Sequence (5’-3’) Target site a Hybridisation stringency (%)b AlongiB16 GCGCTGTTCCAAGGGTCT 345-363 35 % a S. cerevisiae numbering; b in percent formamide in the hybridisation buffer

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Figure 24 A-G

85 20 µm F Results CG

Figure 24: FISH of fungal cultures with specific fungal oligonucleotide probes. A CLSM photomicrograph showing FISH of Tetracladium marchalianum with specific 28S rRNA targeted Cy3-labeled probe TmarchC1_1. B CLSM photomicrograph of Alatospora acuminata hybridised with 18S rRNA targeted Cy3-labelled probe ALacumi1698. C CLSM photomicrograph of Tricladium angulatum after FISH with 18S rRNA targeted Cy3-labelled probe TRIang322. D CLSM photomicrograph of germinating conidium of Heliscus lugdunensis after FISH with 18S rRNA targeted Oregon Green labeled probe Hlug1698. E CLSM photomicrograph of Anguillospora longissima after FISH with 18S rRNA targeted Cy3 labeled probe Alongi340. F Phase contrast photomicrograph of Anguillospora longissima and G corresponding CLSM photomicrograph after FISH with 28S rRNA targeted Oregon Green-labeled probe AlongiB16.

1.3. Summary of all newly designed fungal oligonucleotide probes and their binding sites An overview of the probes is given in Table 31. Two division, nine genus and species-specific fungal oligonucleotide probes are shown, together with their target organisms, sequences and target positions. The attempt of designing probes that target the ITS rDNA region did not yield any useable FISH signals. They are not detailed here.

Table 31: Fungal oligonucleotide probes their target organisms, sequences and target positions

Hybridisation Target Probe sequence rRNA, helix b, Probe c stringency organisms 5’ – 3’ binding position FUN1429 35 Eumycota GTGATGTACTCGCTGGCC 18S; 36; 1429-47 MY1574 20 Eumycota TCCTCGTTGAAGAGC 18S; 47; 1474-89 Genus TCLAD1395 35 CGCACTTCCATTTGCTTG 18S; 44; 1395-1413 Tetracladium Tetracladium TmarchB10 40 GCTTAAGGTCAGGGGTAT 28S; B10; 182-200 marchalianum Tetracladium TmarchC1_1 20 TCACCGGATGATCAACTG 28S; C1_1; 605-23 marchalianum Tetracladium TmarchC1_2 20 GCCCATTCCCAGGCCTTT 28S; C1_2; 679-97 marchalianum Alatospora ALacumi1698 40 CCGCGCTAGGTGGTCGTT 18S; 49; 1698-1716 acuminata Tricladium TRIang322 35 GCCCACTACGCTACAATC 18S; 12; 322-40 angulatum Heliscus Hlug1698 50 GCAACCACCACUCAGGGC 18S; 49; 1698-1716 lugdunensis Anguillospora Alongi340 40 CCCGTTGAAACCATAGGT 18S; 12; 340-58 longissima Anguillospora AlongiB16 35 GCGCTGTTCCAAGGGTCT 28S; B16; 345-363 longissima a in percent formamide in the hybridisation buffer; b according to Ben Ali et al. 1999; Wuyts et al. 2000 c S. cerevisiae numbering

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Figure 25: 18S rRNA secondary structure model of Saccharomyces cerevisiae (GenBank accession number VO1335) showing the binding positions of the newly developed 18S rRNA targeted fungal FISH probes (SSU secondary structure model after Van de Peer et al. 1997).

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E20_1 G5_1

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E19 D18 D19 E18 G16 E11_1 E16 E17 G17 E11 E3 E5 E10 F1 G1 E7 G18 E4 G20 E1 E8 E2 D17 E6 E9_1 G19 E9 H2 H1 H3

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B20 B19 B13 C1_1 B11 B18 C1_2 TmarchC1_1 B12 B14

B13_1 TmarchC1_2 B15 AlongiB16

B17 B16

Figure 26: 28S rRNA secondary structure model of Saccharomyces cerevisiae (GenBank accession number VO1335) showing the binding positions of the newly developed rRNA targeted fungal FISH probes (LSU secondary structure model after Ben Ali et al. 1999).

88 Results C

2. Evaluation of factors influencing FISH, and improvement of fungal FISH signal detection The accessibility of freshwater fungi for fluorescence in situ hybridisation depends on a multitude of parameters. The influence of the following parameters was investigated: • The culture media, • Fixation methods, • Inherent autofluorescence, • Methods for increasing the permeability of cell walls, • The age of the fungal cultures, • The fungal cell type, • Different microscopical methods, • Incubation time, • Composition of the hybridisation buffers.

2.1. Influence of culture media The accessibility of the fungal strains for FISH was not significantly influenced by the use of malt, oatmeal, water agar or liquid growth media.

2.2. Influence of fixation methods The influence of different fixation methods on the accessibility of fungi for hybridisation were investigated using; paraformaldehyde in PBS, paraformaldehyde-ethanol-acetic acid, ethanol and methanol. The fixation methods were tested on conidia, cultures, hyphal tips, biofilms and river snow samples. Fixation times varied from 1 to 4 h. Hybridisation of cultures, hyphal tips, biofilms on leaves, polyethylene slides, cellulose and cage membranes showed good results when fixed with 3.7 % paraformaldehyde in PBS for 2 to 4 h. Shorter fixation times reduced the hybridisation success. The shape of the macroconidia from foam sometimes suffered from paraformaldehyde in PBS fixation. The conidial shape remained undistorted when fixed with paraformaldehyde-ethanol-acetic acid (FAA) solution. Non-germinating conidia from foam samples were not accessible for in situ hybridisation. Therefore the FAA method was used only for the examination of foam samples by phase contrast microscopy. Ethanol fixation did not result in hybridisation success. Methanol fixation increased autofluorescence of fungal structures and background fluorescence and was not further employed. Washing out the fixation agents proved to be essential for successful fungal hybridisation.

2.3. Freshwater fungi and inherent fungal autofluorescence In order to evaluate the inherent autofluorescence signals emitted from the different fungal species, the autofluorescence intensity was measured by performing a scan over the visible light spectrum (300 to 600 nm wavelength) employing CLSM. Two weeks old fungal samples of pure cultures without FISH assay, but with or without a hybridisation buffer and/or with a permeabilisation buffer and/or after the electroporation procedure were tested. As indicated in Figures 27 to 29, the majority of fungi tested, showed the strongest autofluorescence signals using the microscope light filter set in the range of 450-490 nm excitation wavelengths. When performing FISH with Oregon green-labelled oligonucleotides, the probe signals were not

89 Results C distinguishable from autofluorescence signals, if applied to these fungi. Alatospora acuminata, Heliscus lugdunensis and Anguillospora longissima (and Penicillium chrysogenum) showed low or zero autofluorescence in the green light spectrum (Figure 28). They were therefore accessible for Oregon green-labelled probes. In contrast, the autofluorescence signal intensity, in the range of 300-400 and 550-600 nm, was much lower for all fungi tested, with the exception of Helicosporium phragmitis. Therefore labelling of the fungal probes with the indocarbocyanine dye Cy3 (excitation: 568 nm; emission: 565, 615 nm) proved to be preferable. Attempts to employ probes labelled with the fluorochrome Alexa Flour 350 (excitation: 346 nm; emission: 448) or Cy5 (excitation: 647 nm; emission: 670 nm) resulted in weak FISH signals (Figure 30). Figure 31 shows CLSM photomicrographs of the autofluorescence scan in the range of green, red and far-red excitation. Especially noteworthy is Figure 31-C, which shows a bright hybridisation signal of the Cy3-labelled probe MY1574 and a strong green autofluorescence signal at the cell wall of the fungal hyphae.

Treatment with sodiumborohydrid (50 mM NaBH4 in 100 mM Tris-HCl, pH=8; for 30 min) lowered the autofluorescence of leaves and made FISH signal detection of fungi possible. Neither the composition of the hybridisation buffer nor the electroporation procedure used influenced the intensities of fungal autofluorescence.

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Figure 27: Measurements of autofluorescence signal intensities (means ± SD) emitted from Tetracladium marchalianum (‹); Helicodendron giganteum („); Helicomyces roseus (S) and Helicosporium phragmites (z) in the range of 300 to 600 nm wavelengths. Arrows indicate the excitation of fluorochromes Alexa Flour 350, Oregon Green and Cy3.

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Figure 28: Measurements of autofluorescence signal intensities (means ± SD) emitted from Alatospora acuminata (‹); Anguillospora crassa („); Anguillospora longissima (S) and Heliscus lugdunensis (z) in the range of 300 to 600 nm.

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Figure 29: Measurements of autofluorescence signal intensities (means ± SD) emitted from Tricladium angulatum (‹); Tricladium splendens („); Varicosporium elodeae (S) and Penicillium chrysogenum (z) in the range of 300 to 600 nm.

Figure 30: CLSM photomicrograph showing weak FISH signals of Heliscus lugdunensis after hybridisation with Cy5-labelled probe MY1574.

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Figure 31: CLSM photomicrographs of autofluorescence scans.

A Autofluorescence signal of Varicosporium elodeae in the range of 550-600 nm (red) excitation wavelength. B Multichannel scan of Helicosporium phragmitis showing strong autofluorescence signals in the range of 450-490 nm and slightly lower autofluorescence signals in the range of 600-650 nm. C Multichannel scan of Tetracladium marchalianum after hybridisation with Cy3-labelled probe MY1574 showing a probe conferred signal (red) and an autofluorescence signal in the range of 450-490 nm (green), localised at the cell walls ( ) D Autofluorescence signal of Heliscus lugdunensis in the range of 600-650 nm (far red).

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93 Results C

2.4. Comparison of epifluorescence and confocal laser scanning microscopy FISH signal intensities were examined either by epifluorescence microscopy or CLSM. When employing epifluorescence microscopy it was often hard to detect the fungal FISH signals. In addition, autofluorescence signals emitted from out-of-focus areas disturbed the FISH signal detection. Especially the very strong autofluorescence signals emitted from substrata such as leaves or cellulose made FISH signal detection impossible using epifluorescence. In contrast, confocal laser scanning microscopy enabled a much better detection of fungal FISH signals. Additionally, the ability to perform optical sections allowed a more precise examination of thicker structures. Thus, the spatial distribution of freshwater fungi, e.g. growing out of leaves, could be documented more clearly by performing stacks of several pictures along the z-axis.

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s s ta e m a m is a u it um nsi e tum nu ima ss n e den ina d la a ra te m n n lo u li iss n g u le um e g g s roseus ra d p c n iga e h g s a a cha ora c g c p ium ar lon y hrysoge lu m m ra sp n m c s iu iu m lo o m iu u d pora d il r r c spor a m u d ico o lis os o u g n el p e icla ic illospo e s lat ricl u An d H o H Tr A T ladi g o ic Var n ic l enicillium trac A el He P e H T Species

Figure 32: Comparison of fluorescence signal (means ± SD) conferred by Cy3 labelled fungal probe MY1574 and autofluorescence noise using epifluorescence microscopy ( ) and CLSM ( ).

As indicated in Figure 32, the mean background noise from employing CLSM was about 30 units lower than using epifluorescence microscopy. While investigating twelve fungal species hybridised with the Cy3-labelled probe MY1574, the probe conferred signals of eleven species were hardly detectable using epifluorescence microscopy. With the exception of Helicosporium phragmitis, the probe conferred fluorescence signal could be detected unambiguously for all species tested by CLSM.

94 Results C

2.5. FISH signal intensities of different parts of the mycelium In order to determine the FISH signal intensities of different parts of the mycelium, suspensions of conidia, conidiogenous cells, hyphal tips and thin (< 5 µm wide) and thick (> 5 µm wide) hyphae were investigated. Eleven species of freshwater fungi and Penicillium chrysogenum were tested. Conidiogenous cells could only be examined for 7 species. Hybridisations were performed with the probes FUN1429, MY1574 and species specific probes when available. Signal intensities of both probe conferred fluorescence and autofluorescence varied along the different parts of the mycelia (Figure 33). Thin hyphae showed FISH signals of almost 70 arbitrary brightness units. They could easily be detected due to the lower autofluorescence signals. Thick hyphae showed especially strong autofluorescence, resulting in hybridisation signals with half the intensity of thin hyphae. Strong probe conferred signals could be observed within metabolically highly active conidiogenous cells and hyphal tips. In contrast non-germinating conidia could not be hybridised, while germinating conidia are accessible for in situ hybridisation (e.g. Heliscus lugdunensis, Figure 24-D).

100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 H signal intensity [arbitrary units]

S 25 FI 20 15 10 5 0 conidia thin hyphae thick hyphae conidiogenous cells hyphal tips Localization within mycelium

Figure 33: Influence of mycelial loci and conidia on FISH signal intensities (means ± SD). Thin hyphae < 5µm; thick hyphae > 5µm wide. Results are given as mean values of hybridisations with fungal species Heliscus lugdunensis#, Tricladium splendens, Tricladium angulatum, Alatospora acuminata#, Varicosporium elodeae, Tetracladium marchalianum#, Anguillospora longissima, Anguillospora crassa, Helicodendron giganteum#, Helicomyces roseus#, Helicosporium phragmitis# and Penicillium chrysogenum#; # species of which conidiogenous cells were examined.

95 Results C

2.6. Influence of culture age The influence of the culture’s age on autofluorescence and FISH signal intensities was investigated by using 1, 2, 4 and 8 weeks old mycelia. Fluorescence intensities were measured having the microscope filter set to an excitation wavelength of 545 nm. FISH was performed with the Cy3-labelled probe MY1574. The same twelve fungal species as in 2.5 were examined. Autofluorescence increases with age of the culture. The FISH signal intensity also varied strongly depending on the species. The possibility of overlapping autofluorescence and FISH signals is given (indicated in Figure 34 by shadowed rectangles) in all ages. In 8 weeks old cultures the autofluorescence signal and the FISH signal were hardly distinguishable.

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m 55 [arbitrar 50 50 y 45 40 40 nal intensit nal

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Figure 34: Autofluorescence ( ) and FISH signal ( ) intensities in relation to culture age. Mean values of 12 fungal species. Standard deviation is given in the error bars. Shadowed rectangles indicate the range where standard deviations of both graphs overlap.

96 Results C

2.7. Permeabilisation FISH of freshwater fungi is often limited by weak fluorescence signals in combination with the inherent autofluorescence of fungi. Weak FISH signals can be caused by low metabolical activity and/or restricted permeability of the cell wall.

2.7.1. Permeabilisation of 2 and 8 weeks old cultures with chitinase treatment Permeabilisation of fungal cell walls with chitinase enhanced the detection of the probe signal. Lysis up to 6 min had no measurable effect of the fluorescence hybridisation signals. Enzymatic treatment with chitinase (pH 5.0) at a final concentration of 1 mg per ml for 10 min at 20 °C increased the probe conferred signal significantly in 2 and 8 weeks old cultures. FISH signal intensities after chitinase treatment were about 20 arbitrary units higher in the 2 weeks and 8 weeks old cultures, respectively. Chitinase treatments longer than 15 min did not enhance signal intensity. This effect was observed for all freshwater fungi tested and is shown as mean values for 11 species in Figure 35. However, the use of chitinase treatment prior to hybridisation sometimes lead to the destruction of finer morphological structures such as conidia, conidiogenous cells and hyphal tips.

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Figure 35: In situ hybridisation fluorescence signal intensities (means ± SD) of 2 weeks ( ) and 8 weeks ( ) old cultures of 11 freshwater fungal species after chitinase treatment (1mg per ml) for 0-20 minutes.

97 Results C

2.7.2. Permeabilisation by electroporation Electroporation was evaluated as a tool to introduce FISH probes into freshwater fungal cells. This was done to ensure proper permeabilisation of fungal cell walls and as an attempt to generate more homogenous FISH signal intensities. Fixed fungal material and oligonucleotide probe solution were transferred to a 0.2 cm gap cuvette and the electroporation procedure was run at 20 ± 3 °C, voltage 1.5 kV, capacitance 25 µF and resistance 200 Ω using a single pulse. The pulse width was 1.34 – 4.18 ms. Following electroporation, 50 µl of the hybridisation buffer replaced the water and hybridisation took place as described before. Electroporation FISH (E-FISH) was evaluated with the freshwater fungi Tetracladium marchalianum, Alatospora acuminata, Anguillospora longissima, Heliscus lugdunensis and with the common fungus Penicillium chrysogenum. The accessibility of these fungi for electroporation FISH was evaluated using the probes MY1574, FUN1429 and the species specific probes TmarchB10, ALacumi1698, AlongiB16 and Hlug1698. EUK516 was used as a positive control probe. The Bacteria specific probe EUB338 was used as a negative control. To ensure that non-binding and misbinding probes can be washed out properly, all probes were tested against target and non-target species, as described in results part C 1. Probe conferred signals have been observed to be weaker on exposed polyethylene (PE) slides as used in field sampling than in pure cultures. Therefore the accessibility of freshwater fungi for electroporation FISH was tested on fungi grown on polyethylene slides, too. The polyethylene slides were covered with water agar and then inoculated with conidia (100 ml-1). Mycelium was harvested after 14 days exposure in autoclaved tap water, scraping it from the polyethylene slide surface and fixing immediately as described before. In pure cultures, fungal probes and Eucarya signals were about 40 units brighter performing FISH after permeabilisation by electroporation than with paraformaldehyde fixation only (Student’s t-test, P=0.0058). The introduction of FISH probes by electroporation yielded about 20 units stronger fluorescence signal intensities than hybridisation after chitinase treatment (P=0.0101; Figure 36). With electroporation enhanced FISH, fluorescence intensity measurements were much more constant, indicated by a low standard deviation value (± 1.5). In contrast FISH signal measurements after permeabilisation by chitinase treatment or fixation only showed lower reproducibility indicated by higher standard deviation values of ± 9.2 (chitinase) and ± 17.5 (fixation only). Electroporation prior to FISH did not influence the specificity of the tested probes. Using the bacterial probe EUB338, no unspecific binding occurred. No unspecific binding of fungal specific probes with non-target organisms was observed. Non-germinating conidia were not accessible for the E-FISH method nor destroyed by the electroporation procedure. On polyethylene slides FISH signal intensities were not significantly lower than in pure cultures if permeabilisation was performed by fixation only or by chitinase treatment. But if E-FISH was employed, probe conferred signals were significantly more than 10 units lower on PE slides than in pure cultures. Compared to fixation only and chitinase treatment, electroporation FISH yielded slightly but significant higher signal intensities on PE slides (P=0.0004; P=0.017). On PE slides the FISH signal intensity measurements were more reproducible with electroporation FISH than with fixation only and chitinase treatment. However, this effect was less distinct on PE slides than it was in pure

98 Results C cultures as indicated by standard deviations of ± 5.9 units for electroporation FISH, ± 11 units for fixation only, and ± 9.1 units for chitinase treatment.

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Figure 36: FISH signal intensities after different types of permeabilisation treatments of pure cultures and of fungal material grown on polyethylene slides (PE). Shown are mean values of six different fungal species after hybridisation with probes EUK516, MY1574, FUN1429, TmarchB10, ALacumi1698, AlongiB16 and Hlug1698 (n=1140). Error bars indicate the standard deviation values.

2.8. Incubation time For hybridisation, the oligonucleotide solution, hybridisation buffer and sample were incubated for 1.5 to 16 h. Incubation times below 2 hours resulted in weak fluorescence signals of all fungi tested. No differences were observed between incubation times that varied between 2 and 16 hours.

99 Results D

D In situ detection of freshwater fungi

1. Critical evaluation of fluorescent stains In order to examine total fungal cell counts in pure cultures and environmental samples, different fluorescent stains were evaluated. The fluorescent stains were tested for their fungal specificity and their suitability for simultaneous application with rRNA-targeted probes. As control for the physiological activity of fungi a specific life-dead staining assay was tested.

1.1. Calcofluor White Staining of fungal cell walls with Calcofluor White was performed with all fungal species used in this study (see Material and Methods, Table 1). Additionally Calcofluor White (CFW) assays were tested on environmental samples such as leaves, foam, river snow, cellulose and polyethylene slides. Fungal cell walls were reproducibly stained by CFW. However, CFW also stained other structures that contain chitin e.g. residues of crustaceans which can be easily distinguished from fungal structures. Unfortunately, the dual staining of samples with CFW and FISH probes sometimes lead to the crystallisation of both the CFW and the fluorochrome label of the probes. This effect was not influenced by the CFW concentration or by the fixation method. Therefore CFW was only of limited suitability as a dual stain for the FISH method.

1.2. Life-Dead stain Life-dead staining was performed using the Live/Dead Yeast Viability kit (Molecular Probes), employing epifluorescence microscopy. Samples of living and inactivated Penicillium chrysogenum, Tetracladium marchalianum, Heliscus lugdunensis and Varicosporium elodeae cultures were tested. The distinct orange-red fluorescence of Cylindrical Intra Vacuolar Structures (CIVS) which are formed in metabolically active fungal cells could be observed for P. chrysogenum, only (data not shown). No positive reactions with germinating conidia of freshwater fungi were found. The Live/Dead Yeast Viability kit was therefore not suitable as fluorescent control for the physiological activity of fungi.

1.3. SYTO stains Seven preselected SYTO stains were evaluated for nuclear, mitochondrial or cytoplasmic staining of Penicillium chrysogenum, Tetracladium marchalianum, Heliscus lugdunensis and Varicosporium elodeae. The SYTO stains 9, 11, 13 and 21 were supposed to be live-cell nucleic acid stains. With the exception of SYTO 13, no reliable staining of fungal nuclei was observed. Using SYTO 24, 64 and 84 no staining of fungal structures was visible. SYTO 13 stained the fungal nuclei of all fungi tested. As shown in Figure 37, strong signals of SYTO 13 stained nuclei only in some cells and mostly at hyphal tips of the mycelium. This correlates with the observation that strongest FISH probe signals of fungal structures can be seen in hyphal tips and growing hyphae. However, none of the SYTO stains are fungi specific.

100 Results D

Figure 37: Fluorescent staining: CLSM photomicrograph of living mycelium of Varicosporium elodeae stained with Calcofluor white (blue) and nuclei stained with SYTO 13 (green).

1.4. Lectin stain Fixed fungal cultures, as well as fixed polyethylene slides that have been exposed to the Elbe river for several weeks were tested for fungal staining with the Alexa350 labelled WGA-lectin. The optimal concentration of the lectin was 50 µg/ml. All ten fungal species tested gave lectin based signals. However, at the surface of PE slides many structures of unclear function were also stained by the lectin. In contrast to staining with Calcofluor White, ambiguously lectin-stained structures could not be assigned to either fungal or other organisms in environmental samples. Clear lectin based fungal stains were obtained for bigger hyphal structures and in cultures, only. Therefore WGA-lectin staining was not suitable as a fungi specific stain.

2. FISH of freshwater fungi within environmental samples For the evaluation of the FISH method for the in situ detection of freshwater fungi in environmental samples, different types of exposed surfaces were examined. Additionally leaves sampled from the Oberer Seebach were subjected to FISH.

2.1. Biofilms on polyethylene slides Polyethylene slides were exposed for 2-4 months throughout the years 1999, 2000 and 2001 in the Elbe River. In order to examine the occurrence of freshwater fungi in biofilms on the PE slides, hybridisations with the probes EUB338 (Bacteria-specific), FUN1429, MY1574 and with the probes Hlug1698, TCLAD1395 and ALacumi1698 were performed. On the PE slides exposed during the months March, April and May (spring), June, July and August (summer) no fungi could be detected by

101 Results D either FISH or by staining with CFW. Only on PE slides exposed during autumn (September, October, November) and winter (December, January, February) could a small number of fungal structures be detected. Biofilms on PE slides exposed in the Elbe River consisted mainly of green algae in spring and summer months and mostly Bacillariophyceae and bacteria in autumn and winter months. The detection of fungal hyphae on PE slides was most successful using the probe MY1574. Hybridisations on PE slides with the fungal probe FUN1429 less frequently yielded FISH signals. This can be due to the more restricted phylogenetic range of probe FUN1429. Using Tetracladium-, Alatospora- and Heliscus-specific FISH probes, a FISH signal was only observed by probe Hlug1698 on a PE slide exposed from September – October 1999. Although conidia of Tetracladium marchalianum and Alatospora acuminata were existent in small numbers in foam samples of the Elbe River, none of these species could be observed on exposed PE slides. In Figures 38 A-B the detection of fungi on PE slides exposed in autumn and winter in the Elbe River using the probes FUN1429, MY1574 and Hlug1698 is shown. Within the Oberer Seebach (Lunz, Austria), PE slides were exposed for 2 weeks and for 3 months in autumn 2000, over winter in 2000/2001 and for 2 weeks in August 2001. The exposure of the PE slides within the RITRODAT took place at the sampling sites 1R1, with a slow current, and at 17G2.2 with a faster current. In contrast to the PE slides exposed within the Elbe River, fungi were more frequent on the PE slides exposed in the Oberer Seebach. After being exposed for 2 weeks in autumn 2000 and August 2001 many conidia of freshwater fungi were attached to the PE slides. They were accessible for FISH as soon as they formed appressoria or were germinating (Figures 38 C-F). Appressoria were formed by tetraradiate and by sigmoid conidia. Non-germinating conidia could be easily detected by CFW staining (Fig. 39 A). In contrast to the samples exposed for 2 weeks, conidia were not existent on the PE slides exposed for three months in September, October and November 2000. Here, among thick layers of bacteria, hyphae were detected by FISH with the probe M1574 (Figure 39 B). On the PE slides exposed during the winter (October 2000 – February 2001) in the Oberer Seebach no conidia were observed nor could hyphae be detected by FISH. No difference in the accessibility of fungal structures for FISH was observed between the PE slides exposed at sampling sites with the slow and fast currents. As in pure cultures, probe conferred signal intensities varied in different parts of the mycelium on the PE slides. Fungal structures that were suspected to have high metabolic intensities, for example appressoria, showed very bright FISH signals. In contrast, hyphae often showed both cells with FISH signal and cells without FISH signal.

102 Results D

Ap

Figure 38 A-F

103 Results D

Figure 38: FISH of freshwater fungi on exposed polyethylene slides: A Biofilm obtained on PE slide exposed for 3 months (December 1999-February 2000) within the Elbe River, probed with CY3-labelled probe MY1574 (red) and probe EUB338 (green). B Biofilm obtained on PE slide exposed from September-November 1999 within the Elbe River, probed with Oregon green-labelled probe Hlug1698 (bright green) and CY3-labelled probe EUB338 (red). C Conidium of Tetracladium marchalianum attached to PE slide exposed for 2 weeks within the Oberer Seebach (Lunz, autumn 2000) hybridised with CY3-labelled probe MY1574 and simultaneously stained with Calcofluor White; Ap = appressorium. D Conidium of cf. Anguillospora filiformis attached to PE slide exposed for 2 weeks within the Oberer Seebach (Lunz, autumn 2000) hybridised with CY3-labelled probe MY1574 and simultaneously stained with Calcofluor White and Bacteria hybridised with Oregon green-labelled probe EUB338. E Conidium of Alatospora acuminata attached to PE slide exposed for 2 weeks within the Oberer Seebach (Lunz, August 2001) hybridised with CY3-labelled probe ALacumi1698 and simultaneously stained with Calcofluor White. F Germinating conidium of Anguillospora cf. crassa attached to PE slide (2 weeks exposure, autumn 2000) after FISH with CY3-labelled probe MY1574 and simultaneously staining with Calcofluor White.

Figure 39: Freshwater fungi observed in the Oberer Seebach on exposed polyethylene slides.

A Conidium of Clavariopsis aquatica stained with Calcofluor White on PE slide exposed for two weeks (August 2001) within the Oberer Seebach B Biofilm obtained on PE slide exposed for 3 months (September –November 2000) within the Oberer Seebach, probed with Oregon green-labelled probe EUB338 (green) and CY3-labelled probe MY1574 (red). Also visible: auto-fluorescing diatoms.

104 Results D

2.2. Fungi directed FISH on leaves and cellulose 2.2.1. Leaves Leaves of Acer pseudoplatanus, Acer campestre, Fagus sylvatica and Salix caprea were exposed in the Oberer Seebach for two weeks in autumn 2000. Additionally, decaying leaves were collected from the water. Prior to FISH the leaves had to be subjected to treatment with sodiumborohydrid to lower the leaf’s inherent autofluorescence. On all types of leaves growing hyphae and germinating conidia could be detected by FISH. In situ probing was performed with the Eumycota probe MY1574 and with genus and species specific fungal probes. Although many non-germinating conidia of aquatic hyphomycetes were abundant on the leaves, most FISH signals were obtained by using the Eumycota probe MY1574. To a lesser extent using specific probes for freshwater fungi yielded in hybridisation signals. This does not mean that aquatic hyphomycetes, according to the definition of Webster and Descals (1981), were absent on the leaves: Probing the leaves with probe MY1574 showed FISH signals with Clavariopsis aquatica (Fig. 40 A) and an unidentified fungus producing filiform phialoconidia (Fig. 40 B). Using the fungal species-specific probes available in this study, only Alatospora acuminata was detected on a beech leaf by hybridisation with probe ALacumi1491 (Fig. 40 C). As found out in December 2001, the probe ALacumi1491 also matched some bacteria. However, the organism detected by probe ALacumi1491 is unambiguously a fungus. In computer-assisted evaluations against all available sequence databases, probe ALacumi1491 did not match any other fungi than A. acuminata. Remarkably, the fungi detected by FISH signals of probe MY1574 often showed Acremonium- and Phialophora-like phialidic structures (Fig. 40 D, E). About one third of hyphae detected by Calcofluor White staining were not accessible for FISH using the fungal probes available in this study. An example for non-hybridised but visible by CFW stained hyphae is shown in Figure 40 F. As on the PE slides, no difference in the accessibility of fungal structures for FISH was observed on leaves exposed at the RITRODAT sampling sites 2C1.1 (slow current) and 16F3.3 (fast current).

2.2.2. Cellulose Autoclaved pieces of native cellulose were exposed for 10 days at the RITRODAT sampling sites 2C1.1 (slow current) and 16F3.3 (fast current) in autumn 2000. As already mentioned in the results in chapter A, no fungi were present on exposed cellulose pieces.

105 Results D

Figure 40

106 Results D

Figure 40: FISH of freshwater fungi on leaves from the Oberer Seebach. A Conidium and conidiophore of Clavariopsis aquatica growing out of a Fagus sylvatica leaf collected from the Oberer Seebach after hybridisation with CY3-labelled probe MY1574. B Unidentified fungus producing filiform phialoconidia on leaf of F. sylvatica exposed for 2 weeks in the Oberer Seebach (autumn 2000) after hybridisation with CY3-labelled probe MY1574. C Alatospora acuminata on leaf of Acer pseudoplatanus exposed for 2 weeks in the Oberer Seebach (autumn 2000) after hybridisation with CY3-labelled probe ALacumi1491. Remarkable strong FISH signal at hyphal tips ( ). D Fungal hyphae and phialids on F. sylvatica leaf exposed for 2 weeks in the Oberer Seebach (autumn 2000) after hybridisation with CY3-labelled probe MY1574 simultaneously stained with CFW. E Fungal hyphae and phialids on Acer campestre leaf exposed for 2 weeks in the Oberer Seebach (autumn 2000) after hybridisation with CY3-labelled probe MY1574 simultaneously stained with CFW. Also visible: Conidium of Alatospora acuminata ( ). F Fungal hyphae on leaf of A. campestre with septa stained by CFW but without FISH signal after probing with CY3-labelled probe MY1574.

2.3. Fungal FISH of foam samples Many freshwater fungi’s conidia were abundant in foam samples taken from the Oberer Seebach. Foam samples fixed with formaldehyde-acetic acid-ethanol solution had to be thoroughly washed with PBS because fixation residues tend to hinder the FISH procedure. Hereby it is essentially important to carefully centrifuge the samples at 3000 to 6000 rpm. Higher speeds destroy the macroconidia of the aquatic hyphomycetes. Hybridisations were performed with the CY3-labelled probes MY1574, FUN1429, and genus and species specific fungal probes. Conidia in foam were never germinated (Figure 41). The conidia found in foam never showed any probe conferred signals.

Figure 41: Interference contrast photomicrograph of conidia of freshwater fungi in foam sample taken from the Oberer Seebach in autumn 2000. The scale bar corresponds to 20 µm.

107 Results D

2.4. River snow The organisms in the snow in the Elbe River were mainly algae and bacteria in the spring and summer and only bacteria in the autumn and winter. Filamentous structures within in river snow aggregates were never accessible for FISH with fungal probes. Other filamentous structures could be clearly determined as green algae or cyanobacteria due to the characteristic fluorescence of chlorophyll pigments. Examination of river snow aggregates by phase contrast microscopy also gave no evidence for the presence of fungal structures. The small number of three river snow particles in 1l Oberer Seebach water comprised algae (diatoms) and detritus. No filamentous structures nor conidia were observed in the river snow of the Oberer Seebach.

3. Investigation of the germination of conidia and spores within the Oberer Seebach by FISH In order to examine the germination of conidia and spores under natural conditions, conidia, basidiospores, ascospores and hyphal tips were exposed in cages within the RITRODAT area of the Oberer Seebach in autumn 2000. The cages were exposed at the slow and fast current sites for 2, (29.9.-1.10.2000) 5 (1.10.-6.10.2000), and 14 (30.9.-13.10.2000) days in 5, 10 and 25 cm water depth. The water temperature of the Oberer Seebach was stable at 11° C over the entire time of the germination experiments. Following the harvest, germinating conidia and spores were counted directly on the cage membranes. Hyphal tips were measured directly on the membranes. Branching, new septa and elongation > 100% were counted as growth. The accessibility of the exposed conidia and spores for FISH was tested by using the probes Hlug1698; MY1574 and TmarchB10. As shown in Table 32 only conidia of the freshwater fungi showed germination after being exposed in the cages. None of the exposed ascospores or basidiospores or hyphae showed germination or growth. One cage was lost in the Oberer Seebach at site 15F3.3 fast. After 2 days of exposure 80 % of the total number of exposed conidia were still visible on the cage membranes. In contrast, the longer exposure time resulted in increased losses of conidia, spores and hyphae. It could not clearly be determined if the missing parts were washed out or somehow decayed. Decaying activities could have been due to increased numbers of bacteria observed on the entire surface of the cage membranes after the longer exposure period. Bacteria were also observed close, but not directly on remaining conidia after 5 days exposure time of the cages. The germination rates were measured in relation to the total number of conidia originally exposed. The highest germination rate of conidia showed Heliscus lugdunensis (Figure 42-A, B) in cages after exposure for 2 days at site 15F3.3 fast current (80-100 %) and after 5 d exposure at both sites with slow (75-100 %) and fast current (80-100 %). A dense layer of extracellular material displaying a dark red autofluorescence (Figure 42-C) was observed around the hyphae of germinated H. lugdunensis conidia on the cage membrane. After 2 days exposure at the slow current site 2C1.1, H. lugdunensis showed a germination rate of only 15-20 %. Germination of Varicosporium elodeae (Figure 42 D) conidia was observed at slow current exposure sites, only. The germination rate was 9-10 % after 2 days of exposure and 16-20 % after 5 days. Conidia of Tetracladium marchalianum (Figure 42 E) showed germination rates of 5 % after 2 days and 8-10 % after 5 days of exposure at slow current sites.

108 Results D

Germination of conidia of Anguillospora crassa was observed at both slow and fast current sites. After 2 days of exposure, only one and two of only 10 exposed conidia were germinating at slow and fast currents, respectively. After 5 days of exposure the germination rate was also 10 % in the slow current, but the cage in the fast current was gone.

109 Results D

Table 32: Species, number of conidia and spores exposed, exposition time and site and germination rates in percent of total numbers originally exposed within the Oberer Seebach in autumn 2000 Conidia or spores Exposition- time Germination rate Species -1 Exposition-site exposed (ml ) (days) (%) Heliscus 680 2C1.1 slow 2 15-20 lugdunensis Heliscus 680 15F3.3 fast 2 80-100 lugdunensis Varicosporium 320 2C1.1 slow 2 9-10 elodeae Varicosporium 320 15F3.3 fast 2 0 elodeae Tetracladium 1000 2C1.1 slow 2 5 marchalianum Tetracladium 1000 15F3.3 fast 2 0 marchalianum Nectria cinnabarina 1000 2C1.1 slow 2 0 Nectria cinnabarina 1000 15F3.3 fast 2 0 Anguillospora crassa 10 2C1.1 slow 2 10 Anguillospora crassa 10 15F3.3 fast 2 20 Heliscus 680 2C1.1 slow 5 75-100 lugdunensis Heliscus 680 15F3.3 fast 5 80-100 lugdunensis Varicosporium 400 2C1.1 slow 5 16-20 elodeae Varicosporium 400 15F3.3 fast 5 0 elodeae Tetracladium 1000 2C1.1 slow 5 8-10 marchalianum Tetracladium 1000 15F3.3 fast 5 0 marchalianum Nectria cinnabarina 1000 2C1.1 slow 5 0 Nectria cinnabarina 1000 15F3.3 fast 5 0 Anguillospora crassa 10 2C1.1 slow 5 10 Anguillospora crassa 10 15F3.3 fast 5 gone Melastiza scotica 100 2C1.1 slow 5 0 Melastiza scotica 100 15F3.3 fast 5 0 Climacocystes hyphal tips 15F3.3 fast 14 0 borealis Climacocystes hyphal tips 1C1.2.slow 14 0 borealis Undetermined hyphal tips 15F3.3 fast 14 0 Aphyllophorales Undetermined hyphal tips 1C1.2 slow 14 0 Aphyllophorales Baeospora 300 15F3.3 fast 14 0 myriadophylla Baeospora 300 1C1.2. slow 14 0 myriadophylla Gymnopilus sp. 50 15F3.3 fast 14 0 Gymnopilus sp. 50 1C1.2. slow 14 0 Bjerkandera adusta hyphal tips 15F3.3 fast 14 0 Bjerkandera adusta hyphal tips 1C1.2. slow 14 0 Entoloma hirtipes 1000 15F3.3 fast 14 0 Entoloma hirtipes 1000 1C1.2. slow 14 0 Xylaria longipes 1000 15F3.3 fast 14 0 Xylaria longipes 1000 1C1.2. slow 14 0

110 Results D AA B

Figure 42: In situ detection of germinating conidia of freshwater fungi after exposure in the Oberer Seebach.

A Germinating conidium of Heliscus lugdunensis after 2 days exposure, probed with probe Hlug1698. B Hyphae and conidia of H. lugdunensis after 5 days exposure, probed with probe Hlug1698. C Magnification of hyphae from B with layer of extracellular mucilaginous material displaying dark red autofluorescence ( ). D Germinating conidium of Varicosporium elodeae after 2 days exposure, probed with probe MY1574. E Germinating conidium of Tetracladium marchalianum after 2 days exposure, probed with probe TmarchB10.

111 Discussion

DISCUSSION

A) Sampling and isolation of fungi in the river Elbe and the stream Oberer Seebach

For the first time, the fungal community of the river Elbe close to the German town Magdeburg was characterised employing general sampling and isolation strategies. In the Austrian stream Oberer Seebach, two short term studies of freshwater fungal assemblages to investigate their qualitative abundance and phylogenetic affiliation were conducted following earlier work of Marvanová and Gulis (2000). The occurrence of freshwater fungi in both lotic systems was studied on different substrata, such as leaves, wood, alder roots, polyethylene (PE) slides, foam samples and by examination of water and river snow samples. With the exception of river snow sampling and exposition of PE slides, the sampling and isolation strategies employed here are widely used for the characterisation of fungal communities in lotic systems (Webster & Descals 1981; Shearer & Lane 1983b; Bärlocher 1992). River snow samples were investigated as an important microhabitat of microorganisms in larger lotic systems (Böckelmann et al. 2000; Neu 2000) and because of their abundance at the river Elbe sampling sites. PE was used as a hydrophobic material enhancing bacterial attachment and growth (van der Kooj & Veenendaal 1993; Kalmbach 1998) and its aptitude for conidial attachment was tested. Conventional methods were applied for the characterisation of the river Elbe and Oberer Seebach freshwater fungal communities. However, the main purpose of sampling and isolation of fungal species in the present study was: (1) Utilising environmental specimen for phylogenetic investigations (2) Utilising environmental specimen for the development and evaluation of the FISH method for aquatic hyphomycetes. Therefore, this part of the study cannot be considered as an accurate ecological assessment. Nevertheless, the bimonthly sampling schedule in the river Elbe, which included different substrata and seasons, allows at least a restricted preliminary ecological assessment of fungal incidences in the lowland river. The short term sampling conducted in the Oberer Seebach, conveyed a qualitative and, to a lower extend, a quantitative impression of the fungal assemblages in this habitat.

1. Fungal assemblages in two different lotic habitats The low diversity of aquatic hyphomycetes in the lowland river Elbe in contrast to the high diversity in the alpine creek Oberer Seebach may be correlated with the availability of allochthonous leaf litter.

The majority of fungi abundant in the river Elbe on the substrata examined were terrestrial and airborne species. Only 11 species of aquatic hyphomycetes could be distinguished during 24 sampling events in three years at two sampling sites. Most of the freshwater fungi could only be detected in autumn foam samples. Only four times, one species (Clavariopsis aquatica) occurred on decaying alder leaves in spring and summer. This is remarkable as aquatic hyphomycetes are described as the main contributors to the breakdown of leaf litter (Suberkropp 1992a). Altogether, foam samples yielded 45 conidia of aquatic hyphomycetes which corresponds to 3.75 conidia on average in 10 ml Elbe

112 Discussion foam. This is an extremely low number if one considers tetraradiate and sigmoid spores accumulate in foam (Bärlocher 1992a) in lotic habitats. Furthermore, no freshwater fungal conidia in transport could be filtered from the Elbe water, even though this is one of the most frequently used methods for the estimation of aquatic fungal activities in lotic systems (e.g.: Gönczöl et al. 1999a). The attachment of freshwater fungal conidia to exposed PE slides was never observed.

In contrast to the river Elbe, the 100 m stretch of the RITRODAT study area within the Oberer Seebach provided obviously better conditions for aquatic hyphomycetes. Here, sampling and isolations yielded 36 different species of freshwater fungi in two 3-week campaigns in autumn 2000 and summer 2001. This is in accordance with the findings of Marvanová and Gulis (2000) who reported 16 species from one sample of plant debris and one of foam in the same area. With one exception (Isthmotricladia britannica DESCALS), they found the same conidia as in this study. Examining the same size of foam samples as taken in the river Elbe, the samples comprised 5653 conidia 10 ml-1 in autumn and 2654 conidia per foam sample in summer from the Oberer Seebach. Also on leaves, twigs and in water samples, conidia of aquatic hyphomycetes were highly abundant. Great numbers of different conidia were found to be attached on PE slides exposed for several weeks in the Oberer Seebach. Ten aquatic hyphomycete species occurred in both the Elbe river and the stream Oberer Seebach. Lotic systems are heterogeneous in their biota because of complex interactions between - riparian and catchment characteristics - water chemistry patterns - physical parameters - retention abilities - substrate availability - biotic interactions. The big differences of the fungal conidial assemblages of the river Elbe and the Oberer Seebach are not surprising when compared with earlier studies of aquatic fungal communities. Most studies were carried out in small streams and mountain creeks (Bärlocher 1992a) but few investigations were performed in rivers as large as the Elbe. For example, Chauvet (1989) found 16 aquatic hyphomycete species during 18 weeks in the 80 m wide and 2 m deep river Garonne in France. This is similar to the results in the Elbe. The Elbe at Km 322 and Km 318 is even larger with a width of approximately 250 m and a water discharge of 510 m3s-1 (Km 318). In contrast to this, the Oberer Seebach in Lunz is a small stream with a discharge of 0.72 m3s-1. The Oberer Seebach is completely canopied by the treetops of diverse shrubs and trees. Many leaves fall into the stream and provide large amounts of allochthonous substrate supply for aquatic hyphomycetes. The riparian vegetation on the Elbe banks is much less dense and often characterised by willows and dikes. The combination of the riparian vegetation and the huge water discharge leads to a drastic decline of the amount of terrestrial plant detritus available per volume (Bärlocher 1992b). Therefore, sufficient food supply for aquatic hyphomycetes might not be present in the river Elbe. The few leaves that could be collected from the Elbe water and river banks had often turned black due to the formation of FeS under anaerobic conditions. The fungal community examined microscopically on leaves was dominated by Fusarium

113 Discussion sp., Chalara-like fungi and other known phylloplane inhabitants such as Aureobasidium pullulans. On black leaves, collected from the water, hardly any fungi were visible by microscopical examination whereas bacteria were abundant. Additionally, the river Elbe at Magdeburg has little retention abilities. In the Oberer Seebach, natural dams of twigs, gravel and roots build many compartments where downstream transport is slowed. The middle reaches of the Elbe, however, resemble the typical potamon – zone (Illies 1961; Husmann 1970) with a sandy and silty riverbed and therefore fewer possibilities for longer residence of particles and organisms in a certain area. Under these circumstances, the question arises if the low numbers of leaves and the few conidia will allow aquatic hyphomycete colonisation of the substrate. Conidia in the Elbe may be swept downstream and, as observed in this study, get stuck in unfavourable locations without substrate availability, such as foam accumulated at the wall of the ferryship at the sampling site at Km 318. This leads to the question where the conidia observed in the Elbe originate. Only one species occurred on leaves collected from the river banks. The other ten species were solely observed in foam. If one regards the possible refuges and reservoirs for aquatic hyphomycete inoculation and recolonisation, the Elbe shows only beach-like boundaries (Naiman 1988) between riverbed and terrestrial environment. In contrast, the Oberer Seebach provides many microenvironments in the shape of mossy layers and biofilms on stones, woody debris, twigs and leaves where fungi could reproduce. Here also teleomorphic states of aquatic hyphomycetes might be the source for a new inoculum. On the other hand, it is unlikely that conidia observed in the Elbe are washed in from upper reaches of the river. Macroconidia of freshwater fungi are generally too fragile for long distance dispersal either in water or air. Half-life distances of conidial transport in streams have been estimated at 700-800 m (Thomas et al. 1991). However, the smaller sexual spores of teleomorphs of aquatic hyphomycetes may be transported over very long distances (Ingold 1975; Ranzoni 1979). Such a transport might be realised by air, aerosols or by animals such as birds. Another possibility would be that conidia are washed in from smaller streams that flow into the Elbe. Among others, Fisher & Petrini (1989) reported that aquatic hyphomycetes could survive in soils and remain viable for a month or more. Also, Bandoni (1972) wrote about the terrestrial occurrence of e.g. V. elodeae and A. acuminata on “high, well drained areas at some distance from streams”. Here possible transportation of conidia towards the river could take place over water films on leaves. However, some conidia were observed in the Elbe and the question arises if they have any chance to attach to a useful substrate in this habitat. If leaves are not available in sufficient amounts, the allochthonous substrate supply for freshwater fungi is insufficient. Other organic material, abundant in the Elbe, consists of detritus, river snow and great numbers of algae (Böckelmann 2001). But will any of these autochthonous carbon sources be suitable for aquatic hyphomycetes ? In river snow of the Elbe, no fungi were observed. Biofilms, other than river snow, cover the small particles in the river sediment. At these interfaces, fungi might adhere and even penetrate deeply into the sediment (Bärlocher & Murdoch 1989). It is known from studies of Metzler & Smock (1990) that leaves and detritus can be buried in the hyporheal zone in significant amounts (Hynes 1983) and some aquatic hyphomycetes can survive for some time under anaerobic conditions (Field & Webster 1983).

114 Discussion

2. Sampling and isolation strategies Classic sampling and isolation strategies revealed information about the conidial assemblages in the river Elbe and the Oberer Seebach but are dependent on isolation success and sometimes lack the possibility of species determination.

Although the isolation strategies used in this study were successful for the qualitative isolation of aquatic hyphomycetes in both lotic systems, most fungi isolated from the river Elbe were soil fungi (Domsch et al. 1980). Direct microscopic observation of foam and the different substrata revealed low numbers of aquatic hyphomycete macroconidia. From that, about one third could be isolated by transferring the conidia with a fine glass needle to malt agar. The isolation of the remaining two thirds often failed due to overgrow by fast-growing soil fungi. Soil fungi (e.g. Penicillium spp.) produce masses of very small conidia, which can adhere to the macroconidia of the targeted freshwater fungi. Transferred to malt agar, the soil fungi grow faster than the aquatic hyphomycetes despite low culturing temperatures. From foam, almost half of all isolated fungi were aquatic hyphomycetes. Terrestrial fungi observed to be trapped in foam were not only the larger obclavate conidia of Alternaria and the sickle-shaped macroconidia of Fusarium but also many small conidia. Spore suspensions from leaves, wood and alder roots of the river Elbe revealed solely isolates of soil and phylloplane fungi. Senescent leaves are colonised by a range of endophytes and phylloplane inhabiting terrestrial fungi. Some of the terrestrial fungi are known to persist after submersion of the leaves in water (Bärlocher 1992c). However, several studies (Bärlocher & Kendrick 1974; Suberkropp & Klug 1976a; Chergui & Pattée 1988) showed that, after submersion of the leaves, the terrestrial fungi were replaced by aquatic hyphomycetes. When sterilised leaves were exposed in a stream, they were colonised by terrestrial fungi only after 4-5 months (Bärlocher & Kendrick 1974). The above studies were carried out in small streams which provide good conditions for aquatic hyphomycetes. It is not clear whether terrestrial fungi take over the function of the main decomposers of leaves and wood in large rivers where aquatic hyphomycetes might be absent. Due to smaller conidia and often faster growth, soil fungi might be easier to isolate than aquatic hyphomycetes. However, according to the isolation frequencies, one might presume that soil fungi also represent the major group on the substrata collected from the Elbe. On the other hand, conidia and spores of soil fungi can be washed in from the banks of the river and many isolations probably have been performed from dormant stages (Gams et al. 1998). Also in the Oberer Seebach, isolation of aquatic hyphomycetes was difficult. But the huge amount of freshwater fungal conidia in foam and on every substrate examined made isolations successful more often than similar testing on the Elbe. River snow was hardly abundant in the Oberer Seebach. This could be due to more dissolved DOM and the smaller size of POM in combination with higher decomposing rates and stronger shredder activities (G. Bretschko pers. comm. 2001). Besides the isolation difficulties, because of contamination by soil and airborne fungi, many isolates did not grow sufficiently on various culture media. The induction of sporulation, even in aerated submerged cultures, was sometimes not successful. Conidia are not always sufficient for species identification. In many cases, especially for sigmoid conidia, the specifics of conidiogenesis and possible development of synanamorphs are necessary for a reliable determination.

115 Discussion

General sampling methods for aquatic hyphomycetes mainly capture their units of dispersal, the conidia. Maharning & Bärlocher (1996) showed that one cannot conclude with reasonable certainity from high conidia production on the amount of metabolic active mycelium present in an microenvironment. Also, the spatial distribution of a particular species cannot be determined with general sampling and isolation methods. Actually, the same is true for the investigations in the Oberer Seebach, where the great abundance of aquatic hyphomycetes was also confirmed from conidia, even though the life conditions for aquatic hyphomycetes in this habitat seem to be ideal. The topic of the last chapter of this discussion deals with the question whether the FISH method investigated during this study can detect active stages of freshwater fungi in lotic systems.

B) Phylogenetic characterisation of freshwater fungi

The ability of fungi to reproduce in water arose multiple times within the Ascomycota (Ingold 1975, Webster 1987, Webster 1992). The fact that the morphological similarity of tetraradiate or sigmoid conidia is not a sign of monophyly of aquatic hyphomycete taxa goes back to the findings of (e.g.) Webster 1959b; Webster 1961; Tubaki 1966;. Willoughby & Archer 1973 and Abdullah et al. 1981. The few already known connections of aquatic hyphomycetes anamorphs to their teleomorph states pointed to convergent evolution of conidia of fungi adapted to aquatic life. The teleomorphs can be found in different classes within the Ascomycota and, to a smaller extent, within the Basidiomycota (overview: Webster 1992; Shearer www). In the present study, the phylogenetic relationships of 11 species of aquatic hyphomycetes, including 7 species of anamorphic genera, were investigated. The results of sequencing different rDNA regions support the findings of a polyphyletic origin at least of aquatic hyphomycete genera. Phylogenetic analysis of 18S rDNA data placed 9 species (Tetracladium marchalianum, Lemonniera terrestris, L. aquatica, Varicosporium elodeae, Tricladium angulatum, T. splendens, Alatospora acuminata and Anguillospora crassa, A. furtiva) in a clade containing orders of the Leotiomycetes and Myxotrichaceae formerly classified in Onygenales. The species Heliscus lugdunensis and Anguillospora longissima were placed in the classes Sordariomycetes and Dothideomycetes, respectively. The grouping of the orders Cyttariales, Erysiphales, Leotiales, Helotiales, and Rhytismatales within the class Leotiomycetes has been noted several times (Saenz et al. 1994; Tehler et al. 2000; Winka & Eriksson 2000) and, undoubtedly, there are occurrences of paraphyly in this clade. Also, the placement of Myxotrichaceae in the Leotiomycetes class was observed earlier in SSU rDNA studies by Sugiyama et al. (1999). He concluded that the Myxotrichaceae are more closely related to the Erysiphales and Leotiales than to the Onygenales. Although the class Leotiomycetes is not supported in recent studies (Tehler et al. 2000; Winka & Eriksson 2000), it is used for the group of mainly non- lichenized inoperculate discomycetes of the order Helotiales. Remarkably, more than half of all known ascomycetous teleomorphs of aquatic hyphomycetes are located within the Helotiales (Webster 1992; Shearer http://fm5web.life.uiuc.edu:23523/ascomycete/default.html). In spite of these findings, the different conidiogenesis within the investigated taxa supports the argument of convergent evolution of conidia as an adaptation to life conditions in running waters. However, the terrestrial genera grouped in the Leotiomycetes-clade comprise fungi with a broad range of physiological abilities: Members of

116 Discussion the Erysiphales are plant pathogens (e.g. Blumeria graminis, powdery mildew), representatives of the Helotiales are saprobic on wood or parasitic on plants (e.g. Bulgaria inquinans; Leotia lubrica) and members of the Myxotrichaceae (e.g. Pseudogymnoascus roseus, Oidiodendron tenuissimum) are known to be saprobic and have cellulose degrading abilities. Therefore, the paraphyletic assemblage of Leotiomycetes provided a pool of adaptations that could have been useful for the evolution of leaf litter degrading aquatic hyphomycetes in lotic systems. The existence of a common ancestor connecting the aquatic hyphomycetes with orders of the Leotiomycetes is doubtful because this class is not resolved. The paraphyly of the Leotiomycetes clearly shows the need for additional sequence data. The public databases contain mainly sequences of economically important fungi, such as human and plant pathogenic fungi, and, to a much smaller extent, saprobic fungi. Therefore in many cases, closely related neighbours for the aquatic hyphomycetes investigated could not be found. This became obvious when the BLAST (Altschul et al. 1997) sequence similarity searches were performed.

1. Tetracladium marchalianum T. marchalianum is classified in the Leotiomycetes and shows most likely relations to plant associated fungi with phylogenetic affinities to the Helotiales.

Despite the different conidium morphologies, T. marchalianum strains were not separated from other Tetracladium species in 18S rDNA analyses. This is not surprising because all Tetracladium species investigated have less than 1 % overall nucleotide difference in the SSU region. In 18S rDNA analyses, the placement of T. marchalianum is either close to Helotiales or to Myxotrichaceae, depending on the tree inferring algorithms used. This is consistent with the results of Nikolcheva & Bärlocher (2002). They investigated phylogenetic relationships of T. marchalianum and 4 other Tetracladium species on the basis of 18S rDNA sequence data. The closest BLAST hits were Bulgaria inquinans (Helotiales), followed by Oidiodendron tenuissimum (Myxotrichaceae). B. inquinans is a wood inhabiting, saprobic inoperculate ascomycete with an uncertain Endomelanconium-like mitosporic state with holoblastic one-celled brown conidia (Döring & Triebel 1998; Verkley & van der Aa 1997). O. tenuissimum is a saprobic fungus with arthroconidia, occurring in soil, on decaying wood and bark (Domsch et al. 1980). In neighbour joining analysis, T. marchalianum was placed next to

Loramyces juncicola WESTON, although without statistical support. L. juncicola is a freshwater ascomycete usually found on decaying plant matter (Digby & Goos 1987). Its phylogenetic placement within Helotiales (Digby & Goos 1987) based on an apically opening ascus remains uncertain. Interestingly, L. juncicola has an Anguillospora-like anamorph state but did not show any close relationship to Anguillospora spp. sequenced in the present study. However, on the basis of 18S rDNA data, it is not possible to connect T. marchalianum to any of these representatives of orders or families mentioned above. The lowest distance value that could be found between T. marchalianum sequences and sequences of other fungi was 3.3 %, with the exception of other Tetracladium species. This is a distance value which appears to be common for sequence distance values observed for different orders in the SSU data matrix used in the present study. The long branched placement of T. marchalianum in SSU analyses indicates the presence of many apomorphic changes (Felsenstein 1978). Additionally, long branch attraction to other non related taxa of other orders cannot be excluded

117 Discussion

(Stiller & Hall 1999). This could explain the variable placement of T. marchalianum either in Helotiales or Myxotrichaceae or even close to Erysiphales, depending on the analytical method used.

In contrast to SSU analyses, the analyses of the more variable sequences of the ITS rDNA region clearly separated T. marchalianum strains from strains of other Tetracladium species. The overall nucleotide differences of 4.2 to 3.6 % in both the ITS1 and ITS2 regions to T. apiense, T. maxilliforme and T. furcatum were sufficient for distinguishing the different species on the basis of molecular data. This separates the different Tetracladium species according to the differences in conidium morphology summarised by Roldán et al. (1989). All T. marchalianum strains were placed in one cluster. No significant differences of the ITS sequences of isolates from different geographic regions were observed. Branching patterns within this cluster were never statistically supported. However, the investigation of different genotypes of geographically diverse strains would require a higher number of isolates and probably would require more variable target sequences. The closest known ITS1 and 2 sequences that could be found for Tetracladium were that of an axenic ectomycorrhizal isolate. The isolate was described by Vrålstad et al. (2002) who obtained it from a study of symbiotic root- associated ascomycetes with phylogenetic affinity to Helotiales. Another BLAST hit was Dactylaria dimorphospora which is a dematiaceous saprobic hyphomycete. All other BLAST hits were more or less darkly pigmented mycorrhizal hyphomycetes (Vrålstad et al. 2002) and one incompletely described member of Helotiales. Oidiodendron tenuissimum, which showed itself close to T. marchalianum in SSU analyses, was distantly related in ITS analyses. The nearest sister group to T. marchalianum in 28S rDNA sequence parsimony and ML analyses was an Antarctic yeast isolate. Both were placed within a cluster comprising Chalara spp., a salal root associated fungus, and Rhytisma acerinum but the grouping received only weak statistical support. As in SSU analyses, members of the Helotiales, Leotiales, Erysiphales and Rhytismatales clustered within a weakly supported Leotiomycetes clade. Thomas-Hall et al. (2001) classified their Antarctic yeast isolate as a member of the Saccharomycetales. This presumes a blastic acropetal or percurrent conidiogenesis. However, on the basis of the results of the LSU sequence analysis, this classification can be rejected. The Antarctic yeast sequence never grouped with Saccharomycetales when S. cerevisiae was included in the analyses (data not shown). It clearly has phylogenetic affinities to the Leotiomycetes. Yeast-like growth has evolved many times in different groups, e.g. as a response to environmental factors such as osmotic stress (Kendrick 1992). The anamorphic genus Chalara is a large morphologically and ecologically diverse group (Nag Raj & Kendrick 1993). Morphological criteria connecting Chalara anamorphs with ascomycete orders are not known (Gams & Phillipi 1992; Nag Raj & Kendrick 1993). Previously described teleomorph connections and molecular studies (Paulin & Harrington 2000) showed that many Chalara spp. have affinities to Helotiales.

The sequence of the salal (Gaultheria shallon, “Scheinbeere”; Ericaceae) associated root fungus showed close relationships to the Hymenoscyphus ericae aggregate (Allen et al. 2000).

The combined results of the investigations of three different rDNA regions were not sufficient to directly connect T. marchalianum to an ascomycete order. However, T. marchalianum could be clearly

118 Discussion classified as a member of the Leotiomycetes. Its repeated placement close to fungi with suspected Helotiales relationships suggests that T. marchalianum might be closely related to this unresolved order. The investigation of other genetic loci such as protein coding genes (e.g. the beta-tubulin gene or the alpha1-elongation factor gene) will probably not resolve the ambiguity caused by the lack of closely related sequences in public databases. They could be useful for the separation of T. marchalianum on the strain level. For separation at the species level, the ITS sequences of Tetracladium species have been shown to be sufficient. It should be noted that there are a relatively large number of insufficiently classified plant-associated environmental samples which showed the closest relationships to T. marchalianum. Searching for a teleomorph of T. marchalianum should therefore not be restricted to discomycetes in the vicinity of streams. The search should also include plant-associated inoperculate Ascomycetes, taking the terrestrial occurrences (Webster & Descals 1981) of T. marchalianum into account.

2. Alatospora acuminata All investigated A. acuminata strains are most likely related to the Leotiaceae but the sensu stricto and sensu lato morphotypes are clearly separated in ITS analyses.

Phylogenetic analyses of SSU sequences indicate that A. acuminata is part of the inoperculate discomycetes cluster and is closely related to Leotia lubrica. The statistical support for the branch placing A. acuminata as a sister taxonomical unit of L. lubrica is rather low (71-92 %). As stated by several authors (e. g. Felsenstein 1985; Swofford et al. 1996; Ludwig & Klenk 2001; Huelsenbeck 2001), resampling (bootstrapping) or posterior probability (Bayes) values below 95-99% give no convincing evidence that particular branching patterns allow phylogenetic conclusions. However, the repeated appearance of the same branching topology inferred by four different tree building methods provides a reasonable basis for the assumption that A. acuminata might be more closely related to the Leotiaceae than to any other family in the Helotiales. According to Eriksson et al. (2002), the order Helotiales is heterogeneous and the isolated genus Leotia is provisionally classified in the family Leotiaceae. Here, as in all other molecular phylogenetic analyses of the Leotiomycetes, more DNA data are needed before the phylogenetic relationships of the families in the Helotiales can be resolved.

The unclassified introns observed in the SSU region were not useful for phylogenetic analysis above strain level, because strain L8 was lacking this additional sequence. Additionally, those insertions vary in occurrence within groups and species and even within individuals (DePriest 1993). At the ITS level, no closely related sequences could be found for A. acuminata in public databases. The closest BLAST hits display high overall nucleotide differences from ITS sequences of A. acuminata which lead to long branched tree topologies. On the other hand, overall nucleotide differences of 3.5 – 5.6% were sufficient to clearly separate the different A. acuminata strains. The branching into two groups resembled the significant differences in conidium morphology. The strains CCMF- 02383 and CCMF- 13089 clustering in the sensu stricto- cluster were isolated in Great Britain and in the Czech Republic, respectively. These strains show the typical relatively small conidia, i.e., mostly less than 50 µm long; non-, one- and two-branched, with unconstricted branch bases that were described as matching the neotype (IMI 222997, B. J. Dyko) by

119 Discussion

Marvanová & Descals (1985, isolate CCMF- 02383 = CCMF- 12383) ). They classified A. acuminata strains displaying such conidium morphology as A. acuminata sensu stricto. The other cluster consists of the strains CCMF- 12186; CCMF-37194 and L8 which were isolated in Slovakia, Canada and Austria, respectively. They all resemble the A. acuminata sensu lato type with larger conidia (60 to over-80 µm long; two-branched, some with 3 or more branches) and distinctly constricted branch bases. No conclusions on geographic species variation can be drawn from the numbers of studied isolates. Reports of different conidium morphology due to water chemistry, water turbulence and availability of substrata (Webster & Davey 1975) do not provide an explanation for the observed presence of the two morphotypes. Conidia observed in the river Elbe were all of the sensu stricto type. In the Oberer Seebach (Austria), the sensu lato type was more abundant but the sensu stricto type was also present in large numbers. Many more isolates from different habitats should be investigated systematically to prove or reject any hypothesis of a preferred habitat for the two morphotypes. The distance values between the A. acuminata sensu stricto and sensu lato strains are similar to those that separate different Tetracladium species. Some of the A. acuminata sensu lato isolates investigated by Marvanová & Descals (1985) share morphological characteristics with A. constricta Dyko (1978) and A. pulchella Marvanová (1977). Before a maybe necessary modification of the contemporary species concept is possible, more isolates should be studied, using ecological, morphological and molecular methods. The further investigation of the ITS region of different morphotypes of A. acuminata and of other Alatospora species is a promising approach.

3. Tricladium splendens and Tricladium angulatum T. splendens is closely related to Anguillospora crassa and Zalerion varium with most likely classification in the Helotiales. T. angulatum shows close relationships to the Hyaloscyphaceae.

The investigation of SSU rDNA sequences of T. splendens and T. angulatum did not lead to an obvious connection to any order, but placed both species within the class Leotiomycetes. With the exception of the parsimony analysis, T. splendens and T. angulatum were never placed as sister clades. However, in the parsimony analyses, long branch lengths indicated many substitutional steps. This may indicate that the species cannot be grouped within one ascomycetous family. The type species of the large and very heterogeneous genus Tricladium is T. splendens, which has a known teleomorph Hymenoscyphus splendens (Abdullah et al. 1981). Unfortunately, a culture derived from ascospores of H. splendens was not available (J. Webster 2001, pers. communication) for inclusion in the analyses. Only the NJ method placed T. splendens with 95 % bootstrap support as a sister clade of Hymenoscyphus fructigenus. As described by Collado et al. (2002), Hymenoscyphus is also a very large and heterogeneous genus in terms of ecology and as revealed by sequence analyses of ITS and LSU rDNA data. Some species are mycorrhizal symbionts, e.g., H. ericae- aggregate (Vrålstad 2001) and some species are fruit associated saprophytes (e.g., H. fructigenus). The holotype specimen of H. splendens, deposited by Abdullah et al. (1981), was collected on cupules of Fagus. In spite of their heterogeneity, Hymenoscyphus spp. are all classified in the family Helotiaceae in current systems (Eriksson et al. 2002; Kirk et al. 2001: http://www. speciesfungorum. org/ Names/ fundic.asp). As many other inoperculate taxa Hymenoscyphus sequences are placed in the

120 Discussion paraphyletic Leotiomycetes cluster without clear internal subgroupings. This, in combination with the lack of closely related Hymenoscyphus SSU sequences, could explain the unresolved placement of T. splendens in SSU analyses. Interestingly, the lowest distance values of T. splendens SSU sequences were that of Anguillospora crassa (0.3 %, SSU distance matrix Fig. A35 in the appendix). In ITS analyses, T. splendens showed a close relationship to the mitosporic fungus Zalerion varium

ANASTASIOU. This marine fungus has irregular conidial coils (ranging from brown to dark brown) which consist of 10-30 cells. (Kohlmeyer & Volkmann-Kohlmeyer 1991). On the basis of molecular studies, Bills et al. (1999) suspected Z. varium as a member of the order Helotiales. Conidial coils are also common in aero-aquatic fungi such as Helicodendron. The Hymenoscyphus spp. included in the ITS analyses proved to be more distantly related to T. splendens than Z. varium. Also, the ITS sequences of T. angulatum were more similar to T. splendens ITS sequences than any available Hymenoscyphus sequence. Interestingly, the second most closely related sequence currently available is that of Anguillospora crassa. Thus, it seems that, of all the ITS sequences available, Zalerion varium and, to a lesser extent, Anguillospora crassa are the fungi most closely related to T. splendens strains. The relation of T. splendens to the unresolved order Helotiales is already proven by the connection to its teleomorph. It is noteworthy that Tricladium indicum has a teleomorph state, Cudoniella indica (Webster et al. 1995), which is also located in the Helotiaceae (Eriksson et al. 2002). The morphological characteristics which T. indicum and T. splendens share are the relatively big conidia with multiseptate, gently curved main axis and constricted branch insertion. Unfortunately, no sequence data of Cudoniella were available. The results of the molecular phylogenetic analyses of this study are not conclusive because of the obvious lack of additional sequence data. However, they support the phylogenetic affinity of T. splendens to the Helotiaceae. The different T. splendens isolates which possess significant difference in their conidial sizes were not separated by the ITS analyses. The occurrence of the extremely large conidia in isolate CCMF-16599 may be due to low nutrient availability in the habitat (Webster & Davey 1975). It should be noted that these large conidia were also developed under optimum culture conditions. Conidial size of T. indicum was dependent on culture age (Webster et al. 1995).

The impossibility of a well defined phylogenetic placement of T. angulatum in SSU analyses was indicated by long branches. This leads to the conclusion that sequences of closely related species were not included in the SSU analyses. BLAST searches indicated Erysiphales as possessing the most similar sequences but, in fact, T. angulatum displayed rather high distance values to the members of the Leotiomycetes. These values were similar to those found for Tetracladium. The distance values between T. angulatum SSU sequences and Tetracladium sequences were also very large. Thus, T. angulatum appeared to be very isolated within the Leotiomycetes.

In ITS NJ, parsimony and ML analyses, Cistella grevillei (BERK.) P. RASCHLE was placed as the closest sister group to T. angulatum. The discomycete C. grevillei is a member of the Hyaloscyphaceae, Helotiales (Cantrell & Hanlin 1997). The next sister group was an ericoid mycorrhizal fungus isolated from Erica plants in a Mediterranean forest (Bergero et al. 2000; Schadt 2002). Vrålstad et al. (2000) reported that some ericoid mycorrhizal fungi are not closely related to the Hymenoscyphus ericae

121 Discussion aggregate but rather to the Hyaloscyphaceae. Although this was not published by Bergero, the grouping of the ericoid mycorrhizal sp. Sm5 with C. grevillei in the present study suggests a closer relation to Hyaloscyphaceae for this fungus. Remarkably, Mycoarthris corralinus MARVANOVÁ & P.J.

FISHER, an arthroconidial fungus isolated from foam, also shows phylogenetic affinities to C. grevillei (Marvanová et al. 2002b). When M. corralina was included in the phylogenetic analyses (see appendix Fig. A15), it appeared to be more closely related to the ericoid mycorrhizal sp. Sm5 than to T. angulatum. Interestingly, Tricladium chaetocladium has a teleomorph, Hydrocina chaetocladia (Webster et al. 1990), which is classified in the Hyaloscyphaceae. In contrast to T. angulatum, T. chaetocladium has a dark mycelium and larger conidia. Additionally, two aero-aquatic hyphomycetes,

Clathrosphaerina zalewskii BEVERW. (Descals & Webster 1976) and Pseudoaegerita sp. (Abdullah & Webster 1983), have teleomorph states in the Hyaloscyphaceae. While the latter two have bulbil-like propagules, M. corallina has arthroconidia and T. angulatum and T. chaetocladium have branched conidia. It is most likely that morphological characters such as conidial shapes have evolved convergently in the Fungi. A fuller understanding of the phylogenetic relationships of T. angulatum becomes possible when more ITS sequences are available in public databases. However, the most promising approach would be to discover a teleomorph state of T. angulatum. It is likely to be found in the Hyaloscyphaceae.

4. Anguillospora longissima and Anguillospora crassa The genus Anguillospora has to be revised because A. crassa is classified in the Leotiomycetes with relation to the order Helotiales while A. longissima is classified in the Pleosporales with phylogenetic affinities to the genus Lophiostoma.

The rDNA molecular analyses of A. longissima and A. crassa confirmed the assumption that both species are related to different classes within the Ascomycota. According to Willoughby and Archer (1973) and Webster (1961), A. longissima and A. crassa are connected to the teleomorphs Massarina sp. and Mollisia uda, respectively. While A. crassa is located in the Leotiomycetes, A. longissima is a member of the Dothideomycetes. Additionally, A. furtiva WEBSTER & DESCALS was investigated in this study. It has a Pezoloma teleomorph (Descals et al. 1998) and it was also confirmed as a member of the Leotiomycetes. As suspected from the morphological characters of the known teleomorphs, all three species were unambiguously placed in two different classes by SSU analyses. Undoubtedly, the genus Anguillospora has to be revised, although further morphological and molecular studies of all currently known Anguillospora spp. should first be performed. Other known anamorph-teleomorph connections of the genus Anguillospora are A. fustiformis (Marvanová & Descals 1996), A. rosea (Descals et al. 1998) and Anguillospora sp. (Digby & Goos 1987). They are connected to teleomorph states classified in the Leotiomycetes and Orbiliomycetes. In contrast to the clear results on class level, A. crassa and A. furtiva could not be definitely connected to a certain order within the Leotiomycetes. There was weak or no bootstrap support for a placement as sister clade to Erysiphales. More closely related sequences were not available in public databases at the time of this study. In SSU analyses which included all aquatic hyphomycetes investigated in the present study (see Fig. A31-A33, appendix), A. crassa, A. furtiva and Tricladium splendens formed

122 Discussion one cluster. Together with the information about the teleomorphs of this three species, one may conclude that A. crassa and A. furtiva are also members of the unresolved order Helotiales.

In ITS sequences analyses, the A. crassa strains were not separated in spite of different conidium morphologies and different origin from isolates with either stalked or sessile apothecia. Here the ITS resolution power was not sufficient to separate the different morphotypes. All strains were placed as sister clade to Zalerion varium. Remarkably, this was the same placement as for T. splendens. The relation of A. crassa to Z. varium was less statistically supported than for T. splendens, although A. crassa showed higher overall nucleotide similarity with ITS sequences of Z. varium than T. splendens. According to the ITS sequence similarity values, A. crassa and T. splendens were more closely related than A. crassa and the teleomorph genus Mollisia. Additional NJ, parsimony and ML analyses of Leotiomycetes species (see Fig. A34, appendix) resulted in a highly supported cluster comprising A. crassa, T. splendens and Z. varium. All three species were clearly separated. On the basis of the teleomorph states of A. crassa and T. splendens, one would expect them to be in different genera. Therefore, one would have expected tree topologies indicating a closer relationship of A. crassa to Mollisia and of T. splendens to Hymenoscyphus. As mentioned above, the genus Hymenoscyphus seems to be very heterogeneous and the species available in databases may not be closely related to H. splendens. The same might be true for the genus Mollisia. However, it is uncertain whether A. crassa and T. splendens are closely related or whether the ITS sequence data are misleading. The resolution power of the ITS region is generally accepted to be sufficient for evaluating variations close to species level (Kohn 1992; Chen et al. 1992; Takamatsu et al. 1998). This is consistent with the results of this study, where Tetracladium spp., Alatospora morphotypes, Tricladium spp. and Anguillospora spp. were clearly separated. One should keep in mind that ITS sequence-based trees, as all other single gene trees, are not species trees (Doyle 1992). Lieckfeldt & Seifert (2000) discussed the use of ITS sequences in the taxonomy of Hypocreales and concluded that the region is not universally applicable as a species level marker. Two phenomena are reported: (i) The variability of the ITS region within some genera is not sufficient for species separation (e. g. Sclerotium, Carbone & Kohn 1993); and (ii) Due to gene duplication or interspecific hybridisation events, divergent types of the ITS 2 region occur (Waalwijk et al. 1996). As a result, in phylogenetic studies based on the whole ITS 1- 5.8 S- ITS 2 region, divergent ITS 2 types lead to presumptions of distant relationships (O´Donnell & Cigelnik 1997). Although the SSU analyses indicated a close relation of A. crassa and T. splendens, this conclusion should be further investigated by sequencing of the teleomorphs and multigene analysis.

A. longissima showed strong phylogenetic affinities to Pleosporales in SSU, ITS and LSU analyses.

The closest sister clade in SSU analyses was Kirschsteiniothelia maritima (LINDER) D. HAWKSW., a marine ascomycete (Hawksworth 1985). Other members of the genus Kirschsteiniothelia are terrestrial species (e.g., K. aethiops (BERK. & CURTIS) D. HAWKSW. with the exception of K. elaterascus, which was found in a freshwater habitat (Shearer 1993). However, K. elaterascus and K. aethiops are not closely related to K. maritima as is shown in the present study (Figures 18 and 19, RESULTS chapter). They never formed a cluster in SSU analyses and K aethiops was not even placed within the Pleosporales group.

123 Discussion

In all three tree building methods, a relation of A. longissima to the teleomorph genus Massarina was indicated by the highly supported sistergroup. The teleomorph genus Massarina has several aquatic anamorphs, including A. longissima, Clavariopsis aquatica and Tumularia aquatica (Webster 1965; Webster & Descals 1979). While T. aquatica is connected to M. aquatica, the teleomorphs of A. longissima and C. aquatica are described but not classified on the species level. In ITS analyses, the Leptosphaeria contecta sequence showed the best alignment and the highest sequence similarity to A. longissima strains. However, the teleomorph Massarina sp. (Willoughby & Archer 1973) is characterised by pseudothecia which cannot be confused with the cleistothecia which are typical for Leptosphaeria. As in SSU analyses, Massarina spp. were placed as a sistergroup to A. longissima. The ITS sequences of the Massarina spp. used were very heterogeneous (see alignment Fig. A40) and led to the exclusion of many ambiguously aligned regions in the tree reconstruction. Obviously, the Massarina sp. that was described as the teleomorph of A. longissima was not present in the analyses. To confirm an anamorph – teleomorph connection on the basis of ITS data, one would have to observe a 100 % similarity of the sequences of matching fungi (Bridge & Arora 1998). At least a 98 % homology of either the ITS1 or ITS2 region would have indicated a matching of teleomorph and anamorph. This was observed for the ITS1 region of Sclerotinia and Sclerotium by Carbone & Kohn (1993). Depending on the resolution of the gene used, sistergroup relationships between anamorph and teleomorph species are suggested, as it is the case for beta tubulin or mitochondrial small rDNA gene trees (O´Donnell et al.1998). Remarkably, not all Massarina spp. that were included in the ITS analyses were grouped in this clade forming the sistergroup of A. longissima. This indicates that Massarina is not a monophyletic genus, as already confirmed by Liew et al. (2000 & 2002). They evaluated 11 Massarina spp. and suspected relatives on the basis of rDNA data and transferred exactly those 5 species which were forming the sistergroup of A. longissima to the genus Lophiostoma. As far as can be concluded from the literature, the Massarina sp. described as the teleomorph of A. longissima, shows morphological characters which match the characters of the currently revised Massarina (now Lophiostoma) spp.. The Massarina spp. currently transferred to Lophiostoma are distinguished from other species remaining in the genus Massarina (e. g. M. eburnea) by the fusiform ascospores and slightly laterally compressed pseudothecial ostioles. Thus, it is probable that A. longissima and the described teleomorph also belong to the genus Lophiostoma in the sense of Liew et al.. This should be considered when revising the genus Anguillospora. The LSU analyses did not provide much additional information than what was previously obtained from SSU and ITS analyses. The results support the placement of A. longissima in the Pleosporales. Generally, the sequenced D1 and D2 regions of the LSU gene are considered to show enough variability at least for generic delimitation on the level of genera (Bruns et al. 1991; Hibbett 1992; Bridge & Arora 1998). However, in the present study, LSU sequences closely related to A. longissima were not available.

5. Heliscus lugdunensis H. lugdunensis is classified as a member of the order Hypocreales in the class Sordariomycetes.

124 Discussion

The teleomorph of Heliscus lugdunensis is Nectria lugdunensis (Webster 1959b). It is currently the best known holomorph of the aquatic hyphomycetes because of the frequent abundance of both the anamorph and the teleomorph state in nature (Webster & Iqbal 1971; Willoughby & Archer 1973; Shearer & Webster 1991). Although N. lugdunensis was never included in the recent studies of the Hypocreales (e.g. Rossman et al. 1999), its affiliation to this order is unquestioned, due to the morphological characters, The SSU analyses of three H. lugdunensis strains confirmed the placement of this fungus in the Ascomycota class Sordariomycetes as a member of the Hypocreales. This was indicated by the unambiguous placement in a cluster containing sequences of fungi belonging to the hypocrealean family Nectriaceae (Rossman et al. 1999; 2000). The closest sister clade of H. lugdunensis was Gibberella pulicaris which also had the lowest overall nucleotide sequence distance. However, the genus Gibberella is characterised by subglobose to globose, bluish purple ascomata and, where known, Fusarium anamorphs. This is in contrast to the broadly pear shaped, bright red perithecia described for N. lugdunensis. Therefore, one would have suspected a closer placement of H. lugdunensis to Nectria cinnabarina than as a second nearest sister clade. However, the genera Gibberella and Nectria both belong to the family Nectriaceae (Rossman et al. 1999) and the resolution power of the 18S rRNA gene is known not to be sufficient for the separation of such closely related fungi (Hibbett 1992; Bridge & Arora 1998). It is remarkable that Nectria teleomorphs are also known for other fungi from aquatic habitats such as Flagellospora penicillioides (Ranzoni 1956), F. curta (Webster 1992) and Cylindrocarpon ianthothele (Booth 1959). Unfortunately, this project ended before a more thorough investigation could be conducted of the phylogenetic relationships of H. lugdunensis on the basis of ITS and LSU sequence data. Nevertheless, the molecular evidence of the affiliation of H. lugdunensis to the Nectriaceae provides a base for further investigations.

6. Lemonniera aquatica and Lemonniera terrestris L. aquatica and L. terrestris are classified in the Leotiomycetes.

In SSU analyses, two frequently reported species, Lemonniera aquatica and L. terrestris, were placed in the paraphyletic assemblage Leotiomycetes (Ingold, 1975). The closest BLAST hit for L. aquatica and L. terrestris was a Phialophora sp. Elec-N-1 4.PN. This fungus was classified as a member of the Sordariomycetes, Magnaporthaceae (Hain et al. 2000). The results of the present study show that this must be rejected because of a clear placement of Phialophora sp. Elec-N-1 4.PN in the Leotiomycetes. As pointed out by Gams (2000), Phialophora is “a model of a poorly defined, little differentiated and highly polyphyletic genus” comprising anamorphs of Leotiomycetes, Sordariomycetes and Dothideomycetes. However, when distance values in the distance matrix were corrected for superimposed substitutions (Saitou & Nei 1987), the two investigated Lemonniera species showed the closest relationship to Loramyces juncicola. The placement of the Lemonniera species as a sister clade to Loramyces was displayed in all three tree building algorithms, albeit with weak statistical support. The uncertain placement of the two species on the basis of SSU sequence data does not allow any connection to a specific order in the Leotiomycetes. As already discussed for Tetracladium, L. aquatica and L. terrestris might be isolated within the currently available sequences of Leotiomycetes. This also became evident when the distance values of their SSU sequences were compared to all other members of Leotiomycetes used in this study: With the exception of the

125 Discussion unclassified Loramyces sp. and Phialophora sp., all other sequences were, on average, 5 % distant (SSU distance matrix Fig. A35, appendix).

7. Varicosporium elodeae The classification of V. elodeae in the Leotiomycetes must be regarded as preliminary because only a part of its SSU sequence was analysed.

Varicosporium elodeae Kegel, the type species of Varicosporium, is one of the few hyphomycetes from a water environment which had been described before the classic paper on a group of fungi colonizing leaves submerged in streams was published (Ingold 1942). However, it is often present in extraaquatic habitats too. Presently, the genus has eight recognized species. V. elodeae itself seems to be heterogeneous at the intraspecific level. The original isolate of Kegel produced green pigment on 2 % Malt agar media as do most of the aquatic isolates, though some terrestrial isolates produce brown pigment (Park 1982 ). For this study, only a partial SSU sequence for V. elodeae strain CCMF- 11783 could be obtained. This strain produces a green pigment in culture. Because of the short sequence, the results obtained for the phylogenetic placement of V. elodeae must be regarded as preliminary. However, the placement as sister clade to Tricladium angulatum and Loramyces juncicola classifies V. elodeae as another aquatic hyphomycete member of the Leotiomycetes.

8. Phylogeny reconstruction using rDNA data and different treeing methods Four different tree building algorithms revealed very similar tree topologies but the phylogenetic analyses suffered from the lack of closely related sequences in public databases. The phylogenetic analyses of aquatic hyphomycetes should be performed by incorporating morphological, molecular, biochemical and ecological characters.

The most critical initial steps of phylogenetic analyses based on sequence data are (i) the sampling of taxa and (ii) the sequence alignment. In this study, representatives of Ascomycota orders were chosen for SSU analyses and several attempts were made to get the ITS and LSU sequences of the same taxa. Unfortunately, this was not always possible. Additionally, nearest BLAST hits were included in the analyses with the aim of providing a group of sequences which were, because of their similarity, presumed to be closely related to the object of investigation. The first problem arose due to the previously mentioned lack of sufficiently close related sequence data in the public databases. This was leading to long, naked branches in some tree topologies, indicating a rather isolated placement of e. g. Tetracladium marchalianum. With the increase of rDNA sequence data in public databases, in the future some of these branches will become shorter because additional taxa will close missing links. The second step is probably most critical. This is the alignment of sequences because it implies homologies of nucleotide characters. Using the secondary structure information for SSU and LSU rRNA, the automatic alignment function in the adapted ARB program gave satisfactory results. However, the alignment required visual inspection and, especially at both termini, the sequences had to be corrected manually. The incorporation of the huge database of aligned fungal sequences, developed during this study, improved the alignment of SSU, ITS and LSU data sets. This was obtained by automatically searching the closest related sequences through the pt-server and using

126 Discussion these data for the finding of homologies. The ITS region showed a high interspecific variability and the incorporation of secondary structure information in the ARB program for this region was not yet practicable. Consequently, the ITS alignment was often difficult and lead to many ambiguously aligned regions and, therefore, reduced the number of characters going into the analyses. With the use of different programs (such as RNAviz [http://rrna.uia.ac.be/rnaviz/], mfold [http://www.bioinfo. rpi.edu/applications/mfold/old/rna/form1.cgi] and loopDloop [http://iubio.bio.indiana. edu/soft/ molbio /loopdloop/]) for predicting and drawing rRNA structure, the secondary structure information can be incorporated in future databases. In general, three tree building methods were used. Bayesian analyses were – due to limited computer resources – used for two taxa only. The treeing methods were: (i) Neighbour Joining. This method uses the similarity respective distance value of the whole sequence. Here, information is lost during the conversion of the original sequence data into the distance matrix (Swofford et al. 1996). Compared to parsimony and ML, the NJ trees often showed a higher resolved branching order, e.g., between strains of Tetracladium marchalianum. Different lengths of sequences due to sequence length polymorphism or the use of different primers are critical for this method. It was necessary to adjust the sequences to the same length. In ARB, this was computed by the generation of a termini filter. (ii) Maximum parsimony. This method takes into account insertions and deletions of nucleotides and allows the construction of the tree with the shortest length. Although branch length estimation is implemented in ARB, long branch attraction could not always be excluded. However, this was often the result of data sets including some sequences which were not closely related. (iii) Maximum Likelihood. This method computes the likelihood for a single base change while (iv) Bayes evaluates the probability of a whole tree topology, both under a certain model of evolution. All treeing methods showed the same or very similar tree topologies and, therefore, indicated the same patterns of the phylogenetic relationships of the investigated fungi. However, often taxa within the Leotiomycetes could not be properly placed by either treeing method. This was due to the previously mentioned missing links and, presumably, many homoplasy effects due to convergent evolution. In summary, it can be said that, for some of the phylogenetic questions discussed above, the investigation of protein coding genes may lead to a finer taxonomic resolution, especially in species level systematics (O´Donnell et al. 1998). In summary, for a reliable characterisation of aquatic hyphomycetes, as many different characters as possible should be used and combined. This procedure should incorporate morphological, molecular, biochemical and ecological characters. For the evaluation of ecological features of certain taxonomical groups or individuals, the phylogenetic analyses of aquatic hyphomycetes provided an important base. Hence, the evaluation and adaptation of the FISH method for aquatic hyphomycetes became possible.

C) Fluorescence in situ hybridisation of freshwater fungi

Based on the results of sampling, isolation and the phylogenetic characterisation, the accessibility of freshwater fungi for Fluorescence In Situ Hybridisation (FISH) was evaluated to provide a molecular tool for the characterisation of fungal communities in lotic systems. Therefore, different regions of the

127 Discussion ribosomal RNA were screened for potential signatures which would allow the construction of taxon- specific oligonucleotide probes. Furthermore, the factors influencing probe-conferred fluorescence signals and the permeabilisation of fungal cell walls for probe access were investigated, resulting in improvements of the FISH method for applications in fungal ecology.

1. Specificity of oligonucleotide probes for fungi The resolution of the 18S rRNA molecule is sufficient for the design of fungal probes on division level while the species specificity of 18S rRNA targeted probes should be regarded with caution. The 28S rRNA provides sufficient variable signatures for the design of species specific probes. The ITS regions are not available targets for FISH.

For a reliable characterisation of microorganisms within their microenvironments by FISH, it is critical to know the degree of specificity of the oligonucleotide probes employed. The specificity of a probe is determined by the variability of the ribosomal RNA target region, the probe features and the hybridisation conditions that lead to the formation of a stable heteroduplex between the oligonucleotide and the complementary rRNA single strand. While hybridisation conditions are adjusted by varying of hybridisation parameters (described in RESULTS, Part C), the variability of the rRNA molecules is a fundamental factor of the FISH approach. The primary structure of rRNA molecules is composed of highly conserved, variable and hypervariable regions. The construction of specific FISH probes requires the localisation of specific rRNA stretches - called signatures - that are unique for different phylogenetic levels (Manz 1994a). For newly designed probes, a check for other organisms that may have the same nucleotide sequence is performed using public sequence databases, e.g., GenBank. However, this provides only a rough estimation of the specificity of a probe. Of approximately 73,000 currently described fungi and an estimated 1.5 million fungal species world- wide (Hawksworth & Rossman 1997), only sequences of 16,000 fungal species are listed in the databases (GenBank Taxonomy Browser statistics, November, 2002). Therefore, it is indispensable to do in situ testing of new probes against fungi displaying one mismatch in the target region and closely related organisms (e.g. members of the same genus as the target organism) of which the rRNA sequences might not be known.. In the present study, it was sometimes impossible to find closely related fungi. This difficulty had become obvious in the phylogenetic studies. Additionally, in some cases, sequences displaying one mismatch in the target region were not available in the databases. This does not mean that the specificity of such probes is reliably restricted to a certain species because one does not know whether organisms which coincidentally have the same target sequence are present in a particular environmental sample. For that reason, theoretical and practical testing of probes should be performed in short time intervals (Manz 1999). As the 18S rRNA probe design for Tetracladium marchalianum showed, the presumed species specificity of a probe can quickly become obsolete if other members of the genus are included into the analyses. No 18S rRNA targeted T. marchalianum specific probe could be designed after the sequences and cultures of other Tetracladium species were available. This leads to the question of the suitability of fungal rRNA genes for the design of specific FISH probes. The 18S rRNA gene of eukaryotes is among the slowest evolving sequences found among living organisms (Hillis & Dixon 1991) with absolutely conserved, conserved and variable regions (Van de

128 Discussion

Peer et al. 1997b). The 18S rDNA is known to be useful for examining ancient evolutionary events (Hillis & Dixon 1991; Hibbett 1992; Bruns et al. 1991). This allows the construction of group specific oligonucleotides as the widely used fungal primers of White et al. (1990). Consequently, the design of the probes FUN1429 and MY1574 specific for a wide range of Eumycota was easily achieved. It is noteworthy that probe MY1574 was designed on the basis of a much larger database than FUN1429. While the phylogenetic target range of probe FUN1429 seems to be restricted to members of the subphylum Pezizomycotina (Eriksson et al. 2002), probe MY1574 matches also with members of Saccharomycotina, Taphrinomycotina and Basidiomycetes and Zygomycetes. Probe MY1574 is therefore recommended for detecting a wide range of Eumycota. The binding sites of FUN1429 and MY1574 are located in regions known to be conserved in most fungi but not in all eukaryotes (Wuyts et al. 2000). Thus, probe MY1574 might be specific for a large number of members of the division Fungi. It remains to be determined whether the 18S rRNA molecule provides enough variability for the design of probes at class, order, genus or species level. The Antwerp database on ribosomal RNA (http://oberon. rug.ac.be:8080/rRNA/) provides rRNA secondary structure and nucleotide variability maps calculated by substitution rate calibration of several hundred eukaryotic SSU sequences (Van de Peer et al. 1996, 1997b; Wuyts et al. 2000). In the Antwerp map, the nucleotides of the SSU rRNA are divided in five variability subsets which indicate each base pair’s variability status over all investigated sequences. According to this map, the probe binding sites of the one genus specific and four species specific 18S rRNA targeted probes, designed in the present study, are localised in high variable and hypervariable regions. The genus specific probe TCLAD1395 and the species specific probes TRIang322 and Alongi340 bind to regions with high variability at helices 44 and 12, respectively. The probes specific for Alatospora acuminata and Heliscus lugdunensis have the same binding site in a hypervariable region at helix 49. Here the SSU rRNA showed sufficient variability for the presence of two different signatures in the sequences of the two fungi phylogenetically located in different classes of the Ascomycota. Additionally, the slightly lower variable probe signatures found in the SSU sequences of Tricladium angulatum and Anguillospora longissima were sufficient to construct species specific probes. Therefore, using current knowledge of fungal 18S sequences, the highly variable regions of the SSU rRNA appear to be sufficient to design species specific probes. Utilising this approach, the highly variable helical regions E23_1; E23_2; E23_5; 10; 29 and 43 should also provide signature sites for specific probe design. On the other hand, it was not possible to design 18S rRNA targeted species specific probes for Tricladium splendens and Anguillospora crassa which came out to be closely related in the phylogenetic analyses. Therefore, one must be cautious with the species specificity estimation of the designed 18S targeted probes. The 18S rDNA of fungi has a more conserved nature than the 16S rRNA of bacteria (Wuyts et al. 2000). Many bacterial species specific 16S rRNA targeted probes are currently in use (e. g. Amann et al. 1995, Manz et al. 1998). Compared to the bacterial 16S rRNA, it is more likely that the fungal 18S rRNA is a useful marker molecule for the design of probes with specificity on higher phylogenetic levels such as orders or classes. This should become evident as more fungal SSU sequences become available.

129 Discussion

The 28S rRNA gene provides more variations in rates of evolution than the 18SrDNA (Hillis & Dixon1991, Bruns et al. 1991 ) but is less available in public databases. The larger gene and relatively more variable regions (De Rijk et al. 1992; Ben Ali et al. 1999) should provide more signatures for species specific probe design. Phylogenetic analyses cast doubt on whether the LSU gene provides enough resolution for species separation (Bruns et al. 1991, Hibbett 1992, Bridge & Arora 1998). The species specific probes for Anguillospora longissima and Tetracladium marchalianum are both binding to different helical loci within the variable V2 region (De Rijk et al. 1994) of the LSU rRNA. Of course, the LSU rRNA targeted probes also have to be thoroughly tested in situ against one mismatch, non - target organisms and closely related organisms. Remarkably, only 20 % hybridisation stringency was sufficient to successfully discriminate the closely related non-target organisms T. setigerum, T. furcatum, T. apiense and T. maxilliforme using the T. marchalianum specific LSU targeted probes TmarchC1_1 and TmarchC1_2. The LSU targeted probe AlongiB16, specific for A. longissima, could only be tested against fungi that are presumed to be present in the same habitat. As the variability map for the LSU rRNA (Ben Ali et al. 1999) indicates, the molecule provides many more potential signature regions for specific probe design. Nevertheless, the suitability of the LSU rRNA for the construction of species specific probes is difficult to estimate. This is primarily due to the restricted number of LSU rDNA sequences available in public databases. However, the LSU rRNA molecule provides at least enough variable sequence stretches for the design of in situ probes specific to the genus level. This will probably be corroborated when more LSU rDNA sequence data become available. On the basis of nucleotide variability, the ITS regions seem to be the most appropriate target for the design of species-specific FISH probes because they are not meant to contribute to structural RNA. Therefore, less selective evolutionary pressure applies and DNA sequence differences, i.e., point mutations, even between species of the same genus, can be found in these regions (Hillis & Dixon 1991). However, the occurrence of divergent ITS2 types and interspecific hybridisation events (Waalwijk et al. 1996, O´Donnell et al. 1998, Lieckfeldt & Seifert 2000) would restrict the probe design to the ITS1 region. In the present study, the attempt to perform FISH with ITS1 targeted probes failed. As detailed in the Results chapter, no probe conferred signals could be observed using probes specific for T. marchalianum or Penicillium chrysogenum, respectively. This is not unexpected as the internal transcribed spacer regions are, as widely stated (e.g. Knippers 1995, Tollervey & Kiss 1997; Graur & Li 2000), without structural significance in the ribosome complex. In contrast, ITS sequences evolve slower than some introns (Shinohara et al. 1999) and show small conserved homologous DNA regions (Armbruster et al. 2000), indicating that some selective pressure exists. The selective pressure might be important to maintain the secondary structure required for post-transcriptional processing. Studies of Saccharomyces cerevisiae suggest that some sequence regions in ITS1 and ITS2 are necessary for processing of the 45S rRNA precursor (Thweatt & Lee 1990; van der Sande et al. 1992; van Nues et al. 1994). However, ribosomes are located in the cytoplasm. In eukaryotic cells the biogenesis of ribosomes is located in the nucleus. The ribosomal RNAs are generated from the 45S precursor RNA (pre-rRNA) containing external (ETS) and internal (ITS1 and ITS2) transcribed spacer sequences. With the generation of functional 18S, 5.8S and 28S rRNAs, the spacer regions of pre- rRNA are removed and degraded (Tollervey & Kiss 1997). Subsequently, the newly synthesised rRNA

130 Discussion is packaged to generate the ribosomes. This packaging takes place in the nucleus, in the structure called the nucleolus, before the ribosomal structures are transported in the cytoplasm (Seifarth 2002). This would imply that ITS1 and ITS2 sequences are not present in the cytoplasm and therefore are not available targets for FISH.

2. Factors influencing FISH of fungi The fixation method, the fungal inherent and substrate mediated autofluorescence, the imaging technique, the age of the mycelium, the different mycelial parts, and the permeability of fungal cell walls were shown to be the most obvious influencing factors for the detection of fungal FISH signals.

Besides the specificity estimation of probes used in this work, the detection and interpretation of fluorescence signals are the critical steps in applying the FISH method for the characterisation of microbial communities. While employing FISH in fungal ecology, weak FISH signals may be lit up by strong autofluorescence of the mycelium. Thus, autofluorescence of hyphae may be mistaken as probe- conferred signals. Additionally, FISH signals might become undetectable against the strong background fluorescence of the colonised substrata, such as leaves. Weak probe conferred signals may be misinterpreted as low metabolical activity of the mycelia if proper permeabilisation of fungal cell walls is not ensured. Incorrect assumptions about FISH signal intensity and the spatial distribution of fungi associated with dense substrata may be drawn depending on the microscopic technique selected. Although these restrictions in the use of the FISH method in fungal ecology were intensively discussed in a number of studies (Li et al. 1996, 1997; Kosse et al. 1997; Spear et al. 1999; Sterflinger & Hain 1999; McArthur et al. 2001), the factors influencing fungal FISH have not been fully investigated. With the exception of McArthur et al. (2001), who also applied FISH to aquatic hyphomycetes, the application of the FISH method in fungal ecology has been limited to studies of yeast-like fungi. Of the many parameters investigated in the present study, the fixation method, the fungal-inherent and substrate autofluorescence, the imaging technique, the age of the mycelium, the different mycelial parts, and the permeability of fungal cell walls were shown to be the most significant factors in the detection of fungal FISH signals. It is important to note that these factors are somewhat related, because the autofluorescence of hyphae can increase with age.

2.1. Fixation of fungi and environmental samples intended for FISH For FISH of aquatic hyphomycetes paraformaldehyde fixation was most successful.

The purpose of fixation of the sample prior to FISH is to maintain morphological structures during the hybridisation process and to increase the permeability of cell walls for the penetration of the probe. The chemical fixatives used during this study were either coagulating, such as ethanol and methanol, or cross-linking fixatives, such as paraformaldehyde (Zhang et al. 2003). Hybridisation signals of freshwater fungi were achieved with paraformaldehyde fixation only. It is important not to exceed 4 hours fixation time because hybridisation of aquatic hyphomycetes failed after four hours. This might be due to cross-linkage of proteins within similar fungal cells, as observed for gram-positive bacteria

131 Discussion by Manz et al. (1994b). With the exception of hybridisations of Penicillium chrysogenum, the fixation with paraformaldehyde was not optimal in obtaining the highest possible FISH signal intensities of the tested fungi. This is probably due to insufficient permeabilisation of the fungal cell walls treated solely with paraformaldehyde. However, for some yeast species, coagulating fixatives resulted in better hybridisation success than paraformaldehyde fixation (Spear et al. 1999). Therefore, different fixation methods should be used according to the fungal cell wall properties of the species or populations that are to be detected.

2.2. Dealing with autofluorescence and the influence of the microscopic technique Many freshwater fungi showed autofluorescence in the green light spectrum often increasing with the age of the mycelium. The use of confocal laser scanning microscopy significantly enhanced FISH signal detection.

Autofluorescence scans by excitation of different fungal species over the visible light spectrum (300 – 600 nm) resulted in emission spectra with a fluorescence peak which, in most cases, was green light. However, some fungi showed autofluorescence over a wide range of the visible light spectrum (Helicosporium phragmitis) or minimal autofluorescence (Alatospora acuminata, Heliscus lugdunensis, Anguillospora longissima and Penicillium chrysogenum). In order to avoid overlapping of the FISH signal with fungal green autofluorescence, probe labelling with CY3 (emitting fluorescence in the red light range of 565 - 615 nm) is preferable for the majority of the fungi investigated. However, this cannot be generalised. The autofluorescence signal quality is dependent upon the fungal species. Depending on the aim of the study, fungal species under investigation should be scanned for their autofluorescence spectrum prior to in situ probing. In the present study, autofluorescence intensities increased with the age of the mycelium and in hyphae exceeding 5 µm width. Especially in 8 week old cultures, a probe conferred signal was often barely detectable due to overlapping with the autofluorescence signal. The increase of autofluorescence in older mycelium might be due to changes in the fungal cell wall composition and cell contents. This can be observed by increasing pigmentation of the cell walls of some species (e.g. Anguillospora spp.) or deposition of glycogen in the cells (e.g. Tricladium splendens). An open question concerns the age of the mycelia we observe in environmental samples. The persistence of a leaf in a stream is dependent on the leaf type and stage of decay, the presence of shredders, bacteria, fungi and abiotic factors such as temperature (Suberkropp & Chauvet 1995; Baldy & Gessner 1997). Leaves in a stream usually disappear within one year (Bärlocher 1992c). However, nutrients useful for aquatic hyphomycetes can be exhausted after several weeks. The persistence of an aquatic hyphomycete on a particular leaf in a stream is difficult to estimate. First, it is not known whether a fungus remains on a leaf by its first mycelium built from the conidium, initially landed on the leaf, and then persists or spreading in colonies. Second, as some species seem to prefer certain stages of leaf decay (Garnett et al. 2000), one cannot exclude the possibility that a mycelium of one fungus is replaced by the mycelium of another fungus at the same location (Swift 1976, Bärlocher 1992c), which would mean succession of different fungi. In environmental samples such as leaves, PE slides, or cellulose packs, the autofluorescence of the

132 Discussion fungal hyphae was low compared to the autofluorescence emitted by some substrates, in particular by leaves.

The sources of autofluorescence are manifold: Residues of culture media can cause autofluorescence (Graf et al. 1998) in fungal samples and require thorough washing with PBS. Also, fixatives such as methanol lead to an increase of autofluorescence of fungal cells. Many cellular metabolites exhibit autofluorescence (overview: Billinton & Knight 2001) such as flavins (Benson et al. 1979; Aubin 1979), nicotinamide-adenine dinucleotide [NAD+ / NADH] and nicotinamide-adenine dinucleotide phosphate [NADP+ / NADPH] (Galeotti et al. 1970; Aubin 1979). Plant parts emit autofluorescence that causes problems for FISH signal detection due to strong background noise. Chlorophyll types a and b are the major photosynthetic chromophores in leaves. Both types strongly absorb the deep blue and red regions of the light spectrum and thus green light is reflected back from the leaf tissue. Additionally, chlorophyll itself is autofluorescent with maximal excitation at 488 nm and maximal emission at 685 nm with a broad shoulder at 740 nm (Gunning & Schwartz 1999). In woody plant parts, lignin emits blue autofluorescence (Van der Geest & Petolino 1998). In old leaves, the accumulation of phenolic compounds lead to autofluorescence over a broad light spectrum (Martins et al. 1999; Agati et al. 2002). In the present study, autofluorescence could be lowered by bleaching the fungi and substrates with sodiumborohydrid. Van der Geest and Petolino (1998) suggested chemicals like toluidine blue, methylene blue and trypane blue for autofluorescence quenching. The principle of autofluorescence quenching is to use a dye that restricts the autofluorescence while allowing visualisation of a narrower band probe label fluorochrome. The microscopy technique employed is a critical factor in the successful detection of FISH signals of fungi. In a fluorescence microscope, the optical filter set (consisting of an excitation filter, a dichroic beam splitter and an emission filter), should be optimal for the probe label used for detection of FISH signals. Filters should be selected that are sensitive to areas of the spectrum where a particular probe label can be excited and the fluorescence transmitted with greater efficiency than the autofluorescence spectrum (Billinton & Knight 2001). For the detection of signals emitted from different spectral variants of green fluorescent proteins (GFP), special filters are commercially available (Chroma Technology Corp.; Omega Optical Inc., Battleboro, VT, USA), allowing the narrow selection of particular spectra (Zylka & Schnapp 1996). The comparison between epifluorescence and confocal laser scanning microscopy (CLSM) for the detection of fungal FISH signals, as well as for the evaluation of the signal-to-background noise ratio, resulted in a clear preference for CLSM. In CLSM, a laser beam is reflected by a dichroic mirror and focused onto a spot in the sample through an objective lens. The emitted fluorescence is collected by the same objective lens and passes through the dichroic mirror to a photomultiplier. In front of the photomultiplier, a pinhole is positioned such that the light emitted from the sample spot is isolated from the light emitted from the out of focus area (Sheppard & Shotton 1997). This feature enables the increase of the signal-to-noise ratio. In contrast to epifluorescence microscopy, with CLSM optical sections can be performed. This allows a more precise examination of thicker structures. Using optical sections of a hyphae it could be shown that the cell wall of Tetracladium marchalianum emits green

133 Discussion autofluorescence (Figure 31c, RESULTS). The spatial distribution of freshwater fungi, e.g., growing out of leaves, could be documented more clearly by obtaining stacks of several pictures along the z- axis. For distinguishing autofluorescence signals from probe conferred signals, fluorescence intensities of unlabeled control samples of substrates and fungi must be compared with FISH signals of fungal samples. The intensity of fluorescence signals above the autofluorescence level can then be counted as reliable probe conferred signals. Steinkamp & Stewart (1986) suggested a technique employing the “dual-wavelength differential fluorescence correction” for reducing background fluorescence in a study using a green (FITC) probe label. They assume that autofluorescence excitation and emission spectra are broad, extending over several hundred nanometers, whereas the excitation and emission spectra of the target fluorochrome, such as a probe label, are narrow, spanning only 50 to 100 nm. The dual-wavelength differential fluorescence correction method was performed by using two different lasers for providing two excitation wavelengths. An argon laser line (488 nm) was used to excite the FITC fluorochrome plus the background autofluorescence, while a krypton laser line (413 nm) was used to excite the autofluorescence only. The emitted fluorescence readings resulting from excitation of both laser lines respectively were measured in the same wavelength region (green, i.e., 515-545 nm). The difference between both fluorescence emission readings was then presumed to be equal to the fluorochrome fluorescence alone. However, this method corrects for background fluorescence at one excitation laser line only. The autofluorescence emitted directly in the range of the target fluorochrome emission spectrum cannot be corrected by this method. In freshwater fungi, autofluorescence excitation and emission spectra of some species (e.g. Helicosporium phragmitis) are very broad and not restricted to a particular excitation laser line. Even if one excites the sample over the entire visible light spectrum, one would not be able to determine the amount of fluorescence emission added by autofluorescence in the range of the label fluorochrome emission that is to be detected. Two-photon laser scanning microscopy (2-PLSM) would provide enhanced collection of emitted fluorescence and seems, therefore, to be an ideal method for the detection of weak fungal FISH signals. In 2-PLSM, short (femto- or picoseconds) pulses of infrared laser light are used for the production of high photon density (Lawrence et al. 2002). Unfortunately, the first few attempts to use 2-PLSM for the detection of hybridised freshwater fungi failed due to destruction (by burning) of the samples. This might be caused by the high peak power of the infrared laser in correlation with the pigmentation of fungal cell walls and the dark colour of decaying leaves. Thus the use of 2-PLSM in fungal ecology has to be regarded as a tool which is subject to further investigation.

2.3. Enhancement of freshwater fungal cell wall permeability for FISH probes The cell wall permeability could be enhanced by chitinase treatment but the most reproducible results for fungal FISH signal detection were yielded by employing the electroporation-FISH method (E-FISH).

Another crucial factor influencing the accessibility of freshwater fungi for FISH is the permeability of the fungal cell wall. Sufficient penetration of the cell walls of the target fungi by the oligonucleotide probe is essential for the detection of FISH signal at intensities that may be reliably correlated with rRNA content (DeLong et al. 1989) and, therefore, with metabolical activity. The rigid cell wall of

134 Discussion

Eumycota is an intricate network of carbohydrate polymers such as chitin, glucan and mannan, and glycoprotein (Bartnicki-Garcia 1987; Ruiz-Herrera 1992). The glucosidic bonds of β 1,4 or β 1,2 chitin and β 1,3 glucan and, on occasion, attached mannoproteins, determine rigidity and flexibility of the fungal cell walls (Hartland et al. 1994; Peter 2001). A certain degree of permeabilisation for fungal FISH can be achieved by fixation with paraformaldehyde, but with low reproducibility of the probe conferred signals. The high standard deviation value,17.5, (as revealed by CLSM and digital image analysis) shows a tendency to randomly distributed stronger and weaker fluorescence intensities within the target organisms. The reproducibility can be enhanced by the use of the enzyme chitinase which leads to degradation of chitin and dissolution of glucan (Hartland et al. 1994). The use of chitinase prior to FISH also increases the probe conferred signal intensity by approximately 20 %, especially in older cultures. However, even with the chitinase treatment, the reproducibility of probe conferred signals still suffers from high variations of FISH signal intensities (SD= ± 9.2). This indicates that the permeabilisation success of fungal cell walls by chitinase treatment will not yield a sufficiently consistent outcome. This is critical for quantifying metabolical activity using the correlation of fluorescence intensity and ribosome content, especially in complex natural samples. Several approaches using cell wall degrading enzymes such as glucanase (Sterflinger & Hain 1999) and the cellulolytic, Novozym™ (Spear et al. 1999), have been developed for the improved penetration of oligonucleotide probes into yeast cells. Unfortunately, the cell wall composition of filamentous fungi seems to be more complex than that of yeasts (Schoffelmeer et al. 1999; Fontaine et al. 2000; Guest & Momany 2000). Further, not all fungi contain chitin, and the polymer may be absent in one species that is closely related to another which does contain chitin (Peter 2001). Also, the amount of chitin may vary, depending on physiological parameters in natural environments or in cultures of fungi (Roberts 1992). Thus the cell wall composition of different fungi might be problematic in achieving sufficient cell wall permeability by the use of an enzyme. Additionally, the shape of the fungal morphological structures can be destroyed by imprecise use of the enzymes and the exact duration of the treatment is difficult to control in complex environmental samples comprising different types of prokaryotic and eukaryotic organisms. These obstacles could be overcome with the introduction of the fluorescently labelled oligonucleotides into the fungal cells by electroporation (E-FISH). Electroporation is a general method for the introduction of molecules into living cells. The lipid matrix can be disrupted by a strong external electric field leading to an increase in transmembrane conductivity and diffusive permeability (Neumann & Rosenheck 1972). These effects are the result of formation of aqueous pores in the membrane, which also alter the electrical potential across the membrane. The occurrence of pores or membrane openings allow the passing of ions and molecules (Weaver et al. 1988). In mycology, electroporation is used for permeabilisation and transformation of living cells (overview: Goldmann et al. 1995). The use of electroporation for permeabilisation and the introduction of FISH probes into fungal cells increased the fluorescence signals by 20% compared to chitinase treatment. Additionally, with E-FISH the reproducibility of fluorescence intensities was higher (SD± 1.5 fluorescence intensity units). Lower FISH signal intensities of fungi grown on polyethylene slides were covered by the high fluorescence intensity deviations employing fixation only or chitinase treatment. Only with E-FISH the observation of

135 Discussion lower probe conferred signals intensities on PE slides could be significantly reproduced. It seems very likely that the freshwater fungi studied possess lower rRNA content and thus lower FISH signal intensities because the nutrient availability on PE slides is much reduced and thus the metabolic activity of the organisms is reduced accordingly. Although E-FISH can be regarded as a promising tool for the reliable detection of fungal FISH signals, the method is not without disadvantages. First, the pulse width and magnitude is dependent of the strain and its morphological and, probably, physiological conditions. Hence, E-FISH has to be tested for each application. Based on the results of this study, benchmarks may be a single pulse width of 1.2 - 4.2 ms and a magnitude of 1.5 kV for fungal material of cultures or scraped from substrates. There is uncertainty about exactly what happens if electroporation is applied to dead, fixed cells and when it is performed with small molecules like fluorescently labelled oligonucleotide probes. Fluorescent molecules used in other fungal studies have often been much bigger, such as FITC-dextran, with a mass of 70 kDa, which was successfully introduced into living Saccharomyces cerevisiae cells (Bartoletti et al. 1989). Therefore, parameters of the E-FISH method are difficult to generalise but the tool is probably worth refining. In complex environmental samples comprising different prokaryotic and eukaryotic organisms, the E-FISH method has yet to be further evaluated. For some prokaryotic organisms, such as gram positive bacteria, E- FISH might be a useful tool for enhanced permeabilisation of the cells. The stability of morphological structures, such as of conidia, was not influenced from the electroporation procedure. The main disadvantage is the destruction of the spatial distribution of environmental samples due to the required scraping during the sampling for the E-FISH method.

2.4. Influence of different functional parts of the fungal mycelium on FISH signal intensities The FISH signal intensity and the rRNA content resembling the metabolical status of different structures of the mycelium are positively correlated.

Unlike bacteria, where FISH signal intensities of a single cell represent the metabolical status of a whole organism, fungi consist of a mycelium divided into different functional structures. Hybridisation of conidia, conidiogenous cells, hyphal tips and thin (less than 5 µm wide) and thick (greater than 5 µm wide) hyphae yielded different intensities of FISH signals. Hyphal tips and conidiogenous cells exhibiting high growth and production activities yielded significantly stronger probe conferred fluorescence signals than normal hyphae. On the other hand, non-germinating conidia yielded no FISH signals. Germinating conidia of freshwater fungi were accessible for FISH in spite of probably strengthened cell wall structures. As soon as germ tubes or appressoria were formed, conidia yielded high FISH signal intensities. This indicates that the FISH signal intensity and rRNA content resembling the growth status of a cell are positively correlated in fungi, as already confirmed for FISH of bacteria (DeLong et al. 1989; Kramer & Singleton 1992; Poulsen et al. 1993). Successfully hybridised fungi sometimes possessed single cells but also hyphal tubes within their mycelia which show no FISH signal response. This could be observed in both pure cultures and in environmental samples. The phenomena can be explained by the way fungi grow. Empty cells are caused by the transfer of cytoplasmic material from older portions of the thalli forward to younger hyphae (Cooke & Rayner

136 Discussion

1984). Additionally, cell content is used up for conidia production, resulting in empty hyphal structures (Aimer 1989). Summarising, the limiting factors determined in the present study which influence the utilisation of FISH on freshwater fungi can be positively influenced by the suggested procedural refinements. Knowledge about the presence and characteristics of the limiting factors should be taken into account when using the FISH method in freshwater fungal ecological studies. Other methodological attempts to enhance FISH signal detection in bacteria, cyanobacteria and fungi frequently lack general applicability for freshwater fungal purposes. Schröder et al. (2000) suggested the application of colorimetric in situ hybridisation (CISH) with digoxigenin labelled oligonucleotides for fungi and Schönhuber et al. (1997) used horseradish peroxidase labelled oligonucleotides and tyramide signal amplification for bacteria. In both methods, large molecular weight molecules (such as enzymes or antibodies) have to be incorporated into the whole fixed cells. However, this has to be regarded critical as the permeability of fungal cell walls is limited. Additionally, Schröder et al. (2000) observed unspecific binding of probes due to the crucial factor of antibody concentration. An alternative to the conventional oligonucleotides used for FISH could be the use of peptide nucleic acid (PNA) probes (Stender et al. 1999). PNA molecules are pseudopeptides which hybridise with complementary RNA or DNA regions within cells. They are characterised by an uncharged neutral backbone. Additionally, compared to DNA oligonucleotides, PNA probes have a relatively hydrophobic character. This quality makes them capable of penetrating hydrophobic cell walls requiring only mild fixation steps. However, the PNA-FISH approach has been performed only on yeasts (Stender et al. 2001) which have been shown to be more easily accessible for FISH than filamentous fungi.

D) In situ detection of freshwater fungi in the river Elbe and in the Oberer Seebach by FISH

With the application of the probes MY1574 and Hlug1698 the presence of metabolically active hyphae on polyethylene slides in the Elbe river could be shown. In the Oberer Seebach aquatic hyphomycetes were successfully detected on PE slides, leaves and in germination experiments using the set of newly developed fungal FISH probes.

The present study started with the qualitatively sampling and isolation of freshwater fungi in a small stream (Oberer Seebach) and a large river, the Elbe, employing established techniques based principally upon the morphological structures of conidia. This gave an indication of the amount of conidia in transport and of fungal species that could be isolated from different substrata in both habitats but provided no information of the metabolically active fungal community and its spatial distribution. After the phylogenetic analyses over a range of most commonly observed aquatic hyphomycetes and the evaluation and adaptation of the FISH method, the set of newly developed taxon-specific oligonucleotide probes was applied to the two lotic habitats.

137 Discussion

1. Estimation of fungal diversity and biomass For the estimation of the phylogenetic diversity and the biomass of freshwater fungi within their microhabitats, a range of chemical, serological and molecular techniques is available. Chemical stains, such as different lectins, often show limited fungal specificity or selective specificity for only a particular group or species (Müller & von Sengbusch 1983; Robin et al. 1986; Brul et al. 1997). This is probably related to the different cell wall composition of fungi, leading, for example, to coverage of chitin binding sites for lectins by other macromolecules (Mirelman et al. 1975; Smith et al. 2001). Staining of freshwater fungi on the surface of different substrates with Calcofluor White (CFW) was satisfactory, although unspecific staining of other organisms gave sometimes artificial signals. However, the simultaneous application of CFW staining and FISH is problematic due to the occasional crystallisation of both probe label fluorochrome and CFW. The crystallisation of CFW was also observed by Harris et al. (2002) while studying soil samples. They suggested the use of the newly-available SCRI Renaissance 2200 dye (Renaissance Chemicals, Selby, UK) as a non-crystallising replacement for CFW. All methods based on total DNA extraction and the subsequent PCR-based studies lack the determination of the metabolical status of the studied organisms. Recently applied PCR-based methods including restriction fragment length polymorphism (RFLP) analysis (Fargues et al. 2002), random amplified polymorphic DNA (RAPD) analysis (Peláez et al. 1996; Charcosset & Gardes 1999) and denaturing gradient gel electrophoresis (DGGE; e.g. Kowalchuk et al. 2002), have been shown to be powerful tools in determining genetic variation in fungal populations. However, these methods are biased by yielded DNA quantity and quality, primer specificity and selectivity, band pattern interpretation and the dependence on sequences available in databases (Head et al. 1998, Bridge & Arora 1998). For the reliable estimation of fungal biomass and reproduction in aquatic hyphomycete ecology, the ergosterol technique (Newell 1992) is widely used (Suberkropp et al. 1993; Gessner 1997; Hieber & Gessner 2002). Ergosterol is a membrane component specific for most Eumycota and, due to its chemical instability, suggested as a quantitative measure of viable cells (Newell 1992). However, Bermingham et al. (1995b) as well as Charcosset and Chauvet (2001), remarked that the relation between fungal ergosterol content and biomass varies within species and due to culture conditions, thus presenting difficulties in determining the optimal conversion factor for environmental samples. In contrast to the above mentioned PCR-based methods, the ergosterol technique provides no information about the species composition of a sample. Immunostaining, employing monoclonal antibodies and ELISA (enzyme-linked immunosorbent assay), when applied to leaf samples, provides the identification of aquatic hyphomycete species in situ (Bermingham et al. 1995a, 1997, 2001). Besides the requirement of pure cultures of all target species and the rather laborious performance requirements of this technique, the authors remark that immunostaining gives no information about the metabolical status of the investigated fungi and that antigen persisting even on dead hyphae cannot be excluded.

138 Discussion

2. FISH of fungi in the lowland river Elbe and the alpine creek Oberer Seebach Combining both specific in situ detection and information about the physiological condition, the application of FISH gave evidence of metabolically active fungi on substrates exposed in the Oberer Seebach stream and in the Elbe river. With the exception of Clavariopsis aquatica, all conidia of aquatic hyphomycetes were observed in foam samples in the river Elbe. Assuming the species- specificity of the probe Hlug1698, with the detection of hyphae of Heliscus lugdunensis by FISH on PE slides, the presence of a metabolically active aquatic hyphomycete in the Elbe river could be proven. Additionally, the application of FISH with probe MY1574 resulted in detection of active hyphae of unspecified fungi on PE slides. However, PE slides are artificial substrates and the presence of metabolically active aquatic hyphomycetes on natural substrates is limited to one instance, the isolation of C. aquatica growing out from a leaf collected at the river banks. Hence, the present application of FISH in the river Elbe indicates (i) FISH, enhanced with methodological refinements and performed with the newly developed taxon specific probes, is a reliable tool that can be used for further investigations of the freshwater fungal ecology in the Elbe river; and (ii) metabolically active fungi are present in the Elbe river if substrates for conidial attachment are available. For further investigation of the role which aquatic hyphomycetes play in the river Elbe, natural substrates should be examined by combining classic methods with FISH. However, this returns to the questions posed at the beginning of this study: Are there natural substrates for freshwater fungi present in the Elbe river ? The size of the Elbe at Magdeburg considerably complicates the search for probable substrates. Natural substrates were barely observable because of the dilution factor of such a large river. Sometimes allochthonous material can be observed floating in the middle of the wide river and future sampling events should include the use of a boat. Also, in situ probing of other microhabitats, e.g. of larger benthic detritus, should be conducted. Active mycelia could be detected by FISH on all of the leaves and PE slides collected and exposed during the summer and autumn on the Oberer Seebach. On this stream, the Eumycota probe MY1574 yielded more hybridisation success than probe FUN1429. As discussed in the probe specificity section, this is due to the broader phylogenetic range of MY1574. Using the newly developed set of probes specific for different aquatic hyphomycetes, only Alatospora acuminata was detected by probes ALacumi1698 and ALacumi1491 on PE slides and on leaves. However, using probe MY1574, other aquatic hyphomycetes than A. acuminata (including Clavariopsis aquatica, Tetracladium marchalianum and an undetermined species with scolecoform conidia) could be successfully detected. Remarkably, many Acremonium-, Chalara- and Phialophora-like fungi were detected on collected and exposed leaves. Strong hybridisation signals of conidiogenous cells indicated that they were capable of sporulation under water. Terrestrial, epiphytic and endophytic fungi, already present on the leaf before abscission, are understood to be of limited importance in leaf decomposition in streams, and are generally replaced by aquatic hyphomycetes as the decomposition process develops (Bärlocher 1992c; Sridhar & Bärlocher 2000; Garnett et al. 2000). The observation of active epiphytic fungi by FISH may be due to the short exposure time of the leaves (two weeks) and aquatic hyphomycetes might not yet have been representing the major component of the leaf fungal community. This assumption is also supported by the observation of many non-germinated conidia attached to the

139 Discussion leaves and by observations of Sridhar & Bärlocher (2000), who also observed low aquatic hyphomycetes diversity and biomass on maple leaves in a small Canadian stream after 2 weeks. On PE slides exposed for 2 weeks, the majority of aquatic hyphomycetes detected by FISH was in the process of attachment, showing appressorium formation and germ tubes. Developed mycelia could be detected only on PE slides exposed for longer periods in the autumn. These PE slides also showed a dense layer of bacteria and algae which may have provided more nutrients for fungal growth than “naked” PE slides which were only exposed for a short time. About one third of fungal mycelia on leaves and PE slides observed by CFW staining could not be hybridised using the fungal probes. In part, these might have been non-active mycelia not accessible by FISH. However, the probes developed with a broad phylogenetic specificity in this study also have a limited phylogenetic range. Another explanation could be the low rRNA content of mycelia exhibiting low growth activities which would make the detection by FISH difficult. In laboratory experiments, aquatic hyphomycetes inoculated on PE slides were sometimes undetectable by FISH signals, unless the PE slides were covered with malt agar. This limitation indicates that one must be cautious with diversity and biomass estimations when using FISH only. In order to examine the germination of conidia and spores under natural conditions, conidia, basidiospores, ascospores and hyphal tips were exposed in cages within the RITRODAT area of the Oberer Seebach in autumn 2000. The cages were exposed at the slow and fast current sites for 2, 5, and 14 days. The metabolic activity of the exposed conidia and spores was tested by FISH, employing the probes MY1574, Hlug1698 and TmarchB10. Germinating conidia of aquatic hyphomycetes could be successfully hybridised on the cage membranes. Hybridisations with probes of both a broad and a more narrow phylogenetic specificity yielded strong fluorescence signals of highly active germ tubes and young hyphae. According to Byrne & Jones (1975) and Read et al. (1992), the majority of aquatic hyphomycetes germinate within 2-6 hours after attachment to a substrate. Accordingly, the germination rates of 5-20 % of T. marchalianum, V. elodeae and A. crassa after 2 days can be regarded as rather low. Only H. lugdunensis showed a 100 % germination rate in the fast current after 2 days, whereas it also presented a low rate in the slow current. Germination and growth of aquatic hyphomycetes is influenced by the availability of nutrients from the substrate and from the water flowing over the surface of the substrate (Read et al. 1992; Suberkropp 1998). In the Oberer Seebach, the limiting factor for an efficient germination of the exposed species might be the low amount of nitrate and, especially, phosphate present in the stream water. Conidia production and fungal growth are stimulated by inorganic N and P (Sridhar & Bärlocher 2000; Robinson & Gessner 2000). Compared to submerged leaves, the water circulation between the two cage membranes may be less and thus the availability of oxygen and nutrients may have been too low for more efficient germination. In cages exposed for 5 and 14 days, evaluations of germination rates were not reliable, because large numbers of conidia originally inoculated have disappeared. This might have been due to decay activities of bacteria which were present on the membranes in large numbers.

140 Outlook

Outlook

With the molecular phylogenetic analysis of eleven morphologically well described and most commonly observed species of aquatic hyphomycetes, an informative basis of their possible relationships within the major classes of Ascomycota and potential orders and families is provided. Future studies should include an improved taxon sampling, considering possible synanamorph and teleomorph connections. Furthermore, as many characters as possible (including ecology) should be used and more complex analyses should be performed. Both phylogenetic analysis and design of specific oligonucleotides are limited by the sequence data available in public databases. Freshwater fungi, and other saprobic fungi frequently observed in the stream habitat, should be isolated, thoroughly described and the databases of ribosomal genes should be extended. Fluorescence in situ hybridisation is a reliable tool for the detection of metabolically active stages and the spatial distribution of aquatic hyphomycetes in lotic systems. However, depending on the aim of the study, FISH has to be carefully applied (as should every method), recognising its limitations and pitfalls which were discovered and evaluated during the present study. The method should be regarded as a tool which is subject to progressive refinement. A main issue of increasing the validity of FISH is the control of the specificity of existing probes and the future designs of specific probes in synchronisation with growing sequence databases. Additionally, FISH should not be used as the sole tool to characterise the freshwater fungal community, as estimates of the total living fungal biomass, conidia production and in situ enzymatic activities give additional important information. Quantifying metabolically active fungal biomass should also be possible using FISH in combination with the use of image analysis software suitable for filamentous structures. While interactions of aquatic hyphomycetes with invertebrates are already well documented (e.g. Suberkropp 1992b), not much is known about interactions with bacteria. One of the most interesting questions concerns the full life cycle of aquatic hyphomycetes. What is known appears to be a small mitotic cycle, taking place on leaves falling in streams. (I) What happens to aquatic hyphomycetes in the stream after the input of allochthonous leaf litter is stopped due to change of season? (II) In temperate climates, what happens to the last conidia released from the last available leaf in a stream before the change to the cold season slows down microbial life? (III) Do aquatic hyphomycetes rest as dormant states in some yet undiscovered microhabitat until the input of organic litter resumes? (IV) Or is the seasonal occurrence of aquatic hyphomycetes renewed from inocula derived from terrestrial teleomorphs? It would be very interesting and it seems to be practicable at this time to follow succession patterns on leaves and to observe an aquatic hyphomycete throughout its life cycle.

141 Summary

SUMMARY

The aim of the present study is the molecular phylogenetic characterisation of aquatic hyphomycetes, the evaluation of the factors influencing their accessibility for fluorescence in situ hybridisation and the application of newly developed taxon specific rRNA-targeted oligonucleotide probes in the German lowland river Elbe and the Austrian alpine creek Oberer Seebach. The main distinguishing characteristics of the two different lotic habitats are the low retention abilities and the low input of allochthonous leaf material in correlation with a high dilution factor of the large Elbe river and the high retention and the high leaf litter input of the 2nd order stream Oberer Seebach. At first, aquatic hyphomycetes were sampled and isolated from water, river snow, foam, leaves, wood and roots from the two different lotic habitats. During 24 sampling campaigns in three years performed in the Elbe river, with only eleven different species a remarkable low diversity of aquatic hyphomycetes could be distinguished while the majority of fungi observed were terrestrial and airborne fungi. With the exception of Clavariopsis aquatica, isolated from an alder leaf, freshwater fungi could only be observed in autumn foam samples. No conidia in transport could be detected in water samples. Conidial numbers in fixed foam samples of 3.75 10 ml –1 were extremely low in the Elbe river compared to conidial numbers of 5635 10 ml –1 (autumn) and 2654 10 ml –1 (summer) in foam samples from the Oberer Seebach. During two short term, three week campaigns 36 different species of aquatic hyphomycetes could be observed and isolated from foam, water and leaf samples in the Oberer Seebach. The phylogenetic relationships of 11 species of aquatic hyphomycetes were analysed; partly obtained during the first part of this study and partly handed over by Dr. Marvanová (CCM, Brno, Czech Republic). In total it comprised seven type species of anamorphic genera. Phylogenetic analyses of sequences of different rDNA regions confirmed the assumption of a polyphyletic origin of aquatic hyphomycetes. On the basis of 18S rDNA data, nine species (Tetracladium marchalianum, Lemonniera terrestris, L. aquatica, Varicosporium elodeae, Tricladium angulatum, T. splendens, Alatospora acuminata, Anguillospora crassa, and A. furtiva) were placed in the Ascomycota class Leotiomycetes. The species Heliscus lugdunensis and Anguillospora longissima were placed in the classes Sordariomycetes and Dothideomycetes, respectively. In ITS and LSU sequence analyses T. marchalianum showed most likely phylogenetic affinities to the Helotiales. On the basis of ITS sequence data, two morphotypes of A. acuminata were separated into two clades containing the sensu lato and the sensu stricto type, respectively. Here a modification of the contemporary species concept might be necessary, but would require a range of further investigations of additional specimen of the genus. T. splendens showed close relationships to A. crassa and to the marine hyphomycete Zalerion varium in ITS analyses, all with most likely affinities to the Helotiales. Furthermore T. angulatum is related to the Helotiales but showed closer relation to members of the family Hyaloscyphaceae as revealed from ITS analyses. The genus Anguillospora has to be revised as soon as molecular studies of all currently known species of the genus are performed. Similar to T. splendens, A. crassa was placed most likely within the Helotiales. In contrast A. longissima is a member of the Pleosporales with closer relation to the genus Lophiostoma than to the suspected teleomorph genus Massarina.

142 Summary

For the phylogenetic analyses and subsequent probe design, the ARB software environment for sequence data was adapted to properly work with fungi by incorporating 18S rRNA and 28S rRNA secondary structure information and extending and generating 18S rRNA, ITS and 28S rRNA sequence databases. The sequence information gained from the phylogenetic analyses was applied for the design of taxon specific oligonucleotides. The newly developed probes termed FUN1429, MY1574 specific for a wide range of Eumycota, and the genus specific probe TCLAD1395 (Tetracladium) as well as the species specific probes ALacumi1698 (A. acuminata), TRIang322 (T. angulatum), Alongi340 (A. longissima) and Hlug1698 (H. lugdunensis) are targeted against the 18S rRNA. The probes TmarchB10, TmarchC1_1, TmarchC1_2 and AlongiB16, specific for T. marchalianum and A. longissima, respectively, are targeted on the 28S rRNA. The specificity of all newly designed probes was successfully tested in whole fungal hybridisations against target and non-target organisms. The fixation method, the fungal inherent and substrate mediated autofluorescence, the imaging technique, the age of the mycelium, the different mycelial parts, and the permeability of fungal cell walls were shown to be the most obvious influencing factors for the detection of fungal FISH signals. Autofluorescence scans revealed that many freshwater fungi showed inherent fluorescence in the green visible light spectrum. This could be successfully overcome by use of probe labels emitting red fluorescence. Generally, autofluorescence intensities increased with the age of the fungal mycelium. Bleaching of autofluorescing organisms and substrates with sodiumborohydrid was useful for enhanced probe signal detection. FISH signal intensities were examined either by epifluorescence or confocal laser scanning microscopy. Employing epifluorescence microscopy, fungal FISH signals were often insufficient to detect due to high background noise. In contrast, confocal laser scanning microscopy significantly enhanced the detection of fungal FISH signals. Additionally, the spatial distribution of freshwater fungi on leaves could be documented more clearly by CLSM. The FISH signal intensity and rRNA content resembling the metabolical status of different structures of the mycelium were positively correlated. The cell wall permeability could be enhanced by chitinase treatment. However, the most reliable results for FISH signal detection were yielded by employing the E-FISH method newly developed in this study. In E-FISH the fluorescently labelled oligonucleotides are introduced by electroporation into the fungal cells. Application of the probes MY1574 and Hlug1698 yielded FISH signals of metabolically active hyphae on polyethylene slides in the Elbe river. In the Oberer Seebach aquatic hyphomycetes were successfully detected on PE slides, leaves and in germination experiments using the set of newly developed fungal FISH probes.

143 Zusammenfassung

ZUSAMMENFASSUNG

Das Ziel der vorliegenden Arbeit war die molekulare phylogenetische Charakterisierung aquatischer Pilze, die Evaluierung und Optimierung der Fluoreszenz in situ Methode (FISH) für den Nachweis filamentöser Pilze sowie die Entwicklung taxonspezifischer Oligonukleotidsonden für aquatische Pilze und deren Anwendung in dem Tieflandfluß Elbe und dem österreichischen Gebirgsbach Oberer Seebach. Die beiden Fließgewässer unterscheiden sich um Größenordnungen in ihren Retentionsstrukturen, ihren Abflüssen Wassermenge und dem saisonalen Eintrag an Blattlaub in die Gewässer.

Zunächst wurden aus beiden Fließgewässern Pilze von unterschiedlichen Substraten (Blättern, Holz, Wurzeln) sowie aus Schaumproben isoliert und kultiviert. In der Elbe war bei 24 Probenahmen innerhalb von drei Jahren mit elf verschiedenen Spezies nur eine geringe Diversität an aquatischen Pilzen nachweisbar. Die Mehrzahl der aus der Elbe isolierten Pilze waren der ökologischen Gruppe der Bodenpilze oder durch die Luft verbreiteten Arten zuzuordnen. Von Blättern konnte nur Clavariopsis aquatica isoliert werden, während alle anderen in der Elbe gefundenen aquatischen Pilze aus den im Herbst gesammelten Schaumproben stammten. In Wasserproben und Schwebstoffen der Elbe waren keine Pilze nachweisbar. Fixierte Schaumproben aus der Elbe wiesen mit durchschnittlich 3,75 Konidien pro 10 ml sehr niedrige Konidienzahlen auf, während in Schaumproben aus dem Oberen Seebach mit 5635 (Herbst) und 2654 Konidien (Sommer) pro 10 ml weitaus höhere Konidenzahlen gefunden werden konnten. Während zweier dreiwöchiger Probenahmen am Oberen Seebach wurden 36 verschiedene Spezies aquatischer Pilze von Blättern und aus Schaum- und Wasserproben gewonnen. Die aus dem ersten Teil der Untersuchung gewonnenen Isolate sowie weitere, von Fr. Dr. Marvanová (CCM, Brünn, Tschechische Republik) zur Verfügung gestellte Kulturstämme aquatischer Pilze wurden hinsichtlich ihrer phylogenetischen Zugehörigkeit innerhalb der Ascomycota analysiert. Insgesamt wurden elf Spezies aquatischer Pilze untersucht, von denen sieben die Typ-Spezies von anamorphen Gattungen repräsentieren. Die Ergebnisse der phylogenetischen Analyse auf der Basis von Sequenzdaten unterschiedlicher ribosomaler DNS-Regionen bestätigten einen polyphyletischen Ursprung aquatischer Pilze. Auf der Basis von 18S rRNS-Sequenzdaten konnten neun Spezies (Tetracladium marchalianum, Lemonniera terrestris, L. aquatica, Varicosporium elodeae, Tricladium angulatum, T. splendens, Alatospora acuminata, Anguillospora crassa und A. furtiva) in die Klasse der Leotiomycetes eingeordnet werden. Zwei Spezies wurden in die Klasse der Sordariomycetes (Heliscus lugdunensis) bzw. der Dothideomycetes (Anguillospora longissima) eingeordnet. Die Analysen der ITS und LSU-Sequenzbereiche stellten T. marchalianum in die Nähe der Ordnung Helotiales. Auf der Basis von ITS-Sequenzdaten wurden die sensu stricto und sensu lato Morphotypen von A. acuminata deutlich separiert. Eine eventuell notwendige Revision des bisherigen Spezieskonzepts der Art würde jedoch zusätzliche Untersuchungen von Pilzen der Gattung Alatospora erfordern. Die Ergebnisse der ITS-Sequenzanalysen von T. splendens und A. crassa weisen auf eine enge Verwandtschaft beider Arten untereinander und zu dem marinen Pilz Zalerion varium hin. Alle drei Pilze zeigten phylogenetische Beziehungen zur Ordnung Helotiales. Auch T.

144 Zusammenfassung angulatum wurde in ITS-Analysen den Helotiales zugeordnet und zeigt enge Beziehungen zur Familie der Hyaloscyphaceae. Die Gattung Anguillospora muß aufgrund der Ergebnisse der Sequenzanalysen sowohl des 18S rRNS-Gens als auch der Spacerregionen überarbeitet werden. Während A. crassa der Ordnung Helotiales näher steht, wurde A. longissima zweifelsfrei in die Ordnung Pleosporales eingeordnet. Hier zeigte A. longissima eine nähere Verwandtschaft zu Arten der Gattung Lophiostoma als zu Arten der vermuteten Teleomorph-Gattung Massarina. Für die phylogenetischen Analysen und die nachfolgende Entwicklung taxonspezifischer Oligonukleotidsonden wurde das ARB Softwareprogramm (TU München) für die Verarbeitung von pilzlichen Sequenzdaten angepaßt. Dazu wurden die Information über die Sekundärstruktur des ribosomalen 18S und des 28S rRNS Moleküls anstelle des vorhandenen prokaryotischen 16S rRNS- Moleküls in das Programm implementiert. Die bestehende Datenbank für pilzliche Sequenzdaten wurde um umfangreiche Datensätze von 18S, ITS und 28S rRNS- Pilzsequenzen erweitert. Die von den phylogenetischen Analysen gewonnenen Sequenzdaten wurden im weiteren für die Entwicklung von Oligonukleotidsonden auf Divisions-, Gattungs- und Artniveau verwendet. Die neu entwickelten Sonden FUN1429 und MY1574, spezifisch für einen breiten phylogenetischen Bereich der Eumycota, die gattungspezifische Sonde TCLAD1395 (Tetracladium) sowie die artspezifischen Sonden ALacumi1698 (A. acuminata), TRIang322 (T. angulatum), Alongi340 (A. longissima) und Hlug1698 (H. lugdunensis) sind gegen die 18S rRNS gerichtet. Die artspezifischen Sonden für T. marchalianum (TmarchB10, TmarchC1_1, TmarchC1_2) und A. longissima (AlongiB16) sind 28S rRNS gerichtet. Die Spezifität aller neu entwickelten Oligonukleotidsonden konnte durch computergestützte vergleichende Sequenzanalyse und in situ Hybridisierungen gegen Zielorganismen und Vertreter anderer phylogenetischer Gruppen bestätigt werden. Wie in umfangreichen Testserien gezeigt werden konnte, werden die durch die Sonden in Hybridisierungen vermittelten Fluoreszenzsignale sowohl qualitativ als auch quantitativ durch die Fixierungsmethode, das Alter des Myzels, unterschiedliche Stoffwechselaktivität verschiedener Strukturen im Myzel, Autofluoreszenzen, die verwendete Mikroskopiertechnik sowie die Permeabilität der Pilzzellwände wesentlich beeinflußt. Viele der untersuchten aquatischen Pilze emittierten Autofluoreszenzen im Bereich des sichtbaren, grünen Lichts. Durch Markierung der Sonden mit roten Fluoreszenzfarbstoffen konnte die natürliche Autofluoreszenz der Pilze umgangen werden. Mit steigendem Alter des Myzels erhöhte sich in der Regel auch die Intensität der Autofluoreszenz. Die Autofluoreszenzen von Organismen und Substraten konnte durch Verwendung von Natriumborhydrid erfolgreich reduziert werden, was die Sichtbarmachung von Sondensignalen verbesserte. Durch die Verwendung von konfokaler Laser-Scanning-Mikroskopie wurden die Fluoreszenz-Emissionen außerhalb der Fokusebene weiter minimiert und die dreidimensionale Darstellung von Pilzen auf Blättern ermöglicht. Die Intensität der Sondensignale war positiv korreliert mit dem metabolischen Status unterschiedlicher Strukturen im Myzelkörper. Die Durchlässigkeit der Pilzzellwände für Oligonukleotidsonden konnte durch eine vorgeschaltete Behandlung mit Chitinase verbessert werden. Noch weiter konnte die Intensität sowie die Reproduzierbarkeit der Sondensignale durch den Einsatz der in dieser Arbeit neu entwickelten E-FISH Methode gesteigert werden, bei der die Oligonukleotidsonden mittels Elektroporation in die Pilzzellen eingebracht werden.

145 Zusammenfassung

Abschließend konnte die Anwendbarkeit der neu entwickelten Pilzsonden unter natürlichen Bedingungen gezeigt werden. Durch Einsatz der Sonden MY1574 und Hlug1698 konnten stoffwechselaktive Pilze auf Polyethylen-Objektträgern nachgewiesen werden, die in der Elbe exponiert worden waren. Ebenso konnten aus dem Oberen Seebach Pilze erfolgreich auf Blättern und Polyethylen-Objektträgern mit den in dieser Arbeit entwickelten, pilzspezifischen Gensonden nachgewiesen werden.

146 Acknowledgements

ACKNOWLEDGEMENTS

I would like to express my gratitude to all the people who made this work possible:

Prof. Dr. Ulrich Szewzyk for his excellent supervision, invaluable advice, guidance, and support, as well as for providing time and space for the fungi, and for allowing me the freedom to follow my own ideas. Dr. habil. Werner Manz for his tireless encouragement, excellent supervisory support, expert knowledge, and many illuminating discussions. Dr. Ludmila Marvanová (CCM, Brno, CZ), who patiently taught me about isolation procedures, taxonomy, and ecology of aquatic hyphomycetes, and who kindly provided most of the CCMF- strains, read and discussed the manuscript, and guided me throughout this project in spite of the over 500 km distance between us. Dr. Thomas Neu (UFZ, Centre for Environmental Research) for allowing me to use the facilities at UFZ Magdeburg, in particular the CLSM, as well as for his support, many fruitful discussions, and a congenial atmosphere during my stays in Magdeburg. Ute Kuhlicke for friendly and expert technical assistance both at the CLSM and during sampling events. Prof. Dr. Felix Bärlocher (Mount Allison University, CDN) for his interest in my work, his readiness to act as referee, and for providing cultures of Tetracladium marchalianum CCMF-11391; T. setigerum CCMF-20987, T. apiense; T. maxilliforme CCMF-14286, T. furcatum CCMF-11883; Anguillospora furtiva and Heliscus lugdunensis. Prof. Dr. Walter Gams (Centraalbureau voor Schimmelcultures, Utrecht, NL) for always encouraging me to follow my mycological interests as well as for his friendly support over the years, and for making this project possible in the first place. Prof. Dr. Gernot Bretschko (d. 28.3.2002) and Dr. Maria Leichtfried (Biological Station Lunz, A) for giving me the opportunity to teach and conduct research at BSL, for providing abiotic data of the Oberer Seebach, and for always welcoming me most warmly on my personally and scientifically enriching visits to Lunz. Dr. Sibylle Kalmbach, who introduced me to the first steps in FISH. Dr. Elisabeth Grohmann, who supported me with expert advice in molecular genetics and regularly stocked up my sweets supplies. Celia Möbius, who introduced me to the MrBayes program and provided essential help with it. Dr. Uta Böckelmann for invaluable scientific and personal exchanges between bacteriologist and mycologist. Annett Wolf for excellent technical assistance with culturing and sequencing of many fungal strains, for the pleasant working atmosphere, and for holding the fort in the lab while I was in Lunz. Markus Schick for dedicated technical assistance with the ARB program and its databases.

147 Acknowledgements

Klaus Mühlmann, who continued to furnish me with expert technical assistance with the ARB program after Markus’ contract had expired. Marcella Linke and Roy-Alexander Zsupanek, whose extended essays formed part of the project and who contributed valuable work on Anguillospora and Tricladium phylogeny. Brigitte Ziebarth for very useful technical help with the maintenance of fungal cultures and friendly support with many of the small things of a scientist’s daily routine during the first year of the project. Karin Trojan for friendly help with administrative matters. All members of the laboratory for a good working atmosphere. The British Mycological Society for encouragement and scientific as well as financial support in the form of the Howard Eggins Award 2001. Associate-Prof. Dr. David Pommerenke (University of Missouri-Rolla, USA) for invaluable linguistic advice on the manuscript, his unfailing support over long distances of both time and space, and his caring interest in much more than just my research. Prof. Dr. Christian Pommerenke for his valuable advice on a draft version of the discussion. Andreas Ludwig for computer expertise and assistance and... The Gesellschaft der Freunde der TU Berlin who enabled me to take part in the 8th International Symposium of Marine and Freshwater Mycology 2001 (Hurghada, ET) and in the general meeting of the BMS by providing generous financial assistance. The UFZ, Centre for Environmental Research Leipzig-Halle, for financial support in the form of the UFZ-03/99 grant. My family and friends for thoughtfulness and for manifold kindnesses.

148 References

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166 Appendix

APPENDIX

Phylogenetic trees

Drawings

SSU distance matrix

Complete alignments

167 Appendix

Tetracladium maxilliforme Tetracladium furcatum Tetracladium setigerum Tetracladium apiense Tetracladium marchalianum ELBE50 Tetracladium marchalianum 312 99 Tetracladium marchalianum Tetracladium marchalianum 26299 Tetracladium marchalianum ELBE90 Tetracladium marchalianum 26199 Tetracladium marchalianum L27 Tetracladium marchalianum 19399 Oidiodendron tenuissimum 55 Myxotrichum deflexum Hymenoscyphus virgultorum 51 Loramyces juncicola Phyllactinia guttata Blumeria graminis 78 Cyttaria darwinii 99 Pseudogymnoascus roseus Geomyces pannorum var. pannorum Leotia lubrica Bulgaria inquinans Hymenoscyphus fructigenus Cudonia confusa 85 Spathularia flavida 78 Fabrella tsugae Geosmithia putterillii Nectria cinnabarina Gibberella pulicaris Hypocrea lutea 55 Hypomyces chrysospermus Halosarpheia retorquens Microascus cirrosus 99 Pseudallescheria boydii 99 Petriella setifera 100 Graphium penicillioides Neurospora crassa Sordaria firmicola 91 Chaetomium elatum 99 Kionochaeta ivoriensis Aureobasidium pullulans 67 Hortaea werneckii Kirschsteiniothelia aethiops Cucurbitaria elongata 95 Pleospora betae Ophiobolus herpotrichus 98 Leptosphaeria doliolum 95 Kirschsteiniothelia elaterascu Mycosphaerella mycopappi 89 Herpotrichia juniperi 99 Massarina bipolaris 78 Massarina australiensis 96 Kirschsteiniothelia maritima Monascus purpureus 95 Paecilomyces variotii 95 Onygena equina Arthrobotrys superba Orbilia fimicola Arthrobotrys oligospora 99 Monacrosporium doedycoides Orbilia delicatula Hydnum repandum 100 Peniophora nuda Bulleromyces albus Saccharomyces cerevisiae

0.01 substitutions / site

Figure A1: Phylogenetic tree obtained by maximum likelihood analysis (Ln likelihood = –14292.2146), using 18S rDNA sequence data. Bootstrap values were derived from 1000 resampled data sets. The tree shows the possible placement of four Tetracladium marchalianum strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study.

168 Appendix

Tetracladium marchalianum 26199 Tetracladium marchalianum 19399 Tetracladium marchalianum ELBE50 Tetracladium marchalianum 26399 97 Tetracladium marchalianum L27 56 Tetracladium marchalianum 312 99 Tetracladium marchalianum 26299 Tetracladium furcatum 71 Tetracladium maxilliforme 88 Tetracladium apiense 97 axenic ectomycorrhizal isolate 59 Dactylaria dimorphospora Helotiales sp. ARON3063.S ectomycorrhizal isolate ARONR1165 89 ericoid mycorrhizal sp. Sm5 ericoid mycorrhizal sp. Sd9 Oidiodendron tenuissimum

0.01 changes

Figure A2: ITS rDNA (including 5.8S rDNA) sequence-based phylogenetic tree derived using neighbour joining analysis, showing relationships of Tetracladium strains and closest BLAST hits. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). The scale bar indicates one base change per 100 nucleotide positions.

Tetracladium marchalianum 26199 Tetracladium marchalianum 19399 Tetracladium marchalianum ELBE50 Tetracladium marchalianum 26399 100 Tetracladium marchalianum 312 Tetracladium marchalianum L27 94 Tetracladium marchalianum 26299 91 Tetracladium apiense 90 Tetracladium maxilliforme 92 Tetracladium furcatum axenic ectomycorrhizal isolate 86 Dactylaria dimorphospora Helotiales sp. ARON3063.S ectomycorrhizal isolate ARONR1165 Oidiodendron tenuissimum ericoid mycorrhizal sp. Sd9 ericoid mycorrhizal sp. Sm5

0.01 substitutions / site

Figure A3: Phylogenetic tree reconstructed by maximum likelihood analysis, consensus of 12 equally likely trees (Ln likelihood = -2138,09571), based on the same data as Figure A2, but including parsimony uninformative data. Bootstrap values were derived from 1000 resampled data sets. The tree shows the possible relationships of Tetracladium strains and fungal species with the most known similar ITS rDNA sequences. Names in red indicate DNA sequences newly determined in this study.

169 Appendix

Antarctic yeast CBS 8931 salal root associated fungus U Chalara constricta Chalara longipes Chalara microchona 56 Rhytisma acerinum 93 Oidiodendron tenuissimum Myxotrichum deflexum Tetracladium marchalianum 26199 Hymenoscyphus virgultorum 95 Cudonia lutea 63 Spathularia flavida Geomyces pannorum var. pannorum Pseudogymnoascus roseus 92 Phyllactinia moricola 76 Blumeria graminis f. sp. bromi Leotia viscosa 58 Hypocrea lutea Hypomyces chrysospermus 98 Nectria cinnabarina 100 Geosmithia putterillii Gibberella pulicaris 88 Petriella setifera 89 Pseudallescheria boydii 97 Microascus cirrosus Halosarpheia retorquens 100 Graphium penicillioides Sordaria fimicola 92 Chaetomium globosum Neurospora crassa 55 Bipolaris spicifera Curvularia inaequalis 71 Curvularia lunata 89 Curvularia eragrostidis 98 Pleospora herbarum var. herbar 90 Anguillospora longissima 00980 Leptosphaeria doliolum Ophiobolus herpotrichus 87 Westerdykella cylindrica 90 Mycosphaerella mycopappi Aureobasidium pullulans Peziza vesiculosa Bulleromyces albus

0.01 changes

Figure A4: Phylogenetic tree based on 28S rDNA sequences using neighbour joining, showing the placement of Tetracladium marchalianum 26199 and Anguillospora longissima 00980 within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). Bootstrap values were derived from 1000 resampled data sets. The scale bar indicates one base change per 100 nucleotide positions.

170 Appendix

Chalara microchona 58 Chalara longipes 63 Rhytisma acerinum 93 salal root associated fungus U Chalara constricta 86 Antarctic yeast CBS 8931 87 Tetracladium marchalianum 26199 56 Hymenoscyphus virgultorum 91 Myxotrichum deflexum 83 Oidiodendron tenuissimum Pseudogymnoascus roseus 73 Geomyces pannorum var. pannorum 94 Leotia viscosa Spathularia flavida 70 Cudonia lutea 90 Blumeria graminis f. sp. bromi 78 Phyllactinia moricola 87 Pseudallescheria boydii Petriella setifera 84 Microascus cirrosus Halosarpheia retorquens 89 Graphium penicillioides Geosmithia putterillii 56 Hypocrea lutea 98 Hypomyces chrysospermus 86 Nectria cinnabarina Gibberella pulicaris Chaetomium globosum Sordaria fimicola Neurospora crassa 58 Curvularia inaequalis Curvularia lunata 83 Bipolaris spicifera 95 Curvularia eragrostidis Mycosphaerella mycopappi Westerdykella cylindrica 98 Leptosphaeria doliolum Ophiobolus herpotrichus 86 Pleospora herbarum var. herbar 88 Anguillospora longissima 00980 Aureobasidium pullulans Peziza vesiculosa Bulleromyces albus

0.01 substitutions / site

Figure A5: Phylogenetic tree reconstructed by maximum likelihood analysis (Ln likelihood = - 7886.21025), based on 28S rDNA sequence data. Bootstrap values were derived from 1000 resampled data sets. The tree shows the possible relationships of Tetracladium marchalianum and Anguillospora longissima within the Ascomycota. Names in red indicate DNA sequences newly determined in this study.

171 Appendix

Figure A6: Alatospora acuminata sensu stricto, strain CCM-F 02383 (= CCM-F 12383). A-D sessile conidiogenous cells with conidia in various stages of developments. E Acicular conidia. F-J Branched conidia without constricted branch bases. Drawings from Marvanová & Descals 1985.

172 Appendix

Figure A7: Alatospora acuminata sensu lato strain CCM-F 37194. Conidia with constricted insertion of branches. The scale bar corresponds to 50µm. Drawings by courtesy of Dr. Ludmila Marvanová (CCM, Brno).

173 Appendix

Spathularia flavida 60 Cudonia confusa 86 Loramyces juncicola 56 Hymenoscyphus virgultorum Geomyces pannorum var. pannorum 60 Pseudogymnoascus roseus Bulgaria inquinans Alatospora acuminata 37194 Alatospora acuminata 13089 Alatospora acuminata L8 81 Alatospora acuminata 02383 Leotia lubrica 56 Hymenoscyphus fructigenus Phyllactinia guttata 87 Blumeria graminis Myxotrichum deflexum 93 Oidiodendron tenuissimum 65 Cyttaria darwinii 57 Pseudallescheria boydii 94 Petriella setifera 68 Microascus cirrosus 99 Halosarpheia retorquens Graphium penicillioides 56 Gibberella pulicaris 86 Nectria cinnabarina Geosmithia putterillii Hypomyces chrysospermus 99 Hypocrea lutea 94 Sordaria firmicola 98 Neurospora crassa Chaetomium elatum 73 Kionochaeta ivoriensis 86 Fabrella tsugae 94 Hortaea werneckii 83 Aureobasidium pullulans 79 Kirschsteiniothelia aethiops 56 Cucurbitaria elongata Pleospora betae 86 Ophiobolus herpotrichus 94 Leptosphaeria doliolum 60 Kirschsteiniothelia elaterascus Mycosphaerella mycopappi Herpotrichia juniperi 87 Massarina australiensis 63 Massarina bipolaris 91 Kirschsteiniothelia maritima Paecilomyces variotii Monascus purpureus 100 Onygena equina Arthrobotrys superba Orbilia fimicola Arthrobotrys oligospora Monacrosporium doedycoides Orbilia delicatula 91 Hydnum repandum Peniophora nuda Bulleromyces albus Saccharomyces cerevisiae

0.01 changes

Figure A8: Phylogenetic tree based on 18S rDNA sequences using neighbour joining, showing the placement of four Alatospora acuminata strains within the Ascomycota. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets. The scale bar indicates one base change per 100 nucleotide positions.

174 Appendix

100 Blumeria graminis 51 Phyllactinia guttata 100 Oidiodendron tenuissimum 79 Myxotrichum deflexum 81 Loramyces juncicola Hymenoscyphus virgultorum 100 Cudonia confusa Spathularia flavida Cyttaria darwinii Hymenoscyphus fructigenus Alatospora acuminata L8 Alatospora acuminata 02383 100 Alatospora acuminata 37194 90 Alatospora acuminata 13089 Leotia lubrica Bulgaria inquinans 100 Geomyces pannorum var. pannorum Pseudogymnoascus roseus Halosarpheia retorquens Petriella setifera Microascus cirrosus 99 Pseudallescheria boydii Graphium penicillioides Gibberella pulicaris Nectria cinnabarina 99 Geosmithia putterillii Hypocrea lutea 100 Hypomyces chrysospermus Sordaria firmicola Neurospora crassa 100 Chaetomium elatum Kionochaeta ivoriensis 51 Fabrella tsugae Hortaea werneckii Aureobasidium pullulans 82 Kirschsteiniothelia aethiops Ophiobolus herpotrichus Leptosphaeria doliolum Cucurbitaria elongata Pleospora betae 99 Kirschsteiniothelia elaterascus Massarina bipolaris Massarina australiensis Kirschsteiniothelia maritima Mycosphaerella mycopappi 87 Herpotrichia juniperi 100 Paecilomyces variotii 99 Monascus purpureus 99 Onygena equina Orbilia fimicola Arthrobotrys superba Arthrobotrys oligospora 100 Monacrosporium doedycoides Orbilia delicatula Peniophora nuda Hydnum repandum Bulleromyces albus Saccharomyces cerevisiae

0.01substitutions / site

Figure A9: Phylogenetic tree reconstructed by maximum likelihood analysis (Ln likelihood = - 13346.25987), based on 18S rDNA sequence data. Bootstrap values were derived from 1000 resampled data sets. The tree shows the possible placement of Alatospora acuminata strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study.

175 Appendix

Guignardia philoprina Phoma-like coelomycete 1-L-3-5

Alatospora acuminata 37194 Alatospora acuminata L8 Alatospora acuminata 12186

Alatospora acuminata 13089 Alatospora acuminata 02383

Cudonia lutea

0.01 changes

Leotiales sp. Bjelland 61

Figure A10: Phylogenetic tree based on ITS1, 5.8S, ITS2 rDNA sequences using neighbour joining, showing the possible relationships of five Alatospora acuminata strains with their closest BLAST hits. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets. The scale bar indicates one base change per 100 nucleotide positions .

176 Appendix

Figure A11: Conidia of different Tricladium angulatum and Tricladium splendens strains. Strain T. angulatum CCMF-10200 showed conidia of variable size, ranging from 50 – 100 µm. Drawing of strain T. splendens CCMF-19087 shows also conidiogenous cells (right) and conidiophores with attached developing conidia (middle). Drawings by courtesy of Dr. Ludmila Marvanová (CCM, Brno).

177 Appendix

Tricladium angulatum 10200 Tricladium angulatum 01380 100 Tricladium angulatum139 56 Tricladium angulatum14186 Tricladium splendens 12386 Tricladium splendens 11989 100 Tricladium splendens 19087 Tricladium splendens16599 63 Hymenoscyphus fructigenus 60 Cyttaria darwinii 86 Loramyces juncicola 87 Hymenoscyphus virgultorum Oidiodendron tenuissimum 60 Myxotrichum deflexum Phyllactinia guttata Blumeria graminis Pseudogymnoascus roseus 89 Geomyces pannorum var. pannorum 63 Leotia lubrica 83 Bulgaria inquinans Spathularia flavida 93 Cudonia confusa 87 Fabrella tsugae Pseudallescheria boydii 96 Petriella setifera 55 Microascus cirrosus Halosarpheia retorquens 86 Graphium penicillioides Geosmithia putterillii Nectria cinnabarina 100 Gibberella pulicaris Hypomyces chrysospermus 77 Hypocrea lutea 94 Sordaria firmicola 96 Neurospora crassa Chaetomium elatum 79 Kionochaeta ivoriensis 94 Hortaea werneckii 83 Aureobasidium pullulans 94 Kirschsteiniothelia aethiops Cucurbitaria elongata 99 Pleospora betae Ophiobolus herpotrichus 94 Leptosphaeria doliolum 60 Kirschsteiniothelia elaterascus Herpotrichia juniperi 87 Mycosphaerella mycopappi Massarina australiensis 63 Massarina bipolaris 89 Kirschsteiniothelia maritima Paecilomyces variotii 98 Monascus purpureus 100 Onygena equina Orbilia fimicola Arthrobotrys superba Arthrobotrys oligospora 100 Monacrosporium doedycoides Orbilia delicatula 91 Peniophora nuda Hydnum repandum 86 Bulleromyces albus Saccharomyces cerevisiae

0.01 changes

Figure A12: Phylogenetic tree using maximum parsimony, showing the placement of Tricladium angulatum and T. splendens strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. One of 64 equally parsimonious trees (1833 steps) is shown, based on 659 parsimony informative data of 18S rDNA sequences. Bootstrap values were derived from 1000 resampled data sets

178 Appendix

H. sp. GU30 H. ericae 7

H. ericae 6

H. fructigenus

Ericoid mycorrhizal sp. Sm5

0.01 changes

Cistella grevillei

Triang 10200 Triang 14186 Triang 01380 Triang 139

Figure A13: Phylogenetic tree using maximum parsimony, showing the placement of Tricladium angulatum strains and their closest BLAST hits. Names in red indicate DNA sequences newly determined in this study. One of 3 equally parsimonious trees (92 steps) is shown, based on 66 parsimony informative data of ITS1, 5.8S, ITS2 rDNA sequences. Bootstrap values were derived from 1000 resampled data sets. Triang = Tricladium angulatum; H. = Hymenoscyphus

179 Appendix

Tricladium angulatum 10200 Tricladium angulatum 139 Tricladium angulatum 14186 95 Tricladium angulatum 01380 86 Cistella grevillei Ericoid mycorrhizal sp. Sm5 100 Hymenoscyphus sp. GU30 Hymenoscyphus ericae 7 Hymenoscyphus ericae 6 Hymenoscyphus fructigenus

0.01 substitutions / site

Figure A14: Single phylogenetic tree obtained from maximum likelihood analysis (Ln likelihood = - 2067.66536), based on ITS1, 5.8S, ITS2 rDNA sequence data. The tree shows the relationships of Tricladium angulatum strains with their most similar sequences from BLAST searches. Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets.

Tricladium angulatum139 57 Tricladium angulatum14186 Tricladium angulatum10200 96 Tricladium angulatum 01380 Cistella grevillei ericoid mycorrhizal sp. Sm5 84 Pezicula malicorticis Mycoarthris corallina Solenopezia solenia Lachnum bicolor

0.01 changes

Figure A15: Phylogenetic tree based on ITS1, 5.8S, ITS2 rDNA sequence data using neighbour joining. The tree shows the possible relationships of Tricladium angulatum strains with their closest BLAST hits (C. grevillei; ericoid sp. Sm5) and the aquatic hyphomycete M. corralina as well as other members of the Hyaloscyphaceae. Bootstrap values were derived from 1000 resampled data sets. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). The scale bar indicates one base change per 100 nucleotide positions.

180 Appendix

Tricladium splendens 16599 Tricladium splendens 19087 Tricladium splendens 12386 94 Tricladium splendens 11989 86 Zalerion varium 91 Lachnum bicolor 80 Hymenoscyphus ericae 6 90 Hymenoscyphus ericae 7 Hymenoscyphus sp. GU30 Hymenoscyphus fructigenus Blumeria graminis f. sp. bromi

0.01 changes

Figure A16: The most parsimonious tree based on ITS1, 5.8S, ITS2 rDNA sequence data (120 parsimony informative positions, 456 steps) showing the relationships of Tricladium splendens strains with their closest BLAST hits. Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets.

Tricladium splendens 12386 Tricladium splendens 16599 97 Tricladium splendens 11989 95 Tricladium splendens 19087 91 Zalerion varium 55 Lachnum bicolor 87 Hymenoscyphus sp. GU30 62 Hymenoscyphus ericae 7 Hymenoscyphus ericae 6 Hymenoscyphus fructigenus Blumeria graminis f. sp. bromi

0.01 substitutions / site

Figure A17: Single phylogenetic tree obtained from maximum likelihood analysis (Ln likelihood = - 2911.80797), based on ITS1, 5.8S, ITS2 rDNA sequence data. The tree shows the relationships of Tricladium splendens strains with their most similar sequences from BLAST searches. Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets.

181 Appendix

73 Blumeria graminis 52 Phyllactinia guttata 53 Hymenoscyphus virgultorum Fabrella tsugae 65 Myxotrichum deflexum 83 Oidiodendron tenuissimum Loramyces junicola Spathularia flavida Cudonia confusa 56 Cyttaria darwinii 90 Pseudogymnoascus roseus Geomyces pannorum var. pannorum Hymenoscyphus fructigenus Leotia lubrica Bulgaria inquinans Hortaea werneckii 51 Aureobasidium pullulans Cucurbitaria elongata Leptosphaeria doliolum 83 Ophiobolus herpotrichus 65 Pleospora betae 85 Kirschsteiniothelia elaterascu 99 Mycosphaerella mycopappi 64 Herpotrichia juniperi 99 Kirschsteiniothelia maritima 89 Massarina bipolaris Massarina australiensis 90 Kirschsteiniothelia aethiops Heliscus lugdunensis ELBE98 Heliscus lugdunensis L5 73 Heliscus lugdunensis 245 82 Gibberella pulicaris 82 Nectria cinnabarina 84 Geosmithia putterillii 90 Hypomyces chrysospermus 91 Hypocrea lutea 94 Pseudallescheria boydii 63 Petriella setifera 84 Microascus cirrosus 98 Halosarpheia retorquens 99 Graphium penicillioides 80 Neurospora crassa 96 Sordaria firmicola 99 Chaetomium elatum 71 Kionochaeta ivoriensis 94 Monascus purpureus 98 Paecilomyces variotii 98 Onygena equina 76 Arthrobotrys superba 94 Orbilia fimicola 89 Arthrobotrys oligospora 99 Monacrosporium doedycoides Orbilia delicatula 90 Peniophora nuda Hydnum repandum 89 Bulleromyces albus Saccharomyces cerevisiae

0.01 changes

Figure A18: Phylogenetic tree based on 18S rDNA sequences using neighbour joining, showing the placement of three Heliscus lugdunensis strains within the Ascomycota. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets. The scale bar indicates one base change per 100 nucleotide positions.

182 Appendix

85 Microascus cirrosus 100 Halosarpheia retorquens Petriella setifera 100 Pseudallescheria boydii 100 Graphium penicillioides Heliscus lugdunensis ELBE98 98 Heliscus lugdunensis L5 80 Heliscus lugdunensis 245 59 Gibberella pulicaris Geosmithia putterillii 76 Nectria cinnabarina 100 Hypomyces chrysospermus 98 Hypocrea lutea Sordaria firmicola Neurospora crassa 100 Chaetomium elatum Kionochaeta ivoriensis 96 Fabrella tsugae 100 Geomyces pannorum var. pannorum Pseudogymnoascus roseus Leotia lubrica 81 Bulgaria inquinans Spathularia flavida Cudonia confusa 89 Loramyces junicola Hymenoscyphus virgultorum 98 Oidiodendron tenuissimum Myxotrichum deflexum 100 Phyllactinia guttata 91 Blumeria graminis Cyttaria darwinii 51 Hymenoscyphus fructigenus 82 Aureobasidium pullulans 88 Hortaea werneckii 53 Kirschsteiniothelia aethiops Ophiobolus herpotrichus Leptosphaeria doliolum 99 Cucurbitaria elongata 52 Pleospora betae 82 Kirschsteiniothelia elaterascu Herpotrichia juniperi 100 Mycosphaerella mycopappi 99 Massarina australiensis 96 Massarina bipolaris 97 Kirschsteiniothelia maritima 100 Paecilomyces variotii 98 Monascus purpureus 100 Onygena equina Orbilia fimicola Arthrobotrys superba Arthrobotrys oligospora Monacrosporium doedycoides Orbilia delicatula 89 Peniophora nuda 100 Hydnum repandum 95 Bulleromyces albus Saccharomyces cerevisiae

0.01 substitutions / site

Figure A19: Single phylogenetic tree obtained from maximum likelihood analysis (Ln likelihood = - 12863.61197), based on 18S rDNA sequence data. The tree shows the placement of Heliscus lugdunensis strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets.

183 Appendix

Figure A20: Conidia of Anguillospora longissima strains.

A Differently sized conidia of strain CCMF-00980.

B Conidia of strain CCMF-10691.

C Conidium of strain CCMF-11891.

Drawings by courtesy of Dr. Ludmila Marvanová (CCM, Brno).

184 Appendix

B

A

C

Figure A21: Conidia of Anguillospora crassa strains.

A Strain CCMF-07082, conidia obtained from isolate with stalked apothecia.

B Strain CCMF-15583, conidia obtained from Mollisia, sessile apothecia.

C Strain CCMF-05584, differently sized conidia obtained from sessile apothecia.

Drawings by courtesy of Dr. . Ludmila Marvanová (CCM, Brno).

185 Appendix

B

A

Figure A22: Conidia of Anguillospora crassa strains.

A Strain CCMF-13483, differently sized conidia obtained from sessile apothecia.

B Strain CCMF-15283, conidia and microconidial synanamorph (right) obtained from sessile apothecia.

Drawings by courtesy of Dr. . Ludmila Marvanová (CCM, Brno).

186 Appendix

Anguillospora crassa 07082 Anguillospora crassa 05584 Anguillospora crassa 13483 Anguillospora crassa 15583 86 Phyllactinia guttata Blumeria graminis Hymenoscyphus fructigenus Hymenoscyphus virgultorum Loramyces juncicola Myxotrichum deflexum 89 Oidiodendron tenuissimum Cyttaria darwinii Pseudogymnoascus roseus Geomyces pannorum var. pannorum Leotia lubrica 86 Bulgaria inquinans Cudonia confusa 83 Spathularia flavida 71 Fabrella tsugae Hypocrea lutea Hypomyces chrysospermus 60 Gibberella pulicaris Nectria cinnabarina 87 Geosmithia putterillii Halosarpheia retorquens Microascus cirrosus Pseudallescheria boydii 51 Petriella setifera 100 Graphium penicillioides Neurospora crassa Sordaria firmicola 81 Chaetomium elatum 77 Kionochaeta ivoriensis 86 Aureobasidium pullulans Hortaea werneckii Kirschsteiniothelia aethiops Ophiobolus herpotrichus Leptosphaeria doliolum Cucurbitaria elongata Pleospora betae 74 Kirschsteiniothelia elaterascus Mycosphaerella mycopappi 99 Herpotrichia juniperi Anguillospora longissima L22 Anguillospora longissima 11891 97 Anguillospora longissima 00980 Kirschsteiniothelia maritima 74 Massarina bipolaris 90 Massarina australiensis 98 Monascus purpureus 99 Paecilomyces variotii 99 Onygena equina 98 Orbilia fimicola Arthrobotrys superba Arthrobotrys oligospora 98 Monacrosporium doedycoides Orbilia delicatula 96 Peniophora nuda 98 Hydnum repandum Bulleromyces albus Saccharomyces cerevisiae

0.01 changes

Figure A23: Phylogenetic tree using maximum parsimony, showing the placement of Anguillospora longissima and A. crassa strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. The most parsimonious tree (1786 steps) is shown, based on 659 parsimony informative data of 18S rDNA sequences. Bootstrap values were derived from 1000 resampled data sets.

187 Appendix

Anguillospora crassa 15583 Anguillospora crassa 15283 Anguillospora crassa 07082 Anguillospora crassa 05584 Zalerion varium

Phacidium infestans

0.01 changes

Leotiales sp. Bjelland 61

Mollisia cinerea Hymenoscyphus sp. GU30 Mollisia minutella Hymenoscyphus ericae 7

Cudonia lutea

Spathularia flavida

Figure A24: The most parsimonious tree based on ITS1, 5.8S, ITS2 rDNA sequence data (118 parsimony informative positions, 487 steps) showing the relationships of Anguillospora crassa strains with their closest BLAST hits. Names in red indicate DNA sequences newly determined in this study.

188 Appendix

Spathularia flavida Cudonia lutea

Hymenoscyphus sp. GU30

Hymenoscyphus ericae 7 Leotiales sp. Bjelland 61

Phacidium infestans Mollisia minutella Mollisia cinerea

Zalerion varium 0.01 changes Anguillospora crassa 15283 Anguillospora crassa 15583 Anguillospora crassa 05584 Anguillospora crassa 07082

Figure A25: Phylogenetic tree based on ITS1, 5.8S, ITS2 rDNA sequence data using neighbour joining. The tree shows the possible relationships of Anguillospora crassa strains with their closest BLAST hits. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). The scale bar indicates one base change per 100 nucleotide positions.

189 Appendix

Anguillospora longissima 11891 Anguillospora longissima 00980 100 Anguillospora longissima 10691 Anguillospora longissima L22 60 Anguillospora longissima 11791 Lophiostoma caulium 72 Massarina bipolaris 83 Massarina fronisubmersa 51 Lophiostoma vagabundum 92 Massarina rubi 78 Massarina armatispora 99 Massarina corticola Massarina ramunculicola Massarina eburnea Leptosphaeria doliolum Leptosphaeria contecta Pleospora papaveracea Curvularia eragrostidis Massarina walkeri

0.01 changes

Figure A26: Phylogenetic tree based on ITS1, 5.8S, ITS2 rDNA sequence data using neighbour joining. The tree shows the possible relationships of Anguillospora longissima strains with their closest BLAST hits, and Massarina, and Lophiostoma sequences. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). The scale bar indicates one base change per 100 nucleotide positions. Bootstrap values were derived from 1000 resampled data sets.

Lophiostoma caulium 77 Massarina armatispora Massarina fronisubmersa Massarina bipolaris Massarina corticola 64 Massarina rubi Lophiostoma vagabundum 80 Massarina walkeri 99 Leptosphaeria doliolum Anguillospora longissima 10691 Anguillospora longissima L22 Anguillospora longissima 11791 100 Anguillospora longissima 00980 99 Anguillospora longissima 11891 Leptosphaeria contecta 80 Massarina eburnea Massarina ramunculicola 98 Pleospora papaveracea Curvularia eragrostidis

0.01 substitutions / site

Figure A27: Single phylogenetic tree obtained from maximum likelihood analysis (Ln likelihood = - 7549.88369), based on ITS1, 5.8S, ITS2 rDNA sequence data. The tree shows the relationships of Anguillospora longissima strains with their closest BLAST hits, and Massarina spp., and Lophiostoma spp.. Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets

190 Appendix

A B

Figure A28: Conidia of Lemonniera.

A Lemonniera aquatica strain CCMF-04480.

B Lemonniera terrestris strain CCMF-11486.

Drawings by courtesy of Dr. Ludmila Marvanová (CCM, Brno).

191 Appendix

Lemonniera terrestris Lemonniera aquatica 04480 87 Lemonniera terrestris 11486 56 Loramyces juncicola 93 Hymenoscyphus virgultorum Phialophora sp. Elec-N-1 4.PN Phyllactinia guttata 87 Blumeria graminis 96 Oidiodendron tenuissimum 87 Myxotrichum deflexum Cyttaria darwinii Spathularia flavida Cudonia confusa 63 Fabrella tsugae Geomyces pannorum var. pannorum 83 Pseudogymnoascus roseus Leotia lubrica Bulgaria inquinans 73 Hymenoscyphus fructigenus 86 Aureobasidium pullulans 58 Hortaea werneckii 63 Leptosphaeria doliolum 56 Ophiobolus herpotrichus 94 Cucurbitaria elongata 89 Pleospora betae 56 Kirschsteiniothelia elaterascus Herpotrichia juniperi 77 Mycosphaerella mycopappi 95 Massarina australiensis Massarina bipolaris 96 Kirschsteiniothelia maritima 94 Kirschsteiniothelia aethiops Geosmithia putterillii 60 Nectria cinnabarina 58 Gibberella pulicaris Hypocrea lutea 91 Hypomyces chrysospermus 98 Halosarpheia retorquens 57 Microascus cirrosus Pseudallescheria boydii Petriella setifera Graphium penicillioides 94 Neurospora crassa 96 Sordaria firmicola Chaetomium elatum 98 Kionochaeta ivoriensis Paecilomyces variotii Monascus purpureus Onygena equina Arthrobotrys superba Orbilia fimicola Arthrobotrys oligospora Monacrosporium doedycoides Orbilia delicatula 93 Hydnum repandum Peniophora nuda Bulleromyces albus Saccharomyces cerevisiae

0.01 changes

Figure A29: The most parsimonious tree based on 18S rDNA sequence data (659 parsimony informative positions, 1817 steps) showing the phylogenetic placement of Lemonniera aquatica and L. terrestris strains within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets.

192 Appendix

Lemonniera terrestris Lemonniera aquatica 04480 76 Lemonniera terrestris 11486 Loramyces juncicola Hymenoscyphus virgultorum Phialophora sp. Elec-N-1 4.PN 100 Blumeria graminis Phyllactinia guttata 100 Oidiodendron tenuissimum Myxotrichum deflexum 51 Cyttaria darwinii Spathularia flavida 95 Cudonia confusa Fabrella tsugae 100 Geomyces pannorum var. pannorum Pseudogymnoascus roseus Leotia lubrica Bulgaria inquinans Hymenoscyphus fructigenus 90 Aureobasidium pullulans Hortaea werneckii Leptosphaeria doliolum Ophiobolus herpotrichus Cucurbitaria elongata 91 Pleospora betae 57 Kirschsteiniothelia elaterascus Herpotrichia juniperi 80 Mycosphaerella mycopappi Massarina australiensis Massarina bipolaris 97 Kirschsteiniothelia maritima Kirschsteiniothelia aethiops Geosmithia putterillii 62 Nectria cinnabarina 99 Gibberella pulicaris Hypocrea lutea 86 Hypomyces chrysospermus Halosarpheia retorquens Microascus cirrosus Pseudallescheria boydii 97 Petriella setifera 100 Graphium penicillioides Neurospora crassa Sordaria firmicola 99 Chaetomium elatum 95 Kionochaeta ivoriensis Paecilomyces variotii 98 Monascus purpureus 98 Onygena equina Arthrobotrys superba Orbilia fimicola Arthrobotrys oligospora 99 Monacrosporium doedycoides Orbilia delicatula 100 Hydnum repandum 100 Peniophora nuda Bulleromyces albus Saccharomyces cerevisiae

0.01 substitutions / site

Figure A30: One of three phylogenetic trees obtained from maximum likelihood analysis (Ln likelihood = - 13404.92413), based on ITS1, 5.8S, ITS2 rDNA sequence data. The tree shows the placement of Lemonniera aquatica and L. terrestris within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets

193 Appendix

Tetracladium apiense Tetracladium setigerum 82 Tetracladium maxilliforme 96 Tetracladium furcatum Tetracladium marchalianum 19399 91 Myxotrichum deflexum Oidiodendron tenuissimum 91 Lemonniera aquatica 04480 Lemonniera terrestris 11486 86 Loramyces juncicola Leotiomycetes Varicosporium elodeae 11783 71 Tricladium angulatum 10200 Hymenoscyphus virgultorum 98 Anguillospora crassa 05584 96 Anguillospora furtivaL16 71 Tricladium splendens 11989 80 Phyllactinia guttata Blumeria graminis Phialophora sp. Elec-N-1 4.PN Pseudogymnoascus roseus Geomyces pannorum var. pannorum Bulgaria inquinans 95 Leotia lubrica 81 Alatospora acuminata 02383 99 Cudonia confusa Spathularia flavida Cyttaria darwinii 97 Aureobasidium pullulans Hortaea werneckii 67 Gibberella pulicaris 87 Heliscus lugdunensis 245 Nectria cinnabarina 99 Hypocrea lutea Hypomyces chrysospermus 98 Geosmithia putterillii 97 Pseudallescheria boydii 94 Petriella setifera Sordariomycetes 84 Microascus cirrosus 100 Halosarpheia retorquens 100 Graphium penicillioides 80 Neurospora crassa 95 Sordaria firmicola 100 Chaetomium elatum 82 Kionochaeta ivoriensis Ophiobolus herpotrichus Leptosphaeria doliolum 4 96 Cucurbitaria elongata 59 Pleospora betae Kirschsteiniothelia elaterascus Dothideomycetes 99 Mycosphaerella mycopappi 62 Herpotrichia juniperi 98 Kirschsteiniothelia maritima 100 Anguillospora longissima 11891 100 Massarina bipolaris 70 Massarina australiensis 95 Monascus purpureus Paecilomyces variotii Eurotiomycetes Onygena equina 100 Kirschsteiniothelia aethiops Arthrobotrys superba Orbilia fimicola Arthrobotrys oligospora Orbiliomycetes 100 Monacrosporium doedycoides Orbilia delicatula 98 Hydnum repandum Hemiascomycetes Peniophora nuda and Bulleromyces albus Basidiomycetes Saccharomyces cerevisiae 5 (outgroup)

0.01 changes

Figure A31: Phylogenetic tree based on 18S rDNA sequence data using neighbour joining. The tree shows the possible placement of eleven species of aquatic hyphomycetes within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Distance matrices were constructed from the aligned sequences and corrected for multiple base changes at single positions by the algorithm of Jukes and Cantor (1969). The scale bar indicates one base change per 100 nucleotide positions. Bootstrap values were derived from 1000 resampled data sets.

194 Appendix

56 Tetracladium setigerum 86 Tetracladium maxilliforme Tetracladium furcatum 98 Tetracladium apiense Tetracladium marchalianum 19399 95 Lemonniera terrestris 11486 86 Lemonniera aquatica 04480 89 Varicosporium elodeae 11783 58 Tricladium angulatum 10200 95 Loramyces juncicola 63 Hymenoscyphus virgultorum 77 Oidiodendron tenuissimum Myxotrichum deflexum 95 Anguillospora furtiva L16 Anguillospora crassa 05584 60 Tricladium splendens 11989 Blumeria graminis 95 Phyllactinia guttata 87 Phialophora sp. Elec-N-1 4.PN 87 Cyttaria darwinii Spathularia flavida Cudonia confusa Geomyces pannorum var. pannorum 60 Pseudogymnoascus roseus Bulgaria inquinans Alatospora acuminata 02383 77 Leotia lubrica 98 Geosmithia putterillii 86 Nectria cinnabarina 77 Gibberella pulicaris Hypomyces chrysospermus Hypocrea lutea 97 Heliscus lugdunensis 245 60 Pseudallescheria boydii 98 Petriella setifera 55 Microascus cirrosus Halosarpheia retorquens 100 Graphium penicillioides 94 Sordaria firmicola 94 Neurospora crassa Chaetomium elatum 79 Kionochaeta ivoriensis 94 Hortaea werneckii 83 Aureobasidium pullulans 79 Kirschsteiniothelia aethiops 86 Leptosphaeria doliolum Ophiobolus herpotrichus 56 Pleospora betae 94 Cucurbitaria elongata 60 Kirschsteiniothelia elaterascus Herpotrichia juniperi 100 Mycosphaerella mycopappi 99 Anguillospora longissima 11891 63 Kirschsteiniothelia maritima 98 Massarina australiensis 84 Massarina bipolaris Paecilomyces variotii Monascus purpureus 100 Onygena equina Orbilia fimicola Arthrobotrys superba Arthrobotrys oligospora 100 Monacrosporium doedycoides Orbilia delicatula 99 Peniophora nuda Hydnum repandum Bulleromyces albus Saccharomyces cerevisiae

0.01 changes

Figure A32: Strict consensus tree obtained from parsimony analysis based on 18S rDNA sequence data (659 parsimony informative positions, 1896 steps) showing the phylogenetic placement of eleven species of aquatic hyphomycetes within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets.

195 Appendix

100 Hypomyces chrysospermus Hypocrea lutea Heliscus lugdunensis 245 77 Gibberella pulicaris 97 Nectria cinnabarina 98 Geosmithia putterillii Microascus cirrosus Halosarpheia retorquens Pseudallescheria boydii 100 Petriella setifera 100 Graphium penicillioides Sordaria firmicola Neurospora crassa 100 Chaetomium elatum Kionochaeta ivoriensis 99 Lemonniera terrestris 11486 Lemonniera aquatica 04480 Varicosporium elodeae 11783 93 Hymenoscyphus virgultorum Loramyces juncicola 95 Tricladium angulatum 10200 Tetracladium maxilliforme Tetracladium furcatum Tetracladium setigerum 100 Tetracladium apiense Tetracladium marchalianum 19399 99 Oidiodendron tenuissimum Myxotrichum deflexum Phyllactinia guttata 98 Blumeria graminis Phialophora sp. Elec-N-1 4.PN 99 Anguillospora crassa 05584 90 Anguillospora furtiva L16 Tricladium splendens 11989 Cyttaria darwinii 99 Leotia lubrica Alatospora acuminata 02383 100 Geomyces pannorum var. pannorum Pseudogymnoascus roseus 77 Bulgaria inquinans Spathularia flavida Cudonia confusa Hortaea werneckii 89 Aureobasidium pullulans Pleospora betae 100 Cucurbitaria elongata Leptosphaeria doliolum 4 Ophiobolus herpotrichus Herpotrichia juniperi 100 Mycosphaerella mycopappi 99 Massarina australiensis 90 Massarina bipolaris 98 Anguillospora longissima 11891 93 Kirschsteiniothelia maritima Paecilomyces variotii 100 Monascus purpureus 100 Onygena equina Orbilia fimicola Arthrobotrys superba Arthrobotrys oligospora 100 Monacrosporium doedycoides Orbilia delicatula 100 Hydnum repandum 99 Peniophora nuda Bulleromyces albus Saccharomyces cerevisiae 5

0.01 substitutions/site

Figure A33: Phylogenetic tree obtained from maximum likelihood analysis (Ln likelihood = - 14405.39641991), based on 18S rDNA sequence data. The tree shows the possible placement of eleven species of aquatic hyphomycetes within the Ascomycota. Names in red indicate DNA sequences newly determined in this study. Bootstrap values were derived from 1000 resampled data sets.

196 Appendix

Tetracladium marchalianum 26199 Tetracladium marchalianum 19399 Tetracladium marchalianum ELBE50 Tetracladium marchalianum 312 98 Tetracladium marchalianum 26299 Tetracladium marchalianum L27 67 Tetracladium marchalianum 26399 94 Tetracladium maxilliforme 83 Tetracladium furcatum 88 Tetracladium apiense axenic ectomycorrhizal isolate 80 Dactylaria dimorphospora Oidiodendron tenuissimum 100 Helotiales sp. ARON3063.S ectomycorrhizal isolate (Helotiales) ericoid mycorrhizal sp. Sd9 ericoid mycorrhizal sp. Sm5 Mycoarthris corallina Tricladium angulatum10200 Tricladium angulatum139 100 Tricladium angulatum14186 98 Tricladium angulatum 01380 Cistella grevillei 82 Alatospora acuminata 12186 96 Alatospora acuminata L8 97 Alatospora acuminata 37194 81 Alatospora acuminata 02383 Alatospora acuminata 13089 97 ericoid mycorrhizal sp. Sm5 98 Guignardia philoprina 71 Phoma-like coelomycete 1-L-3-5 Leotiales sp. Bjelland 61 Anguillospora crassa 15283 Anguillospora crassa 07082 99 Anguillospora crassa 05584 89 Anguillospora crassa 15583 98 Zalerion varium Tricladium splendens19087 Tricladium splendens16599 99 Tricladium splendens11989 76 Tricladium splendens12386 Hymenoscyphus fructigenus Hymenoscyphus monotropae 99 Hymenoscyphus sp. GU30 Hymenoscyphus ericae Hymenoscyphus ericae GU27 Mollisia minutella Blumeria graminis

Figure A34: Majority rule consensus tree (1178 steps) resulting from 1000 bootstrap replications of parsimony analysis of the ITS1, 5.8S, ITS2 sequence data set of aquatic hyphomycetes classified in the Leotiomycetes and their most similar sequences derived from BLAST searches. Names in red indicate DNA sequences newly determined in this study.

197 Appendix

SSU distance matrix

198 Herpot juni rho lg rhoorsoiopr ...... 049493899489888497526.5 5.2 7. 9.7 7.1 8.4 5.7 8.8 10.4 8.9 8.9 9.4 9.2 8.9 9.2 10.0 9.3 9.6 8.6 9.4 10.4 8.7 9.9 10.0 7.9 8.2 10.7 8.0 9.2 8.6 7.7 9.3 8.6 8.6 6.4 8.1 4. 8.4 6.1 9.0 8.6 5.8 5.0 9.0 7.8 5.5 6.5 6.6 7.4 5.7 4.6 6.3 8.3 5.6 6.0 5.2 8.1 6.7 6.0 5.1 6.2 7.9 6.7 6.1 5.0 5.7 8.0 5.5 5.6 6.3 5.4 5.6 8.2 6.6 6.7 5.6 5.3 6.0 9.8 5.3 6.2 7.1 5.8 6.0 7.3 5.5 6.4 5.7 5.4 7.0 6.3 6.6 6.0 6.8 6.7 7.2 5.3 6.5 6.0 8.8 5.6 7.4 6.7 6.7 6.5 5.6 7.2 5.7 5.7 6.8 5.2 5.8 6.0 7.5 6.1 5.6 5.9 6.8 4.2 7.3 7.0 5.3 7.3 6.0 4.6 7.7 4.6 7.1 Arthrobotrysoligospora 5.1 Monacro doed Orbiliafimicola 4.3 Arthrob oligo 4.8 Arthrobotryssuperba 5. 4.9 Orbilia firm Onygenaequina 6.1 4.9 Arthrob sup Paecilomycesvariotii 5.9 4.9 Onyge equi 5.8 5.0 Monascuspurpureus 5.2 5.9 Paecilo var 5.2 4.1 Mona purpur 9.1 Anguillosporalongissima11891 8.2 Mycos myco 7.8 Kirschsteiniothelia maritima 8.6 Angu longi Massarinaaustraliensis 9.2 Kirsch mariti 8.4 Mass austra 8.6 8.6 10.3 7.7 7.3 7.8 8.0 7.9 0.4 -0 2.6 4.0 3.2 3.1 3.7 3.2 3.5 3.2 4.1 3.5 3.7 3.6 Cudoniaconfusa Spath flav Cud confusa 1.3 2.6 2.6 -0 Anguillosporacrassa05584 Angu crassa ha lcNPilpoas.Ee-- .N192424172127233125262817353564655858628773737276726564773. 7.7 5. 6.4 4.3 6.5 2.8 5.8 7.2 7.5 6.8 7.6 6.7 6.0 7.2 6.4 4.6 7.3 7.1 8.8 7.3 7.7 7.9 8.7 7.3 7.6 6.2 7.4 8.3 5.8 7.0 8.9 5.8 8.7 8.7 6.5 6.1 8.6 6.4 5.9 8.8 3.5 5.8 9.8 3.5 6.6 7.3 1.7 6.3 7.2 5.9 2.8 3.3 7.2 4.6 2.6 3.3 7.6 5.9 2.5 2.0 7.8 5.1 3.1 2.6 4.7 4.8 5.2 2.3 2.2 3.3 4.7 6.0 2.7 2.0 3.7 3.5 4.6 11.9 2.1 2.8 12.2 2.1 5.0 5.0 12.1 1.7 1.8 11.7 2.1 4.3 2.0 11.8 2.2 2.4 4.4 3.7 12.4 1.9 13.7 2.2 2.4 4.3 3.8 5.0 10.9 1.3 1.9 10.8 3.4 3.5 5.1 10.5 2.3 11.7 3.5 4.8 11.5 2.3 3.3 0 9.2 0.3 3.1 Phialophorasp.Elec-N-14.PN 13.2 1.5 9.3 13.1 Angu furtiv 4.1 13.4 1.0 14.0 Lemonnieraterrestris11486 7.7 13.5 13.2 4.9 4.1 Phial Elec-N 13.9 5.9 12.4 9.0 Lemonnieraaquatica04480 12.7 4.9 12.5 3.9 13. 5.6 Lem terrestr 12.2 8.8 12.8 1.9 13.2 12.5 Tricladiumsplendens11989 12.8 4.4 13.9 12.7 8.7 Lem aquatic 4.1 11.1 12.6 5. 13.2 11.1 9.0 12.1 9.3 10.2 4.1 Hel lugdun 12.6 5.9 10.4 9.5 8.4 8.7 12.9 3.4 10.7 5.6 13.0 Tricladiumangulatum10200 9.3 Tri splen Tetracladiumsetigerum 7.9 10.2 9.3 10.2 10.4 10.1 5.8 10.2 Tetracladiummaxilliforme 8.7 Tri angulatum 10.3 9.0 9.5 10.4 10.2 8.9 8.8 Tetracladiumfurcatum -0 10.7 Tet setiger 9.4 10.4 10.8 9.0 10.0 8.2 3.4 12.0 9.8 10.1 Tet maxilli 9.0 10.4 8.4 10.2 9. 8.8 10.2 9.7 11.3 5.3 8.6 8 Tet furcat 10.4 8.9 8.1 12.1 8.1 9.8 4.2 7.9 Alatosporaacuminata 10.2 9.8 8.8 9.8 7.8 10.2 10.2 7.8 8.0 10.3 7.5 8.5 10.1 12.5 7.8 Varicosporiumelodeae 7.5 9.0 -0 9.4 Ala acumi 6.9 Saccharomycescerevisiae5 7.5 8.1 10.4 4.4 Bulleromycesalbus 10.2 4.4 Var elodeae 8.2 8.1 4.4 1.5 8.1 8.7 Sacch cere Peniophoranuda 9.4 5.0 8 6.1 9.7 -0 Bull albus 9.0 7.5 8.6 Hydnumrepandum 5.9 1.5 7.9 7.6 7.6 8.8 Penio nuda 4.8 Orbiliadelicatula 6.0 7.6 7.5 7.3 8.7 Hyd repand 5.7 5.9 7.4 5.8 7.2 7.3 5.1 5.7 7.6 Orbilia dent 7 6.0 8.6 7.2 5.2 6.0 7.8 3.6 7.2 6.7 6.3 5.7 5.3 7.2 5.9 4.3 7.9 5.8 6.0 -0 5.0 4.9 5.9 4.6 7.4 5.8 6.6 3.2 4.6 5.4 4.4 4.3 6.0 3.9 6.0 3.0 4.3 4.6 5.3 5.1 3.7 5.8 2.0 5.8 3.9 4.6 5.1 4.8 4.3 4.5 3.7 1.5 9.0 5.6 4.7 5.3 4.8 4.8 4.3 3.8 9.1 4.8 -0 7.8 5.6 5.2 4.7 4.3 7.6 4.2 4.0 7.8 5.4 5.0 4.0 4.7 8.6 4.0 4.2 6.3 5.2 3.7 5. 4.8 8.1 5.0 3.9 7.2 5.5 3.6 5.3 5.2 8.0 4.9 3.9 6.4 3.9 4.9 5.0 4.8 7.8 9.1 5.5 4.2 6.3 5.6 5.3 5.2 7.7 9.2 5.7 4.2 4. 7.0 2.0 5.3 3.5 6.9 7.8 7.4 7.3 5.2 Massarinabipolaris 8.8 6.2 5.1 1.2 4.6 7.8 7.4 5.1 9.5 7.9 8.5 8.6 6.1 4.8 4.9 Kirschsteiniotheliaelaterascus 7.3 6.9 -0 4.8 8.4 7.9 7.0 Mass bipola 7.0 6.9 2.9 5.3 7.0 6.7 2.0 5.4 8.0 7.9 Pleosporabetae 6.8 7.9 7.1 8.0 Kirsch elater 4 5.3 7.4 8.2 2.0 5.1 8.1 8.3 7.9 7.6 6.7 6.8 Cucurbitariaelongata 3.2 10.3 Pleosp betae 8.3 8.8 7.7 4.9 7.9 7.5 8.2 -0 7.7 6.6 6.3 Leptosphaeriadoliolum4 8.1 8.5 7.9 7.3 7.8 5.3 Curcub elong 7.6 7.6 7.7 7.8 6.7 6.9 6.9 Ophiobolusherpotrichus 9.6 8.0 7.2 5.6 3.6 8.1 7.5 7.9 7.0 Lepto doliolu 7.9 6.4 8.8 7.2 7.8 7.4 6.9 8.0 7.8 7.2 6.2 Kirschsteiniotheliaaethiops 6.9 7.4 7.0 Ophio herpo 8.9 Hortaeawerneckii 7.0 8.5 6.4 6.7 8.1 7.0 8.2 7.6 7.9 Aureobasidiumpullulans 8.5 3.9 6.7 Kirsch aethio 6.2 6.7 8.0 6.9 8.6 2.6 7.7 7.6 8.5 4.0 6.6 6.8 7.3 7.9 Horta wern 6.7 7.3 2.6 8.9 Kionochaetaivoriensis 7.6 8.5 2.4 6.8 6.0 8.1 7.5 7.1 Aureo pull 6.6 7.6 9.5 -0 7.0 5.9 7.6 -0 7.0 6.4 6.2 8.8 Kionoch ivo 9.0 2.4 7.1 6.7 7.6 2.3 6.5 Sordariafirmicola 6.2 6.6 8.8 1.5 3.5 7.3 6.3 Chaet elatum 2.3 7.4 6.2 6.7 Neurosporacrassa 6.3 8.9 1.4 3.7 8.8 6.4 3.7 Sord firmicol 5.7 5.9 6.7 6.4 9.1 2.4 Petriellasetifera 2.4 6.7 6.1 Graphiumpenicillioides 3.0 6.8 Neuro crass 3.1 6.8 2.0 3.7 6.4 5.8 3.4 6.7 3.1 6.9 Graph penici 2.2 6.4 2.9 3.6 6.4 1.4 3.1 1.9 6.7 2.8 Petri seti 2.9 Microascuscirrosus 2.3 3.2 1.3 6.8 3.5 Pseudall boy -0 0.4 1.5 2.3 3.6 3.8 2.5 Halosarpheiaretorquens Microas cirr 2.3 2.8 3.5 -0 2.8 2.8 2.3 Halos retorqu Nectriacinnabarina 2.1 2.8 -0 3.6 3.5 2.3 2.4 Geosm putte 0.4 2.5 Gibberellapulicaris 2.5 2.5 3.6 2.9 3.2 Nect cinna 2.5 2.4 1.9 2.4 3.3 Gibb pulica 3.2 1.7 2.5 2.7 Hypocrealutea 3.1 2.7 Hypomy chry 2.5 2.7 -0 2.5 2.9 1.9 Hypocr lutea 2.1 2.6 2.3 2.5 2.6 Bulgariainquinans 1.9 3.0 2.4 3.3 Leotialubrica -0 2.5 2.1 2.2 2.1 Bulg inquin 1.8 Geomycespannorumvar. -0 2.6 Leo lubrica Pseudogymnoascusroseus 1.9 Geom pann 2.1 Cyttariadarwinii Pseudo ros Oidiodendrontenuissimum 1.3 Cytt darwinii Oidio tenui Loramycesjuncicola Myxo deflex Hymenoscyphusvirgultorum Loram junc Blumeriagraminis Hym virgulto Blum gram Phylla gutt e pes ercaimaine464848534.4 5.3 4.8 4.8 4.6 3.3 4.1 4.3 4.0 Tetracladiumapiense Tetracladiummarchalianum Tet apiense Tet march Figure A35: DistancematrixSSUsequences eptihajnpr . . 5.0 5.2 5.1 Herpotrichia juniperi oarsoimdeyods525452595556525548485445535577807677771...... 6 4.7 9.3 7.9 8.6 8.8 9.2 8.7 8.6 8.6 10.0 7.7 7.7 7.6 8.0 7.7 5.5 5.3 4.5 5.4 4.8 4.8 5.5 5.2 5.6 7.6 5.5 5.6 5.9 5.4 5.2 4.2 5.4 5.2 5.2 4.6 4.6 5.2 4.2 Monacrosporium doedycoides 4.8 4.9 5.3 4.9 6. 5.1 6.0 4.9 6.0 5.9 4.2 9.0 Mycosphaerella mycopappi 8.1 7.8 8.5 9.1 8.6 8.7 8.8 10.3 7.7 7.2 7.7 8.0 7.7 -0 0.4 2.6 3.9 3.1 3.1 3.5 3.0 3.4 3.0 3.9 3.6 3.9 3.5 Spathularia flavida nulopr utv ...... 5. 5.7 4.9 3.5 8.2 7.2 7.1 7.9 8.2 7.7 7.8 7.4 9.5 6.8 6.3 6.1 7.0 6.8 4.0 4.0 2.4 3.3 2.9 2.7 3.5 2.5 2.9 2.2 1.3 3.0 3.0 0.6 6.0 5.5 Anguillospora furtiva Heliscus lugdunensis 8 8.7 7.6 6.6 6.0 4.0 4.6 5.4 5.3 5.4 5.0 4.8 4.0 5.3 4.0 5.1 4.2 4.9 6.2 5.2 6.7 2.8 3.2 5 6.8 2.1 7.8 7.6 5.5 6.8 2.0 6.5 8.0 5.3 6.1 -0 7.0 5.3 6.7 0.9 6.8 3.6 7.6 8.0 7.9 8.1 6.8 8.0 8.2 6.8 6.8 6.3 7.6 6.6 7.2 6.9 7.6 7.2 Chaetomium elatum 6.8 8.5 7.4 8.0 6.7 6.3 7.5 6.4 7.0 6.2 7.6 6.4 6.5 5.9 7.5 6.5 6.5 8.9 7.5 6.4 6.5 Pseudallescheria boydii 5.9 6.2 3.4 6.7 6.1 3.5 6.9 6.9 2.2 6.5 6.7 3.4 Geosmithia putterillii 3.9 2.7 3.7 2.5 2.8 2.8 3.8 1.3 Hypomyces chrysospermus 3.0 -0 2.9 2.5 3.3 1.9 2.4 2.5 2.6 2.6 3.3 2.4 2.1 1.8 -0 2.6 Myxotrichum deflexum Phyllactinia guttata ...... 04909289899383869939595. 5.9 3.9 9.9 8.6 8.3 9.3 8.9 8.9 9.2 9.0 10.4 7.8 7.6 7.5 7.9 8.0 6.0 5.8 4.0 5.1 3.9 3.8 4.4 3.9 4.4 4.9 4.5 3.9 4.5 4.2 ...... 00908889908477808947616.9 6.1 4.7 6.0 8.9 4.6 8.0 8.8 7.7 8.0 8.4 3. 7.6 9.0 3.6 8.4 8.9 5.1 8.9 8.8 1.6 8.9 9.0 6.8 8.7 10.0 5.6 8.9 7.4 10.0 5.0 7.3 7.4 5.9 7.3 7.2 5.6 7.8 7.3 5.4 8.0 7.8 5.4 4.9 7.9 5.9 4.8 4.8 6.8 3.6 4.8 4.4 5.1 3.6 4.8 4.5 5.0 4.2 4.5 4.4 4.2 4.4 4.5 4.1 3.4 4.4 2.7 3.6 3.5 3.0 3.3 3.5 0.8 3.1 3.3 3.0 4.2 3.1 1.1 4.2 4.1 1.3 4.0 4.2 2.1 3.9 1.1 1.3 2.0 1.1 1.3 1.4 0.9 4. 4.2 3.4 -0 7.5 6.9 6.9 7.6 7.6 6.9 7.0 7.3 8.5 6.4 6.3 6.0 6.6 6.6 4.2 4.1 2.4 3.4 3.2 2.9 3.5 3.2 3.2 2.9 2.1 3.6 3.6 3.2

Angu crassa ...... 4.2 8.5 7.8 7.7 8.3 8.0 7.6 7.9 8.1 9.9 7.7 7.2 7.3 7.2 7.3 5.8 5.7 4.4 5.3 4.7 4.6 5.2 4.3 4.9 4.9 5.1 5.0 5.2 ...... 03888686848679819 8.1 5.5 7.9 5.6 8.6 4.3 8.4 9.0 8.6 8.5 8.6 7.9 8.8 10.3 8.9 7.7 8.6 7.4 8.1 7.3 8.3 7.9 8.6 7.7 9.9 6.4 7.9 6.2 7.5 5.0 7.2 5.9 8.4 5.2 8.2 5.2 6.0 5.7 5.9 5.0 4.7 5.4 5.4 5.1 5.0 5.1 4.8 5.5 5.3 5.8 4.6 5.1 4.6 4.7 5.1 5.5

Phylla gutt ...... 7 7.2 7.6 7.2 6.0 5.6 3.9 5.1 4.0 9.9 4.9 8.6 3.9 8.4 3.4 8.9 3.4 9.5 3.9 9.0 5.5 9.2 2.2 9.2 1.3 10.0 0.7 7.7 2.0 7.8 2.0 7.9 7.5 5.0 8.6 7.5 5.9 8.5 5.3 5.9 5.3 6.5 4.5 5.3 6.0 9.4 4.7 5.8 8.4 6.1 6.3 8.7 5.4 5.9 9.0 5.3 5.9 8.9 5.1 6.2 8.7 4.4 5.7 9.0 4.6 6.1 8.8 3.4 10.2 2.9 7.9 5.0 7.9 7.5 8.4 8.0 5.2 5.2 3.8 4.8 3.9 3.9 5. 4.7 4.5 4.4 8.9 5.0 8.3 5.5 8.1 6.1 8.9 5.0 8.3 7.9 8.1 8.6 10.3 8.1 7.4 7.2 8.2 8.0 7. 7.4 6.2 8.1 7.5 6.1 7.9 7.8 4.6 6.4 7.6 5.7 6.3 6.3 5.2 4.8 5.8 4.8 4.9 4.3 5.5 5.7 4.6 4.8 4.8 5.4 5.3 4.2 4.2 5.0 4.2 3.8 5.1 4.2 3.6 5.4 6.0 3.9 5.6 -0 1.9 1.9 2.0 -0 5.0 2.8 1.2 5.0 3.0 2.1 3.2 7.7 2.3 7.7 7.7 7.2 6.5 6.1 7.3 6.2 7.1 5.7 6.9 6.6 6.9 7.5 6.6 6.3 7.0 6.7 6.2 7.0 6.1 6.6 7.0 6.3 6.1 8.5 6.8 6.2 6.1 6.1 6.9 6.1 6.7 6.7 6.0 6.4 6.5 6.4 3.0 3.2 2.0 3.0 2.4 2.3 2.5 -0 1.3 1.9 1.3 2.7

Blum gram ...... 0587858280887880894456552 5.5 5.6 4.4 8.9 8.0 7.8 8.8 8.0 8.2 8.5 8.7 10.5 7.8 7.4 7.4 8.0 7.8 5.7 5.6 4.5 5.2 4.8 4.6 5.3 4.2 4.8 5.0 5.3 ...... 0488909294877883814 8.3 8.1 8.5 8.3 8.3 7.8 8.1 8.7 6.9 9.4 4.4 9.2 -0 9.0 1.5 8.8 10.4 2.5 8.1 5.8 7.8 5.7 7.9 5.5 8.7 5.7 8.4 7.5 6.0 4.8 5.8 4.3 4.6 3.7 5.8 4.0 5.1 4.2 5.1 8.1 5.0 8.2 5.0 6.4 4.9 7.5 5.2 6.9 4.5 6.8 7.3 6.5 6.5 7.3 7.6

Hym virgulto ...... 14959695981. . . 006173767. 7.6 7.3 6.1 10.0 9.1 9.6 10.0 9.8 9.5 9.6 9.5 11.4 9.7 9.0 8.6 8.9 9.0 7.8 7.7 6.6 6.8 6.5 6.4 7.6 7.4 7.4 7.2 5 4.6 3.2 7.9 6.8 6.7 7.4 7.9 7.2 7.4 7.0 9.1 6.4 5.9 5.8 6.5 6.4 3.5 3.6 2.1 2.8 2.5 2.3 3.1 2.1 2.4 2.2 ...... 028383838487808088436062595 5.9 6.2 6.0 4.3 8.8 8.0 8.0 8.7 8.4 8.3 8.3 8.3 10.2 7.7 7.3 7.1 7.8 7.7 4.3 4.5 2.7 3.0 3.5 3.5 3.9 3.4 3.8 3.8 ...... 02898788908476808846616. 6.1 4.6 8.8 8.0 7.6 8.4 9.0 8.8 8.7 8.9 10.2 7.3 7.2 7.3 7.7 7.9 4.8 4.7 3.6 5.0 4.4 4.4 4.4 3.3 3.5 3.3

Loram junc ...... 4 4.9 2.1 8.4 7.6 7.1 8.8 7.8 8.2 8.2 8.6 9.0 6.1 6.7 6.3 5.9 5.7 3.9 4.1 1.3 2.9 1.9 1.7 2.5 1.3 1.9 ...... 0796949596908286955367756. 7.5 6.7 5.3 9.5 8.6 8.2 9.0 9.6 9.5 9.4 9.6 10.7 8.0 7.8 7.9 8.4 8.5 5.5 5.4 4.2 5.7 5.1 5.1 5.0 4.0 4.2

Myxo deflex

Oidio tenui ...... 0688908787938184943657502.3 5.0 5.7 3.6 9.4 8.4 8.1 9.3 8.7 8.7 9.0 8.8 10.6 7.6 7.5 7.3 7.5 7.5 5.6 5.4 3.7 4.8 3.9 3.7 4.3

Cytt darwinii ...... 5 4.3 8.7 7.7 7.4 7.9 8.4 8.4 8.2 8.1 9.6 7.2 7.1 7.0 7.7 7.8 4.6 4.6 3.4 4.6 3.9 3.9 5.3 5.0 3.9 8.8 7.8 7.6 8.4 8.2 7.5 7.8 8.3 10.0 7.6 7.3 7.1 7.9 7.9 5.5 5.3 4.3 5.2 4.5 4.3

Pseudo ros ...... 06869089949387859558696568706.4 7.0 6.8 6.5 6.9 5.8 9.5 8.5 8.7 9.3 9.4 8.9 9.0 8.6 10.6 8.7 8.0 8.2 8.7 8.8 6.5 6.5 5.4 6.4 5.5 ...... 015255076858789898 8.9 8.9 7 8.7 7.9 8.5 8.1 7.6 9.2 5.0 8.1 9.0 2.5 6.9 8.1 1.5 7.6 7.0 -0 5.7 7.3 6.7 5.9 5.5 6.5 6.5 5.7 6.4 3.2 6.4 6.6 3.0 -0 7.7 0.7 0.7 5.7 2.0 -0 5.4 4.2 1.5 4.7 4.2 3.9 5.3 3.8 4.2 5.4 3.7 3.6 8.5 4.0 3.6 8.6 4.1 4.0 7.0 8.6 3.9 8.0 8.4 8.7 7.0 6.8 8.6 8.2 7.1 5.0 7.6 8.1 4.6 7.6 5.2 5.5 2.9 9.5 7.3 6.9 8.2 8.1 7.9 7.9 8.2 9.5 6.8 6.9 6.7 7.5 7.4 3.0 3.2 1.9 3.6 2.7

Geom pann

Leo lubrica

Bulg inquin

Cud confusa ...... 089596979792879199557.1 5.5 9.9 9.1 8.7 9.2 9.7 9.7 9.6 9.5 10.8 8.4 8.4 8.5 9.1 9.0 5.9 9.7 8.5 8.6 7.5 7.2 7.7 5.6 4.2 3.9 4.0 -0 6.0 5.6 5.6 6.2 6.3 10.3

Spath flav . . . . . 0792929293958990975565666 6.6 6.5 5.5 9.7 9.0 8.9 9.5 9.3 9.2 9.2 9.2 10.7 9.0 8.3 8.1 8.7 9.0 ...... 4.6 4.7 4.6 4.4 2.4 7.6 6.4 6.3 7.0 7.3 6.8 7.1 7.0 8.5 6.1 5.7 5.6 6.3 6.2

Hypocr lutea . . . . 01858581788677798742535. 5.3 4.2 8.7 7.9 7.7 8.6 7.8 8.1 8.5 8.5 10.1 7.4 7.3 7.3 7.9 ...... 7.9 8.0 8.2 8.4 7.8 6.6 5.8 4.2 4.7 5.4 4.9 4.1 3.9 4.2 6.3 3.0 2.3 2.0 0.9

Hypomy chry ...... 5 5.2 5.7 5.4 5.8 5.3 3.4 8.6 7.5 7.2 8.0 8.5 8.2 8.1 7.9 9.6 7.1 6.9 6.9

Gibb pulica

Nect cinna

Geosm putte 061...... 05577273626461656753586567606386767508- . . . . 061...... 6.4 6.4 7.2 6.8 6.5 5.4 8.4 10.7 10.6 9.8 2.7 2.2 1.1 -0 0.8 7.5 7.6 8.6 6.3 6.0 6.7 6.5 5.8 5.3 6.7 6.5 6.1 6.4 6.2 7.3 7.2 5.7 10.5 9.0 9.4 9.6 9.9 9.5 9.9 10.0 10.6

Halos retorqu

Microas cirr

351. 301. 251. . 111. 051. 041. 081. 021. 111. 071. 151. 04981. 0498- . . 229610 9.6 12.2 7.0 5.6 -0 9.8 10.4 10.6 9.8 10.4 11.2 11.5 12.4 10.7 10.5 11.1 10.8 10.2 10.1 10.8 10.9 10.4 10.8 10.5 11.0 11.1 9.8 13.5 12.5 12.7 13.0 13.8 13.5 6 Pseudall boy 311. 321. 271. 001. 141. 031. 121. 041. 041. 071. 241. 191. 071. 07977072- 18911 10. 9.1 9.6 11.8 12.1 7.2 -0 7.2 -0 7.0 5.6 10.2 9.7 10.8 10.7 11.0 11.0 10.6 10.7 11.4 11.5 11.5 11.9 11.4 11.8 12.2 12.4 10.2 10.8 10.2 10.7 10.5 11.0 10.8 10.4 10.6 10.2 10.8 10.4 10.7 9.9 11.2 10.4 10.8 10.3 9.8 10.7 10.3 11.4 10.0 11.3 11.2 10.0 10.8 13.8 12.7 9.8 12.9 13.7 13.2 12.2 13.1 12.6 13.1 12.8 13.4 13.0

Petri seti

Graph penici

Neuro crass

Sord firmicol 131. . 011...... 091...... 221. 18- . . 031. . 001. 118410. 8.4 11.1 10.1 10.0 9.9 10.5 10.3 8.8 8.7 -0 11.8 12.1 12.2 8.0 8.3 8.7 8.4 8.9 9.4 10.3 10.9 9.6 9.4 9.7 9.7 9.7 9.7 9.5 9.8 9.4 9.1 9.6 10.4 10.1 9.0 13.2 11.3

Chaet elatum 045565921241922- ...... 08991...... 5.8 5.4 6.6 7.6 7.3 5.1 7.2 6.2 6.2 6.1 6.8 6.3 6.5 4.2 9.5 10.7 9.9 10.8 5.4 6.1 6.3 6.7 7.2 7.5 6.9 7.7 2.5 2.3 3.0 2.7 2.7 2.4 -0 2.2 1.9 2.4 2.1 5.9 5.6 4.5 .0 Kionoch ivo 258- ...... 101. 141...... 5.7 5.3 6.7 7.9 7.6 5.0 7.3 6.9 6.9 6.8 7.5 6.9 6.2 3.6 10.4 11.4 11.2 11.0 5.9 6.7 6.9 7.3 7.7 6.5 6.6 7.6 5.5 5.3 5.5 5.2 5.2 5.0 5.9 5.9 5.3 5.8 5.5 -0 5.8 .2 ...... 013202170636669656259531. 081...... 5.5 4.9 6.2 7.2 6.8 4.8 7.1 6.0 5.9 5.8 6.6 6.0 5.7 3.9 9.7 10.4 10.8 10.8 5.3 5.9 6.2 6.5 6.9 6.6 6.3 7.0 2.1 2.0 1.3 -0 2.2 2.2 2.7 2.9 2.5 2.6 2.6 5.2 5.2 1 ...... 02.2 -0 3.2 4.3 5.7 1.8 4.7 4.0 3.9 3.9 4.6 4.0 3.3 1.1 7.9 9.9 9.5 9.8 4.3 4.8 5.1 5.9 5.7 6.3 6.7 7.5 4.9 4.7 5.0 4.9 4.2 4.0 5.4 5.2 4.8 5.1 5.2 5.3 4.4 0 Aureo pull ...... 0202169636570676259551. 051...... 5.5 5.0 6.0 7.1 6.9 4.8 7.1 6.0 6.0 5.8 6.6 5.9 5.8 4.1 9.7 11.0 10.5 11.1 5.5 5.9 6.2 6.7 7.0 6.5 6.3 6.9 2.1 2.0 -0 1.3 2.1 1.9 3.0 2.9 2.5 3.0 2.8 5.5 5.1 ...... 161. 231...... 045775745475.1 4.7 4.5 5.7 7.7 4.5 -0 5.5 5.4 5.4 6.0 5.5 6.1 1.7 11.1 12.3 11.4 11.6 6.6 6.9 6.6 7.2 7.3 8.3 8.6 9.1 6.8 6.7 7.1 7.1 5.5 5.2 7.2 7.2 6.6 7.2 6.9 7.3 6.6 867646659646644466260596182757971716560601. 101...... 0323.8 3.2 -0 1.5 7.0 3.1 4.5 4.6 4.5 4.5 5.2 4.4 5.0 0.9 9.6 11.5 11.0 10.7 6.0 6.0 6.5 7.1 7.1 5.5 7.9 4.8 7.5 6.0 8.2 7.3 6.1 7.4 5.9 4.8 6.0 6.9 6.2 5.9 4.6 5.9 4.4 5.8 6.6 6.7 6.4 6.0 5.9 5.8 6.6 5.0 6.4 8.3 10.7 6.7 10.8 .8 10.4 2.2 -0 1.3 2.2 2.6 6.8 7.0 7.9 5.6 5.4 5.9 5.9 6.0 5.6 6.1 5.7 5.5 5.9 5.6 6.7 .0 8515052455152393946474648655355575751483998951...... 2.9 2.6 3.9 5.1 6.0 2.2 5.4 4.5 4.4 4.3 5.1 4.4 3.5 1.1 8.8 10.2 9.5 9.8 3.9 4.8 5.1 5.7 5.7 5.5 5.3 6.5 4.8 4.6 4.7 4.6 3.9 3.9 5.2 5.1 4.5 5.2 5.0 5.1 .8 . . 007152427282630222076707069646059541. 031...... 5.5 5.1 6.6 7.6 7.1 5.1 7.2 6.2 6.2 6.1 6.8 6.3 5.9 4.1 9.1 10.3 10.3 10.8 5.4 5.9 6.0 6.4 6.9 7.0 7.0 7.6 2.0 2.2 3.0 2.6 2.8 2.7 2.4 1.5 0.7 -0 0.9 5.8 9 Horta wern ...... 013221. 101...... 5.5 5.1 6.5 7.6 7.7 5.0 6.6 6.1 6.0 5.9 6.8 6.2 6.0 5.3 8.7 11.0 11.0 10.6 2.2 1.3 -0 1.1 1.6 7.4 7.3 8.4 5.8 5.6 6.2 6.2 5.8 5.4 6.3 6.0 5.7 6.0 5.7 6.9 ...... 181. 251...... 015434.9 4.3 1.5 -0 8.2 4.4 5.7 5.7 5.7 5.6 6.3 5.7 6.2 2.4 10.9 12.5 12.1 11.8 7.2 7.3 7.6 8.0 8.2 9.2 8.8 9.5 7.2 6.9 7.1 7.2 4.7 4.5 7.6 7.5 7.0 7.6 7.4 7.9 5. 8.7 10.0 10.0 10.0 9.8 10.7 10.2 10.2 6.8 13.7 14.0 13.9 13.9 10.2 10.0 10.4 10.6 10.7 10.6 10.7 11.4 10.5 10.1 9.9 9.8 10.4 10.6 10.3 9.8 10.0 10.3 9.9 10.4 ...... 121. 191...... 0025435715745394.1 3.9 4.5 5.7 7.1 3.5 5.4 0.2 -0 0.1 0.8 0.4 5.1 2.3 10.0 11.9 11.2 11.2 5.7 5.9 6.0 6.4 6.6 7.8 7.2 7.9 5.9 5.5 6.0 5.9 4.4 4.2 6.2 6.0 5.7 6.2 5.9 6.9 5475147515143454951535373758867716759611. 211. 02111933533131312913571921171.3 1.7 2.1 1.9 0.6 5.7 1.9 1.3 3.5 2.9 4.8 3.1 5.5 3.1 0.3 3.1 4.9 5.3 4.0 3.3 3.9 1.9 3.9 1.1 4.6 10.2 12.0 4.0 12.1 12.5 3.4 6.1 0.9 5.9 8.6 10.4 6.7 10.1 10.2 7.1 4.2 6.7 5.2 8.8 5.2 7.5 6.0 7.3 5.9 5.3 6.1 5.3 6.0 5.1 7.1 4.9 5.1 4.5 4.9 4.3 5.2 5.1 5.2 5.1 4.2 4.7 3.8 5.1 5.5 4.7 5.4 .5 4.8 5.2 5.1 .3 ...... 00269626262605654511. 021...... 5.2 4.7 5.9 6.9 7.1 4.5 6.7 5.6 5.5 5.5 6.2 5.6 5.7 4.1 9.4 10.7 10.2 10.5 5.1 5.4 5.6 6.0 6.2 6.2 6.2 6.9 0.2 -0 2.0 2.0 2.2 2.1 2.3 2.2 1.7 2.2 2.0 3 ...... 0081626271. 141...... 6.2 5.7 7.1 8.2 8.1 5.6 7.3 6.6 6.6 6.5 7.3 4.1 6.8 4.2 6.6 4.6 5.0 4.7 8.9 7.2 11.5 4.2 11.4 10.4 5.5 2.7 4.4 2.6 4.4 1.6 4.3 0.8 5.6 -0 4.4 7.9 5.9 7.8 4.1 8.8 9.7 10.2 6.4 10.6 10.2 6.2 5.0 7.0 6.0 6.9 5.8 5.6 5.8 4.9 5.6 7.2 7.5 6.7 5.7 6.3 6.1 6.9 2.3 6.4 2.2 7 2.1 2.2 -0 0.9 2.7 2.6 2.8 2.8 2.6 2 ...... 02840834364235353645- . . . . 0.7 1.8 3.1 4.4 5.4 -0 4.5 3.6 3.5 3.5 4.2 3.6 3.4 0.8 8.4 10.2 9.7 9.7 4.1 4.8 5.0 5.6 5.6 5.8 5.8 6.7 4.8 4.5 4.8 4.8 4.2 3.9 5.1 5.1 4.5 5.1 4.9 0 ...... 151. 231. . . 0090404045536705744404.3 4.0 4.4 5.7 7.0 3.6 5.5 0.4 0.4 0.4 0.9 -0 5.1 2.3 10.3 12.3 11.5 11.5 5.9 6.0 6.2 6.8 6.8 7.9 7.2 7.9 5.9 5.6 5.9 6.0 4.4 4.2 6.3 6.2 5.7 6.3 6.1 9 Kirsch aethio . . 0091925282525171869636463615755521. . 0894385857635657576645697059485.1 4.8 5.9 7.0 6.9 4.5 6.6 5.7 5.7 5.6 6.3 5.7 5.8 3.8 9.4 10.8 9.8 10.4 7.8 5.2 7.3 5.5 8.2 5.7 9.6 6.1 3.4 6.3 7.4 6.4 9.2 6.3 8.8 6.9 8.7 1.8 8.6 1.7 9.4 2.5 8.7 2.5 8.3 2.8 5.4 11.8 2.5 13.2 13.4 1.9 13.6 0.9 9.0 -0 8.6 0.7 9.3 0.5 9.9 9.9 9.0 9.2 9.6 8.5 8.5 7.9 8.0 9.2 9.0 8.6 8.4 7.8 8.1 8.3 ...... 131. 201...... 05536715746404.1 4.0 4.6 5.7 7.1 3.6 5.5 -0 0.2 0.1 0.8 0.4 5.1 2.3 10.1 12.0 11.3 11.3 5.7 5.9 6.1 6.5 6.6 7.9 7.3 8.0 5.9 5.6 6.0 6.0 4.4 4.2 6.2 6.1 5.7 6.2 6.0 5.5 5.2 6.4 7.4 7.2 4.9 6.9 6.0 5.9 5.8 6.6 6.1 8.2 5.9 7.6 3.9 8.4 9.6 9.7 10.7 10.0 3.9 10.5 7.7 5.0 9.5 5.6 9.0 5.7 8.9 6.2 8.9 6.4 9.6 6.8 9.0 6.8 8.4 7.5 5.6 2.0 12.1 13.1 2.0 13.4 13.8 2.8 8.9 2.6 9.2 2.6 9.4 2.3 9.9 2.1 10.0 1.0 9.4 0.5 9.3 0.9 9.8 -0 8.0 7.8 8.0 8.1 8.9 8.7 8.4 8.8 8.2 8.3 8.6 ...... 021. 0487113433403435344418594633232.5 2.3 3.3 4.6 5.9 1.8 4.4 3.4 3.5 3.4 4.0 3.3 3.4 1.1 8.7 10.4 10.1 10.2 4.4 5.2 5.3 6.1 6.0 6.2 6.5 7.4 4.2 4.2 4.3 4.3 3.9 3.6 5.0 5.0 4.3 4.8 4.6 0201824252223212102- ...... 071. 0896425659655859596848727261495.4 4.9 6.1 7.2 7.2 4.8 6.8 5.9 5.9 5.8 6.5 5.9 5.6 4.2 9.6 10.8 10.2 10.7 5.2 5.6 5.8 6.3 6.4 6.4 6.4 7.0 -0 0.2 2.1 2.1 2.3 2.2 2.5 2.4 1.8 2.0 .0 87063696958576363626421- ...... 151. 181...... 6.4 6.7 7.5 8.8 8.3 5.8 8.6 7.3 7.2 7.1 7.9 7.2 7.6 5.4 10.3 11.8 11.4 11.5 6.5 7.0 7.3 7.6 7.8 3.1 -0 2.1 6.4 6.2 6.3 6.3 5.7 5.8 6.9 6.9 6.3 7.0 .8 48279827975797472798089878790928680841. 271. 17427877847678788666209177657.0 6.5 7.7 9.1 2.0 6.6 8.6 7.8 7.8 7.6 8.4 7.7 7.8 4.2 11.7 12.8 12.7 12.9 8.4 8.0 8.6 9.2 9.0 8.7 8.7 8.9 8.0 7.9 7.2 7.4 7.9 7.5 7.9 8.2 7.9 8.2 .4 ...... 0214788868479741. 221. 09688679867879809167889582757.4 7.5 8.2 9.5 8.8 6.7 9.1 8.0 7.9 7.8 8.6 7.9 8.6 6.8 10.9 12.4 12.2 12.4 7.4 7.9 8.4 8.6 8.8 4.7 2.1 -0 7.0 6.9 6.9 7.0 6.1 6.2 7.7 7.7 6.9 7.6 5 ...... 031. 058814354347404141510759493822-0 2.2 3.8 4.9 5.9 0.7 5.1 4.1 4.1 4.0 4.7 4.3 3.5 1.4 8.8 10.5 1.4 10.2 1.1 10.3 0.9 4.6 2.4 5.5 3.8 5.5 0.8 6.2 1.7 6.2 2.3 6.4 2.3 6.4 2.3 7.4 3.8 5.4 2.3 5.2 2.6 5.5 5.5 -0 4.1 8.7 3.8 9.1 5.8 9.6 5.8 9.6 5.1 4.5 5.5 5.0 5 5.3 5.4 5.0 6.6 5.4 6.8 4.2 4.1 4.1 3.9 4.1 3.9 4.2 4.2 3.5 3.8 3.0 4.1 4.3 9 5.5 8.2 6.0 7.7 2.8 8.7 5.1 9.9 4.7 5.6 4.6 7.5 4.6 9.9 5.3 8.9 4.6 8.8 4.3 8.8 1.6 9.5 9.0 10.0 8.8 9.8 8.8 9.8 6.8 13.2 4.5 13.8 4.7 13.7 13.5 5.2 9.4 5.7 9.3 5.7 9.7 5.8 10.5 10.4 5.5 9.5 6.1 2.7 9.7 4.4 10.0 2.5 4.2 8.9 3.9 4.2 8.7 5.2 4.1 8.5 5.8 3.9 8.6 2.0 3.6 9.9 5.3 4.5 9.4 4.5 4.4 9.0 4.5 3.9 9.0 4.4 4.5 8.8 5.1 3 8.9 4.4 0 3.5 1.3 8.7 10.1 9.4 9.7 3.9 4.8 5.0 5.6 5.6 5.6 5.5 6.4 4.6 4.6 4.6 4.6 3.8 3.7 5.2 5.2 4.3 4.8 8 ...... 201. 251. . . . 00808086042776352464.7 4.6 5.2 6.3 7.7 4.2 6.0 0.8 0.8 0.8 -0 0.9 5.7 3.8 10.5 12.5 12.0 12.0 6.6 6.7 6.8 7.2 7.3 8.5 7.9 8.6 6.5 6.2 6.6 6.6 5.6 5.4 6.8 6.8 6.3 6.8 6 6.8 6.2 7.2 8.4 2.2 6.1 7.7 7.4 7.4 7.3 8.0 7.3 7.7 4.4 10.9 13.2 12.7 12.6 7.9 7.7 7.9 8.6 8.6 8.7 9.0 9.7 7.8 7.4 7.7 7.3 7.8 7.6 7.7 8.0 7.6 8.1 9 Ophio herpo . . . . 0092219212262587449535456461. 081...... 3.8 4.0 4.4 4.5 6.9 3.9 5.2 4.2 4.2 4.1 5.4 4.2 5.6 3.9 9.7 10.4 10.8 10.1 4.6 5.6 5.4 5.3 4.9 7.4 5.8 6.2 2.2 2.1 1.9 2.2 0.9 -0 2.4 2.5 2.5 2.7 ...... 111. 189923500408- ...... 4.0 3.9 4.5 5.6 7.0 3.5 5.4 0.1 0.1 -0 0.8 0.4 5.0 2.3 9.9 11.8 11.1 11.1 5.7 5.8 5.9 6.4 6.5 7.7 7.1 7.8 5.8 5.5 5.8 5.8 4.3 4.1 6.1 6.0 5.6 6.1 ...... 0981...... 4.6 4.3 6.0 7.2 7.4 4.1 6.6 5.7 5.7 5.7 6.6 5.9 5.0 4.5 8.0 9.7 10.2 9.8 -0 2.2 2.2 2.7 2.7 6.8 6.5 7.4 5.2 5.1 5.5 5.3 5.0 4.6 5.4 5.4 5.2 5.4 176797981837477777896898792948886871. 261. 19508076827676778464408774657.1 6.5 7.4 8.7 4.0 6.4 8.4 7.7 7.4 7.6 7.3 7.6 8.1 8.2 9.5 7.6 3.9 6.1 8.0 7.0 5.8 5.0 9.2 7.0 11.9 12.9 9.0 8.5 12.6 12.7 8.9 0.7 8.7 8.8 5.8 8.6 9.6 7.9 8.8 8.9 7.3 9.4 8.3 7.3 9.2 5.9 7.2 12.4 8.7 13.1 7.9 13.5 8.9 7.3 13.2 9.6 7.1 8.8 7.8 4.2 8.6 10.5 7.7 9.4 12.5 10.0 12.5 7.7 10.0 12.6 7.4 8.6 7.5 8.3 9.2 7.6 8.1 9.5 7.7 3.0 7.9 8.7 8.2 2.4 7.9 8.5 8.1 3.8 7.6 8.1 8.2 5.1 .1 8.2 8.1 6.0 9.0 8.6 2.3 8.8 7.4 4.9 8.8 7.3 4.2 8.9 7.3 4.2 8.3 7.2 4.1 .6 7.5 4.8 7.3 4.3 7.3 4.1 7.5 1.4 7.1 9.0 10.2 .2 10.2 10.4 5.3 5.4 5.6 6.0 6.0 6.8 7.0 7.7 5.2 5.1 5.2 5.0 4.5 4.5 5.8 5.6 5.2 .7 169737369726869717288838281847774741. 251. 06386870777071717754- . . . 5.9 5.7 7.0 8.2 -0 5.4 7.7 7.1 7.1 7.0 7.7 7.0 6.8 3.8 10.6 12.3 12.5 12.4 7.4 7.4 7.7 8.4 8.1 8.2 8.3 8.8 7.2 7.1 6.9 6.8 7.2 6.9 7.3 7.3 6.9 .1 958636556595758575686767466656058501. 061. . . 051575051516134686250333.5 3.3 5.0 6.2 6.8 3.4 6.1 5.1 5.1 5.0 5.7 5.1 -0 2.6 8.8 10.2 10.6 10.2 5.0 5.8 6.0 6.5 6.6 7.4 7.6 8.6 5.6 5.7 5.8 5.7 5.9 5.6 6.5 6.3 5.8 .9 ...... 021. 1192274348554848495333755946354.0 3.5 4.6 5.9 7.5 3.3 5.3 4.9 3.5 4.8 3.1 4.8 4.7 5.5 6.0 4.8 6.3 4.3 2.8 2.7 5.1 9.2 4.4 11.1 10.2 4.4 10.2 4.3 5.2 5.0 5.5 4.4 5.8 3.9 6.4 2.1 6.6 9.3 6.5 10.3 10.0 6.7 9.8 7.8 4.7 5.7 5.5 5.6 5.4 5.8 6.0 5.8 6.0 6.0 6.7 5.6 6.6 6.4 7.6 6.1 5.3 5.5 5.2 2 5.2 5.1 4.4 4.3 5.7 5.3 5.1 5 Lepto doliolu ...... 2.4 1.7 3.4 4.6 5.3 2.0 4.7 3.6 3.6 3.5 4.2 3.6 2.7 0.8 7.7 9.5 9.3 8.9 3.8 4.5 4.6 5.0 5.2 5.4 5.5 6.6 4.5 4.2 4.4 4.2 4.0 3.7 5.0 4.8 4.3 ...... 251. 271...... 7.2 6.4 7.7 9.1 3.9 6.7 8.6 8.0 8.0 7.9 8.6 8.0 8.0 5.6 11.3 12.7 12.2 12.5 8.4 7.9 8.4 9.0 8.9 8.5 9.0 9.1 8.0 7.9 7.8 7.8 8.6 8.4 8.1 8.4 7.8 ...... 031. 0890203833443333333422622021212.2 2.1 2.1 2.0 6.2 2.2 3.4 3.3 3.3 3.3 4.4 3.3 3.8 2.0 9.0 10.8 11.3 10.3 5.5 5.5 5.4 5.6 5.3 6.8 5.7 7.2 5.0 4.9 4.9 4.9 4.9 4.6 5.1 4.6 4.3 ...... 211. 241...... 6.3 5.8 7.1 8.4 1.3 5.9 7.8 7.3 7.2 7.2 7.8 7.2 7.3 4.8 10.8 12.4 12.2 12.1 7.9 7.7 8.0 8.7 8.6 8.0 8.3 9.0 7.4 7.3 7.2 7.0 7.6 7.5 7.4 7.5 7.3 487869393838386881...... 301. 321...... 7.9 7.2 7.9 7.7 9.2 7.2 4.9 8.4 7.1 9.7 8.9 3.4 8.4 7.3 8.4 9.0 8.3 8.9 9.0 8.9 8.4 8.7 8.7 9.5 5.9 8.8 12.2 8.3 13.2 12.8 5.4 13.0 11.7 13.1 9.0 13.0 8.8 13.5 8.9 8.7 9.6 8.7 9.2 8.9 9.3 9.5 9.5 9.6 10.0 8.9 8.8 9.2 8.6 9.5 8.3 8.2 8.3 8.1 9.3 7.6 9.3 7.9 8.6 8.9 8.7 8.7 .4 8.6 8.2 .5 9- ...... 091. 1298426362686060617251737564525.8 5.2 6.4 7.5 7.3 5.1 7.2 6.1 6.0 6.0 6.8 6.2 6.3 4.2 9.8 11.2 10.4 10.9 5.4 5.7 6.0 6.5 6.7 7.1 6.9 7.7 2.4 2.2 2.9 2.9 2.6 2.5 2.2 -0 .9 ...... 021. 1193304547544748485333755946354.0 3.5 4.6 5.9 7.5 3.3 5.3 4.8 4.8 4.7 5.4 4.7 4.5 3.0 9.3 11.1 10.4 10.2 5.2 5.3 5.5 6.1 6.3 6.5 6.7 7.7 5.6 5.4 5.7 5.6 5.8 5.4 6.2 5.9 3 ...... 111. 131...... 4.9 4.4 5.8 7.1 7.2 4.3 6.6 6.1 6.0 6.0 6.7 6.1 6.0 5.1 10.1 11.3 10.8 11.1 5.9 6.0 6.5 7.2 7.1 6.9 6.5 7.3 5.6 5.3 5.1 5.2 5.9 5.7 5.6 5.6 0 Curcub elong ...... 079757468681. 151...... 6.4 6.3 7.9 9.2 8.2 5.8 8.3 7.9 7.8 7.7 8.5 7.9 7.4 6.6 9.4 11.9 11.5 11.2 6.8 6.8 7.4 7.5 7.9 -0 3.1 4.7 6.4 6.2 6.5 6.6 7.5 7.4 7.5 7.1 ...... 301. 281...... 6.8 6.4 7.8 9.0 2.0 6.3 8.5 8.0 7.9 7.8 8.5 7.9 7.7 4.1 11.5 3.0 12.8 13.2 2.4 13.0 3.7 8.0 5.0 7.7 6.1 8.4 2.3 9.3 5.0 9.0 4.2 8.8 4.1 9.0 4.1 9.0 4.8 7.8 4.1 7.6 4.1 7.3 1.3 7.2 9.0 8.0 10.4 7.5 9.8 7.7 9.4 8.1 5.0 5.2 5.6 5.7 5.7 6.5 6.8 7.3 5.0 4.9 5.0 4.9 3.9 3.7 5.5 5.3 75948515353525268666462656054481. 041...... 3.3 2.8 4.6 5.9 6.5 2.6 6.1 5.1 5.0 5.0 5.7 5.0 3.0 3.0 9.0 10.7 10.4 10.4 4.8 5.4 6.0 6.5 6.2 6.4 6.6 6.8 5.2 5.2 5.3 5.3 5.1 4.8 5.9 .7 Pleosp betae

Kirsch elater ...... 021. 0793133835423535364622595037272.9 2.7 3.7 5.0 5.9 2.2 4.6 3.6 3.5 3.5 4.2 3.5 3.8 1.3 9.3 10.7 10.2 10.2 5.0 5.6 5.8 6.3 6.3 6.4 6.7 7.4 4.8 4.8 4.9 4.8 4.4 4.3

Mass bipola

Mass austra

Kirsch mariti

Angu longi

Mycos myco

Herpot juni

Mona purpur

Paecilo var

Onyge equi

Arthrob sup

Orbilia firm

Arthrob oligo

Monacro doed

Orbilia dent

Hyd repand

Penio nuda

Bull albus

Sacch cere

Var elodeae . 231. 181. 201. 021. 251. . 10.5 9.9 11.5 12.5 12.3 10.2 12.3 12.0 11.9 11.8 12.5 12.3 0.2 21. 201. 121. 16971. 181. . 10.3 9.8 10.7 11.8 12.4 9.7 11.6 11.3 11.2 11.1 12.0 11.5 .2 151. 111. 131. . 251. 109510.2 9.5 11.0 12.1 12.5 9.7 11.4 11.3 11.2 11.1 12.0 11.5 6 Ala acumi

Tet march

Tet apiense

Tet furcat

Tet maxilli ...... 6.2 5.9 7.1 8.0 8.4 5.6 7.2 6.5

Tet setiger

Tri angulatum

Tri splen 0896879.5 8.7 9.6 10.8 5 0996798.8 7.9 9.6 10.9 6 Hel lugdun

Lem aquatic

Lem terrestr

Phial Elec-N

Angu furtiv Appendix

Alignment SSU sequences

1 11 21 31 41 51 61 71 81 91 100 | | | | | | | | | | | 1 ...... A-UCCUGCC AGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AUCUAUACUG UGAAACUGCG 81 TcdMaxil 1 ...... A-UCCUGCC AGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AUCUAUACUG UGAAACUGCG 81 TcdSetig 1 ...... A-UCCUGCC AGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AUCUAUACUG UGAAACUGCG 81 TcdFurca 1 ...... A-UCCUGCC AGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AUCUAUACUG UGAAACUGCG 81 TcdApien 1 ...... U-CAAAGAU. UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AUCUAUACUG UGAAACUGCG 55 Tmarch2639 1 ...... GCUUG-U-C U-CAAAGAU- UAAGCC-UGC -AUGU-UA-A GUAUAAGCA- AUAUAUACAG UGAAACUGCG 60 LemAqu1 1 ...... C AGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AUAUAUACAG UGAAACUGCG 64 LemTerr1 1 ...... 0 var11783 1 ...... A AGCUUG-U-C U-CAAAGAU- UAAGCCAUGC .AUGUCUA-A GUAUAAGCA- AUCUAUACGG UGAAACUGCG 64 TriAng1020 1 ...... UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AUCUAUACGG UGAAACUGCG 47 Mc8Coral 1 ...... 0 HVZ81382 1 ...... UAUACCG UGAAACUGCG 17 Loram 1 ...... GAAACUGCG 9 986Defle 1 ...... GAAACUGCG 9 OidTenui 1 ...... UAAGCA- A-UUAUAUAG UGAAACUGCG 25 Acrassa055 1 ...... AGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AU-UAUAUAG UGAAACUGCG 50 AngFurt 1 ...... UG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUUAA GUAUAAGCA- AU-UAUAUAG UGAAACUGCG 59 TriSpl1198 1 ...... CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AAUUAUACCG UGAAACUGCG 54 472Gutta 1 ...... CA- AUUUAUACCG UGAAACUGCG 22 7BGrami 1 ...CU-GGUU GA-UUCUGCC AGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AUCUAUACGG UGAAACUGCG 88 PiaSpec3 1 ...... AUGC -AUGUCUA-A GUAUAAGCA- AUCUAUACGG UGAAACUGCG 41 164Darwi 1 ...... UA-A GUAUAAGCAA A-CUAUACCG UGAAACUGCG 32 CudConfu 1 ...... GCUUA-A GUAUAAGCAA A-CUAUACCG UGAAACUGCG 35 ShaFlavi 1 ...... GAAACUGCG 9 989Roseu 1 ...... GAACUUGCG 9 39GPanno 1 ...... AGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AUCUAUACUG UGAAACUGCG 70 58BInqui 1 ...... 0 0LLubri 1 ...... G-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-CUAUACCG UGAAACUGCG 57 13089Alacu 1 ...... GUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACNG CGAAACUGCG 71 NerCinn5 1 ...... UAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACAG CGAAACUGCG 70 GsaPutte 1 ...... CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACAG CGAAACUGCG 53 GibPuli5 1 ...... GUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG CGAAACUGCG 71 HpaLute3 1 ...... CAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACGG CGAAACUGCG 41 HymChrys 1 ...... UGCUUG-U-C U-CAAAGAU- KAA-CCAUGC GAUGUCUA-A GUAUAAGCA- A-UUAUACAG CGAAACUGCG 62 Hlug13783 1 ...... CCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG CGAAACUGCG 42 144Seti3 1 ...... CCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG CGAAACUGCG 42 PsaBoyd6 1 ...... GUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACGG CGAAACUGCG 71 98HRetor 1 ...... AUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG CGAAACUGCG 40 MiaCirro 1 ...... UCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG CGAAACUGCG 67 GmmPeni3 1 UACCU-GGUU GA-UUCUGCC AGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUUUAAGCA- A-UUAAACCG CGAAACUGCG 90 NeuCrass 1 UACCU-GGUU GA-UUCUGCC AGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUUUAAGCA- A-UUAAACCG CGAAACUGCG 90 SorFirmi 1 ...... AUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG CGAAACUGCG 40 ChtElatu 1 ...... UCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG CGAAACUGCG 67 KicIvori 1 AACCU-GGUU GA-UCCUGCC AGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-CUAUACGG UGAAACUGCG 90 AuePullu 1 ...... AAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-CUAUACGG UGAAACUGCG 52 Hr0Werne 1 ...... U GA-UUCUGCC AGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AAGUCUA-A GUAC-AGC-- A-UUGUACCG UGAAACUGCG 80 KirAethi 1 ...... UA -G--UG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG UGAAACUGCG 61 OpbHerpo 1 ...... U... .GUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG UGAAACUGCG 72 LehDoli6 1 ...... CAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG UGAAACUGCG 41 156Elong 1 ...... U..U. UGCUUG-UUC U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG UGAAACUGCG 65 PlpBetae 1 ...... U--- -GUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- -UUUAUACCG UGAAGCUGCG 72 KirElate 1 ...... UGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG UGAAACUGCG 72 42MMycop 1 ...... AUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG UGAAACUGCG 40 21HJunip 1 ...... U--- -GUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG UGAAACUGCG 72 KirMarit 1 ...... A AGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG UGAAACUGCG 63 alongi1189 1 ...... CAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG UGAAACUGCG 41 996Bipol 1 ...... CAUGC -AUGUCUA-A GUAUAAGCA- A-UUAUACCG UGAAACUGCG 41 996Austr 1 ...... AUGC -AUGUCUA-A GUGUAAGCA- AUUUAUACUG UGAAACUGCG 41 MonPurpu 1 ...... CUGCG 5 243Vari2 1 ...... U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- ACUUGUACGG UGAAACUGCG 57 OngEquin 1 ...... AGCCAUGC -AUGUCUA-A GUAUAAGCA- A-CUAUACAG UGAAACUGCG 44 750Supe2 1 ...... UGCC AGUAGUCAUA UGCUUGUU-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- A-CUAUACAG UGAAACUGCG 77 ObaFimic 1 ...... AGCCAUGC -AUGUCUA-A GUAUANGCA- A-CUAUACAG UGAAACUGCG 44 750Olig2 1 ...... AGCCAUGC -AUGUCUA-A GUAUAAGCA- A-CUAUACAG UGAAACUGCG 44 897Doedy 1 ...... U--- -GUAGUCAUA UGCUUG-U-C U-CAAAGAU- GAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AUCUAUACAG UGAAACUGCG 73 ObaDelic 1 ...C-AGCUU GAA---U-UC -GUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAACAA AUUUAUACUG UGAAACUGCG 85 90HRepan 1 .....AGCUU ---GAAU-UC .GUAGUCAUA UGNGUG-U-C UCCAGAGAN- UAAGNCAUGC -AUGUCUA-A GNAUAACCAC GUUUGUACUG GGAAACUGCG 85 130Nuda2 1 UACCU-GGUU GA-UCCUGCC AGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAACAA AUUCAUACUG UGAAACUGCG 92 BulAlbus 1 UAUCU-GGUU GA-UCCUGCC AGUAGUCAUA UGCUUG-U-C U-CAAAGAU- UAAGCCAUGC -AUGUCUA-A GUAUAAGCA- AUUUAUACAG UGAAACUGCG 91 SayCe108 101 111 121 131 141 151 161 171 181 191 200 | | | | | | | | | | | 82 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 172 TcdMaxil 82 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 172 TcdSetig 82 AAUGGCUCAU UA-ANUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 172 TcdFurca 82 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 172 TcdApien 56 AAUGGCUCAU UA-AAUCA.G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 146 Tmarch2639 61 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 151 LemAqu1 65 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 155 LemTerr1 1 ...... 0 var11783 65 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU. AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 155 TriAng1020 48 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 138 Mc8Coral 1 ...... 0 HVZ81382 18 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACAUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 108 Loram 10 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 100 986Defle 10 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 100 OidTenui 26 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 116 Acrassa055 51 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 141 AngFurt 60 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 150 TriSpl1198 55 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-AUCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAG-CC 145 472Gutta 23 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAG-CC 113 7BGrami 89 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 179 PiaSpec3 42 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCC-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUGAAAA-CC 132 164Darwi 33 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACUU-U-A CUACAUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 123 CudConfu 36 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACUU-U-A CUACAUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 126 ShaFlavi 10 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 100 989Roseu 10 AAUGGCUCAU UA-AAUCA-G U-AUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 99 39GPanno 71 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 161 58BInqui 1 ...... A-G -UA-CGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 73 0LLubri 58 AAUGGCUCAU UAUA-UCA-G UCUAAAUAU- ACUUGA-UAG U.ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA--C 147 13089Alacu 72 AAUGGCUCAU UA-UAUAA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUUAAAA-UC 162 NerCinn5 71 AAUGGCUCAU UA-UAUAA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UU 161 GsaPutte 54 AAUGGCUCAU UA-UAUAA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 144 GibPuli5 72 AAUGGCUCAU UA-UAUAA-G UUAUCGUUU- AUUUGA-UAA U-ACUU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 162 HpaLute3 42 AAUGGCUCAU UA-UAUAA-G UUAUCGUUU- AUUUGA-UAA U-ACUU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 132 HymChrys 63 AAUGGCUCAU UA-UAUAA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 153 Hlug13783 43 AAUGGCUCAU UA-UAUAA-G UUAUCGUUU- AUUUGA-UAG C-ACAU-U-A CUACAUGG-A UAACUGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 133 144Seti3

Figure A36: Alignment of complete SSU sequences.

199 Appendix

Alignment SSU sequences continued

43 AAUGGCUCAU UA-UAUAA-G UUAUCGUUU- AUUUGA-UAG C-ACAU-U-A CUACAUGG-A UAACUGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 133 PsaBoyd6 72 AAUGGCUCAU UA-UAUAA-G UUAUCGUUU- AUUUGA-UAG C-ACCU-U-U UUACUCGG-A UAACUGUGGU AAUUCUAGAG CUAAUACGUG CUGAAAA-UC 162 98HRetor 41 AAUGGCUCAU UA-UAUAA-G UUAUCGUUU- AUUUGA-UAG C-ACGU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 131 MiaCirro 68 AAUGGCUCAU UA-UAUAA-G UUAUAGUUU- AUUUGA-UAG C-ACCU-U-A CUACAUGG-A UAACUGUGGU AAUUCUAGAG CUAAAACAUG CUAAAAA-UC 158 GmmPeni3 91 AAUGGCUCAU UA-AAUCA-G UUAUAGUUU- AUUUGA-UAG U-ACCU-U-A CUACAUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 181 NeuCrass 91 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACAUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 181 SorFirmi 41 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACAUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 131 ChtElatu 68 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-UUC CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 159 KicIvori 91 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 181 AuePullu 53 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACAUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 143 Hr0Werne 81 AACGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCC-U-A CUACAUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAG-CC 171 KirAethi 62 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 152 OpbHerpo 73 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUGAAAA-CC 163 LehDoli6 42 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACAUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 132 156Elong 66 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUGAAAA-CC 156 PlpBetae 73 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 163 KirElate 73 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CCACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 163 42MMycop 41 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CCACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 131 21HJunip 73 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUGAAAG-CC 163 KirMarit 64 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 154 alongi1189 42 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 132 996Bipol 42 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUGAAAA-CC 132 996Austr 42 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACAUGG-A UACCUGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 132 MonPurpu 6 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-G CUACAUGG-A UACCUGUGGU AAUUCUAGAG CUAAUACAUG CUGAAAA-CC 96 243Vari2 58 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCUUUUU CCACAUGG-A UACCCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-CC 150 OngEquin 45 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 135 750Supe2 78 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 168 ObaFimic 45 -AUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 134 750Olig2 45 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-ACUU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 135 897Doedy 74 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGAAUAG U-ACCU-U-A CUACUUGG-A UAACCGUGGU AAUUCUAGAG CUAAUACAUG CUAAAAA-UC 165 ObaDelic 86 AAUGGCUCAU UA-AAUCA-G UUAUAGUUU- AUUUGA-UGG U-ACCU-U-G CUACAUGG-A UAACUGUGGU AAUUCUAGAG CUAAUACAUG CAUCAAAGCC 177 90HRepan 86 AAUGGUUCAU UA-AAUCA-G UUAUAGUUU- AUUUGA-UGG U-CCCU-U-G UUACAUGG-A UAACUGUGGU AAUUCUAGAG CUAAUACAUG CAAUCAAGCC 177 130Nuda2 93 AAUGGCUCAU UA-AAUCA-G UUAUAGUUU- AUUUGA-UGG U-AUCU-U-G CUACAUGG-A UAACUGUGGU AAUUCUAGAG CUAAUACAUG CUGAAAAGCC 184 BulAlbus 92 AAUGGCUCAU UA-AAUCA-G UUAUCGUUU- AUUUGA-UAG U-UCCU-UUA CUACAUGGUA UAACUGUGGU AAUUCUAGAG CUAAUACAUG CUUAAAA-UC 184 SayCe108

201 211 221 231 241 251 261 271 281 291 300 | | | | | | | | | | | 173 -CC-GACU-U U-UGG--A-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 262 TcdMaxil 173 -CC-GACU-U U-UGG--A-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 262 TcdSetig 173 -CC-GACU-U U-UGG--A-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 262 TcdFurca 173 -CC-GACU-U U-UGG--A-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 262 TcdApien 147 -CC-GACU-U U-UGG--A-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 236 Tmarch2639 152 -UC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 241 LemAqu1 156 -UC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 245 LemTerr1 1 ...... 0 var11783 156 .CC-GACU-U -ACGG--A-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CCUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 245 TriAng1020 139 -CC-GACU-U -ACGA--A-G-- GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 228 Mc8Coral 1 ...... 0 HVZ81382 109 -UC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 198 Loram 101 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CCUGGU GAUUC-ACAA UAACUCAACG AAUCGCAUGG 190 986Defle 101 -UC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-UCUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 190 OidTenui 117 -GC-GACU-U --CGG-GA-G CGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 206 Acrassa055 142 -GC-GACU-U --CGG-GA-G CGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 231 AngFurt 151 -GC-GACU-U --CGG-GA-G CGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 240 TriSpl1198 146 -CU-GACG-U --CAG-AA-G GGGUGUAUUU AUUAGAUUAA AAACCAAU-G CCCUUCGGGG CUC-GAUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 235 472Gutta 114 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUCCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 203 7BGrami 180 -UC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUU-CAUAA UAACUUAACG AAUCGCAUGG 269 PiaSpec3 133 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUCCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUCACG AAUCGCAUGG 222 164Darwi 124 -CC-GACU-U --CGG-AA-G G-GUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGNN CUC-CCUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 212 CudConfu 127 -CC-GACU-U --UGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G NCCUUCGGGG CUC-CCUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 216 ShaFlavi 101 -CC-AACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CUCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 190 989Roseu 100 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CUCUUCGGGG CUC-CUUGGU GGUUC-AUAA UAACUUAACG AAUCGCAUGG 189 39GPanno 162 -UC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUCAACG AAUCGCAUGG 251 58BInqui 74 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUAAACG AAUCGCAUGG 163 0LLubri 148 -CCAGACU-U --CGG- AAUG GG-UGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUGA UAACUUAACG AAUCGCAUGG 238 13089Alacu 163 -CC-GACU-U --CGG-AA-G GGAUGUAUUU AUUAGAUUAA AAACCAAU-G CCCUUCGGGG CUC-UUUGGU GAUUC-AUGA UAACUUCUCG AAUCGCAUGG 252 NerCinn5 162 -CC-GACU-U --CGG-AA-G GGAUGUAUUU AUUAGAUUAA AAACCAAU-G CCCUUCGGGG CUC-UCUGGU GAUUC-AUGA UAACUUCUCG AAUCGCAUGG 251 GsaPutte 145 -CC-GACU-U --CGG-AA-G GGAUGUAUUU AUUAGAUUAA AAACCAAU-G CCCUUCGGGG CUC-ACUGGU GAUUC-AUGA UAACUCCUCG AAUCGCAUGG 234 GibPuli5 163 -CC-GACU-U --CGG-AA-G GGUUGUAUUU AUUAGAUUAA AAACCAAU-G CCCU-CGGGG CUC-UCUGGU GAAUC-AUGA UAACUAGUCG AAUCGACAGG 251 HpaLute3 133 -CC-GACU-U --CGG-AA-G GGUUGUAUUU AUUAGAUUAA AAACCAAU-G CCCUCUGG-G CUC-UCUGGU GAAUC-AUAA UAACUUGUCG AAUCGACAGG 221 HymChrys 154 -CC-GACU-U --CGG-AA-G GGAUGUAUUU AUUAGAUUAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUGA UAACUCCUCG AAUCGCAUGG 243 Hlug13783 134 -CC-GACU-U --CGG-AA-G GGAUGUAUUU AUUAGAUUAA AAGCCAAC-G CCCUUCGGGG CUU-CCUGGU GAUUC-AUAA UAACUUUUCG AAUCGCAUGG 223 144Seti3 134 -CC-GACU-U --CGG-AA-G GGAUGUAUUU AUUAGAUUAA AAGCCAAC-G CCCUUCGGGG CUU-CGUGGU GAUUC-AUGA UAACUUUUCG AAUCGCAUGG 223 PsaBoyd6 163 -CC-GACU-U --CGG-AA-G GGCUGUAUUU AUUAGAUUCA AAACCAAU-G CCCUUCGGGG CUU-CAUGGU GAUUC-AUAA UAACUGCUCG AAUCGCAUGG 252 98HRetor 132 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUUAA AAGCCAAC-G CCCUUCGGGG CUC-UGUGGU GAUUC-AUGA UAACUUUUCG AAUCGCAUGG 221 MiaCirro 159 -CC-GACU-U --CGG-AA-G GGAUGUGUUU AUUAGAUUAA AAACCAAU-G CCCUUCGGGG CUG-CUUGGU GAUUC-AUGA UAACCUCUCG AAUCGCACGG 248 GmmPeni3 182 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUUAA AAACCAAU-G CCCUUCGGGG CUA-ACUGGU GAUUC-AUAA UAACUUCUCG AAUCGCAUGG 271 NeuCrass 182 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUUAA AAACCAAU-G CCCUUCGGGN CUU-CCUGGU GAUUC-AUGA UAACUUCUCG AAUCGCACGG 271 SorFirmi 132 -CC-GACU-U --CGG-AA-G GGAUGUAUUU AUUAGAUUAA AAACCAAU-G CCCUUCGGGG CUC-UCUGGU GAUUC-AUAA UAACUUCUCG AAUCGCACGG 221 ChtElatu 160 -CC-GACU-U --CGG-AA-G GGAUGCAUUU AUCAGAUACA GAACCAAUUG CCCUCCGGGG CUC-CCUGGU GAAUC-AUGA UAACUCCGCG GAUCGCACGG 250 KicIvori 182 -CC-AACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAC-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUAAACG AAUCGCAUGG 271 AuePullu 144 -CC-GACU-U --CGG-GA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAAUC-AUAA UAACUUAACG AAUCGCAUGG 233 Hr0Werne 172 -CC-GACU-G --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUGA UAACUCAACG GAUCGCAGGG 261 KirAethi 153 -CC-AACU-U --CGG-GA-G GGGUGUAUUU AUUAGAUAAA AAACCAAC-G CCCUUCGGGG CUU-CUUGGU GAUUC-AUGA UAACUUUACG GAUCGCAUGG 242 OpbHerpo 164 -CC-AACU-U --CGG-GA-G GGGUGUAUUU AUUAGAUAAA AAACCAAC-G CCCUUCGGGG CUU-CUUGGU GAUUC-AUGA UAACUUUACG GAUCGCAUGG 253 LehDoli6 133 -CC-AACU-U --CGG-GA-G GGGUGUAUUU AUUAGAUAAA AAACCAAC-G CCCUUCGGGG CUU-CUUGGU GAUUC-AUAA UAACUUUACG GAUCGCAUGG 222 156Elong 157 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAC-G CCCUUCGGGG CUU-CUUGGU GAUUC-AUAA UAACUUUACG GAUCGCAUGG 246 PlpBetae 164 -CC-AACU-U --CGG-GA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-UUUGGU GAUUC-AUGA UAACUUCUCA GAUCGCAUGG 253 KirElate 164 -CC-GACU-U --CGG-AA-G GGGUGUGUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-UUUGGU GAUUC-AUGA UAACCUAACG GAUCGCAUGG 253 42MMycop 132 -CC-GACU-U --CGG-AA-G GGGUGUGUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-UUUGGU GAUUC-AUGA UAACCUAACG GAUCGCAUGG 221 21HJunip 164 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAGCCAAU-G CCCUUCGGGG CUC-UUUGGU GAUUC-AUGA UAACUUAACA GAUCGCAUGG 253 KirMarit 155 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAGCCAAU-G CCCUCCGGGG CUC-UUUGGU GAUUC-AUAG UAACUUAACG GAUCGCAUGG 244 alongi1189 133 -CU-GACU-U --CGG-AA-A GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUCCGGGG CUC-UUUGGU GAUUC-AUGA UAACUUAACG GAUCGCAUGG 222 996Bipol 133 -CU-GACU-U --CGG-AA-A GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUUGGGG CUC-UUUGGU GAUUC-AUGA UAACUUAACG GAUCGCAUGG 222 996Austr 133 -CC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAC-G CCCUUCGGGG CUC-CUUGGU GAAUC-AUAA UAACUAAACG AAUCGCAUGG 222 MonPurpu 97 -UC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUAACG AAUCGCAUGG 186 243Vari2 151 -UC-GACU-U --CGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G CCCUUCGGGG CUC-CUUGGU GAUUC-AUAA UAACUUUUCG AAUCGCAUGG 240 OngEquin 136 -CC-GACC-U C-NGG-AA-G GGAUGUAUUU AUUAGAUAAA AAACCAAU-G C-CUUCGG-G CUC-CUUGGU GAUUC-AUGA UAACUUAACG AAUCGCAUGG 224 750Supe2 169 -CC-GACC-U C-CGG-AA-G GGAUGUAUUU AUUAGAUAAA AAACCAAU-G C-CUUCGG-G CUC-CUUGGU GAUUC-AUGA UAACUUAACG AAUCGCAUGG 257 ObaFimic 135 -CC-GACC-U C-CGG-AA-G GGAUGUAUUU AUUAGAUAAA AAACCAAU-G C-CUUCGG-G CUC-CUUGGU GAUUC-AUGA UAACUUAACG AAUCGCAUGG 223 750Olig2 136 -CC-GACC-U C-UGG-AA-G GGAUGUAUUU AUUAGAUAAA AAACCAAU-G C-CUUCGG-G CUC-CUUGGU GAUUC-AUGA UAACUUAACG AAUCGCAUGG 224 897Doedy 166 -CC-GACC-U C-UGG-AA-G GGAUGUAUUU AUUAGAUAAA AAACCAAU-G C-CUUCGG-G CUC-CUUGGU GAUUC-AUGA UAACUUAACG GAUCGCAUGG 254 ObaDelic 178 -CC-GACU-U C-UGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAC-G CGGUUCGCCG CUCCUUUGGU GAUUC-AUAA UAACUUCUCG AAUCGCAUGG 269 90HRepan 178 -CC-GACU-U C-UGG-GA-G GGGUGUAUUU AUUAGAUAAA AAACCAAC-G CGGCUUGCCG CUCGUUUGGU GAUUC-AUAA UAACUUCUCG AAUCGCAUGG 269 130Nuda2 185 -CC-GACU-U C-UGG-AA-G GGGUGUAUUU AUUAGAUAAA AAACCAAU-G GGUGAAAGCC CUC-UAUGGU GAUUC-AUGA UAACUUCUCG AAUCGCAUGG 275 BulAlbus 185 -UC-GACCCU U-UGG-AA-G AGAUGUAUUU AUUAGAUAAA AAAUCAAU-G U-CUUCGG-A CUC-UUUGAU GAUUC-AUAA UAACUUUUCG AAUCGCAUGG 274 SayCe108

Figure A36 continued

200 Appendix

Alignment SSU sequences continued 301 311 321 331 341 351 361 371 381 391 400 | | | | | | | | | | | 263 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUUAGGU CUUGGCUAAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 359 TcdMaxil 263 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUUAGGU CUUGGCUAAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 359 TcdSetig 263 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUUAGGU CUUGGCUAAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 359 TcdFurca 263 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUUAGGU CUUGGCUAAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 359 TcdApien 237 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUUAGGU CUUGGCUAAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 333 Tmarch2639 242 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUACGGU CUUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 338 LemAqu1 246 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUACGGU CUUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 342 LemTerr1 1 ...... 0 var11783 246 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA.U CAACUUUCG- AUUGUAGCGU AGUGGGCUAC A-AUGGUUUC AACGGGUAAC GGGGAAUUAG 342 TriAng1020 229 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAUGGU CUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 325 Mc8Coral 1 ...... 0 HVZ81382 199 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAUGGU CUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 295 Loram 191 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUUGUAUGGU CUUGGCUUAC A-AUGGUUUC AACGGGUAAC GGGGAAUUAG 287 986Defle 191 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUUGUAAGGU CUUGGCUUAC A-AUGGUUUC AACGGGUAAC GGGGAAUUAG 287 OidTenui 207 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 303 Acrassa055 232 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 328 AngFurt 241 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 337 TriSpl1198 236 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGUAU AUGGGACUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 332 472Gutta 204 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGUAU AUGGGACUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 300 7BGrami 270 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 366 PiaSpec3 223 CCUUGUGCNG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 319 164Darwi 213 CCUUGCGC-G GUGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 308 CudConfu 217 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 313 ShaFlavi 191 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 287 989Roseu 190 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 286 39GPanno 252 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 348 58BInqui 164 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 260 0LLubri 239 CCUUGUGCCG GCGAUGGUUC AUUCUAUAUU CUGCCCUA-U CAACUUUCG. AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 335 13089Alacu 253 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUUCCCUA-U CAACUUUCG- AUGUUUGGGU AUUGGCCAAA C-AUGGUUGC AACGGGUAAC GGAGGGUUAG 349 NerCinn5 252 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUUCCCUA-U CAACUUUCG- AUGUUUGGGU AUUGGCCAAA C-AUGGUUGC AACGGGUAAC GGAGGGUUAG 348 GsaPutte 235 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUUCCCUA-U CAACUUUCG- AUGUUUGGGU AUUGGCCAAA C-AUGGUUGC AACGGGUAAC GGAGGGUUAG 331 GibPuli5 252 CCUUGUGCCG GCGAUGGCUC AUUCAAAUUU CUUCCCUA-U CAACUUUCG- AUGUUUGGGU AUUGGCCAAA C-AUGGUGGC AACGGGUAAC GGAGGGUUAG 348 HpaLute3 222 CCUUGUGCCG GCGAUGGCUC AUUCAAAUUU CUUCCCUA-U CAACUUUCG- AUGUUUGGGU AUUGGCCAAA C-AUGGUGGC AACGGGUAAC GGAGGGUUAG 318 HymChrys 244 CCUUGUGCCG GCGAUGGUUC AUUCAAAUUU CUUCCCUA-U CAACUUUCG- AUGUUUGGGU AUUGGCCAAA C-AUGGUUGC AACGGGUAAC GGAGGGUUAG 340 Hlug13783 224 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUUCCCUA-U CAACUUUCG- AUGCGAAGGU AUUGUCUUCG C-AUGGUUGC AACGGGUAAC GGAGGGUUAG 320 144Seti3 224 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUUCCCUA-U CAACUUUCG- AUGCGAAGGU CUUGUCUUCG C-AUGGUUGC AACGGGUAAC GGAGGGUUAG 320 PsaBoyd6 253 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUUCCCUA-U CAACUUUCG- AUGCGAGGGU CUUGUCCUCG C-AUGGUUAC AACGGGUAAC GGAGGGUUAG 349 98HRetor 222 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUUCCCUA-U CAACUUUCG- AUGCGAGGGU CUUGUCCUCG C-AUGGUUGC AACGGGUAAC GGAGGGUUAG 318 MiaCirro 249 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUUCCCUA-U CAACUUUCG- AUGCGAAGGU AUUGUCUUCG C-AUGGUUAC AACGGGUAAC GGAGGGUUAG 345 GmmPeni3 272 CCUUGCGCUG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- ACGGCUGGGU CUUGGCCAGC C-AUGGUGAC AACGGGUAAC GGAGGGUUAG 368 NeuCrass 272 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- ACGGCUGGGU CUUGGCCAGC C-AUGGUGAC AACGGGUAAC GGAGGGUUAG 368 SorFirmi 222 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- ACGGCUGGGU CUUGGCCAGC C-GUGGUGAC AACGGGUAAC GGAGGGUUAG 318 ChtElatu 251 CCUUGCGCUG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- ACGGCUGGGU CUUGGCCAGC C-GUGGUGAC AACGGGUAAC GGAGGGUUAG 347 KicIvori 272 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUAUC AACGGGUAAC GGGGAAUUAG 368 AuePullu 234 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGGAUC AACGGGUAAC GGGGAAUUAG 330 Hr0Werne 262 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU CGGGGCCUAC C-AUGGUAUC AACGGGUAAC GGGGAAUUAG 358 KirAethi 243 CCUUGCGCCG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAAGGU AUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 339 OpbHerpo 254 CCUUGCGCUG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAAGGU AUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 350 LehDoli6 223 CCUUGCGCCG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAAGGU AUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 319 156Elong 247 CCUUGCGCCG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAAGGU AUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 343 PlpBetae 254 CCUUGCGCCG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAAGGU AUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 350 KirElate 254 CCUUGCGCCG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAAGGU AUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 350 42MMycop 222 CCUUGCGCCG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAAGGU AUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 318 21HJunip 254 CCUUGCGCCG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAAGGU AUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 350 KirMarit 245 CCUUGCGCCG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAAGGU AUUGUCUUAC CUAUGGUUUC AACGGGUAAC GGGGAAUUAG 342 alongi1189 223 CCUUGCGCCG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAAGGU AUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 319 996Bipol 223 CCUUGCGCCG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAAGGU AUUGGCUUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 319 996Austr 223 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUGGC AACGGGUAAC GGGGAAUUAG 319 MonPurpu 187 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUGGC AACGGGUAAC GGGGAAUUAG 283 243Vari2 241 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUGGC AACGGGUAAC GGGGAAUUAG 337 OngEquin 225 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC UACGGGUAAC GGGGAAUUAG 321 750Supe2 258 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC UACGGGUAAC GGGGAAUUAG 354 ObaFimic 224 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 320 750Olig2 225 CCUUGCGCCG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 321 897Doedy 255 CCUUGCGCCG GCGACGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUUAG 351 ObaDelic 270 CCUUGUGCCG GCGAUGCUUC AUUCAAAUAU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGAGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUAAG 366 90HRepan 270 CCUUGUGCCG GCGAUGCUUC AUUCAAAUAU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGUCCUAC C-AUGGUUUC ACCGGGUAAC GGGGAAUAAG 366 130Nuda2 276 CCUUGCGCCG GCGAUGCUUC AUUCAAAUAU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGAGGCCUAC C-AUGGUAUC AACGGGUAAC GGGGAAUUAG 372 BulAlbus 275 CCUUGUGCUG GCGAUGGUUC AUUCAAAUUU CUGCCCUA-U CAACUUUCG- AUGGUAGGAU AGUGGCCUAC C-AUGGUUUC AACGGGUAAC GGGGAAUAAG 371 SayCe108 401 411 421 431 441 451 461 471 481 491 500 | | | | | | | | | | | 360 GGUUC-UAUU CCGGAG-AGU GAGCCUGAGA AACGGCUAAC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 456 TcdMaxil 360 GGUUC-UAUU CCGGAG-AGU GAGCCUGAGA AACGGCUAAC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 456 TcdSetig 360 GGUUC-UAUU CCGGAG-AGU GAGCCUGAGA AACGGCUAAC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 456 TcdFurca 360 GGUUC-UAUU CCGGAG-AGU GAGCCUGAGA AACGGCUAAC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 456 TcdApien 334 GGUUC-UAUU CCGGAG-AGU GAGCCUGAGA AACGGCUAAC ACAUCCAAGG AAGGCAGCAG G.CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 430 Tmarch2639 339 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 435 LemAqu1 343 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 439 LemTerr1 1 ...... UCAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 59 var11783 343 GGUUC.UAUU CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 439 TriAng1020 326 GGUUC-GACU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 422 Mc8Coral 1 ...... 0 HVZ81382 296 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGAUACGGG GAGGUAGUGA 392 Loram 288 GGUUC-UAUU CCGGAG-AGC GAGCCUGAGA CACGGCUAGC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 384 986Defle 288 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACUACCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 384 OidTenui 304 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 400 Acrassa055 329 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 425 AngFurt 338 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 434 TriSpl1198 333 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 429 472Gutta 301 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 397 7BGrami 367 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUGCC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 463 PiaSpec3 320 GGUUC-GACU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 416 164Darwi 309 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CNGACACGGG GAGGUAGUUA 405 CudConfu 314 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 410 ShaFlavi 288 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 384 989Roseu 287 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 383 39GPanno 349 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 445 58BInqui 261 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 357 0LLubri 336 GGUUC-UAUU CCGGAG.AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 432 13089Alacu 350 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACUCGGG GAGGUAGUGA 446 NerCinn5 349 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACUCGGG GAGGUAGUGA 445 GsaPutte 332 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 428 GibPuli5 349 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 445 HpaLute3 319 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 415 HymChrys 341 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 437 Hlug13783 321 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 417 144Seti3 321 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 417 PsaBoyd6 350 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 446 98HRetor

Figure A36 continued

201 Appendix

Alignment SSU sequences continued

319 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 415 MiaCirro 346 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 442 GmmPeni3 369 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 465 NeuCrass 369 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 465 SorFirmi 319 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 415 ChtElatu 348 GGCUC-GACC CCGGAG-AAG GAGCCUGAGA AACGGCUACU ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 444 KicIvori 369 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 465 AuePullu 331 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGAAGCAG G-CGCGCAAA UUACCCAMUC CCGACAGGGG GAGGUAGAGA 427 Hr0Werne 359 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA GACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACUCGGG GAGGUAGUGA 455 KirAethi 340 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 436 OpbHerpo 351 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 447 LehDoli6 320 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 416 156Elong 344 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 440 PlpBetae 351 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA GACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 447 KirElate 351 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 447 42MMycop 319 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 415 21HJunip 351 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 447 KirMarit 343 GGUUC-GAUU CCGGAG-AGG GAGCCU--GA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 437 alongi1189 320 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 416 996Bipol 320 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 416 996Austr 320 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 416 MonPurpu 284 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGAUACGGG GAGGUAGUGA 380 243Vari2 338 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGAUCCGGG GAGGUAGUGA 434 OngEquin 322 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGAUACGGG GAGGUAGUGA 418 750Supe2 355 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGAUUCGGG GAGGUAGUGA 451 ObaFimic 321 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCAAUUCGGG GAGGUAGUGA 417 750Olig2 322 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCAAUUCGGG GAGGUAGUGA 418 897Doedy 352 GGUUC-UAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGAUACGGG GAGGUAGUGA 448 ObaDelic 367 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 463 90HRepan 367 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGACAGCAG N-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 463 130Nuda2 373 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CCGACACGGG GAGGUAGUGA 469 BulAlbus 372 GGUUC-GAUU CCGGAG-AGG GAGCCUGAGA AACGGCUACC ACAUCCAAGG AAGGCAGCAG G-CGCGCAAA UUACCCAAUC CUAAUUCAGG GAGGUAGUGA 468 SayCe108

501 511 521 531 541 551 561 571 581 591 600 | | | | | | | | | | | 457 CAAUAAAUAC UGAUCCAGGG CUCUUUU------GGGUCU UGG-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 548 TcdMaxil 457 CAAUAAAUAC UGAUCCAGGG CUCUUUU------GGGUCU UGG-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 548 TcdSetig 457 CAAUAAAUAC UGAUCCAGGG CUCUUUU------GGGUCU UGG-AAU-GG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 547 TcdFurca 457 CAAUAAAUAC UGAUCCAGGG CUCUUUU------GGGUCU UGG-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 548 TcdApien 431 CAAUAAAUAC UGAUCCAGGG CUCUUUU------GGGUCU UGG-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 522 Tmarch2639 436 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 527 LemAqu1 440 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 531 LemTerr1 60 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 151 var11783 440 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU.AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 531 TriAng1020 423 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 514 Mc8Coral 1 ...... AGUCU 5 HVZ81382 393 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 484 Loram 385 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 476 986Defle 385 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 476 OidTenui 401 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 492 Acrassa055 426 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 517 AngFurt 435 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 526 TriSpl1198 430 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 521 472Gutta 398 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 489 7BGrami 464 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 555 PiaSpec3 417 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 508 164Darwi 406 CAAUAAAUAC AGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 497 CudConfu 411 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 502 ShaFlavi 385 CAAUAAAUAC UGAUACAGGG CUCUUUU------GAGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 476 989Roseu 384 CAAUAAAUAC UGAUACAGGG CUCUUUU------GAGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 475 39GPanno 446 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 537 58BInqui 358 CAAUAAAUAC UGAUAUUGGG GUCUUUA------GGCUCU AAU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 449 0LLubri 433 CAAUAAAUAC UGAUCUUGGG CCAUUUU------UGGUCU AAG-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 524 13089Alacu 447 CAAUAAAUAC UGAUACAGGG UUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 538 NerCinn5 446 CAAUAAAUAC UGAUACAGGG CUCUUUA------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 537 GsaPutte 429 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 520 GibPuli5 446 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUCGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 537 HpaLute3 416 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUCGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 507 HymChrys 438 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 529 Hlug13783 418 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUCGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 509 144Seti3 418 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUCGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 509 PsaBoyd6 447 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUCGG AAUGAGUACU AUUUAAAUCC CAUAACGAGG AACAAUUGGA GGGCAAGUCU 538 98HRetor 416 CAAUAAAUAC UGAUACAGGG CUCUUUA------GGGUCU UGU-AAUCGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 507 MiaCirro 443 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG GAUGAGUACA AUUUAAAUCC CUUAACGGGG AACAAUUGGA GGGCAAGUCU 534 GmmPeni3 466 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 557 NeuCrass 466 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGUGG AACAAUUGGA GGGCAAGUCU 557 SorFirmi 416 CAAUAAAUAC UGAUACAGGG CUCUUUC------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 507 ChtElatu 445 CAAUAAAUAC UGAUCCAGGG CUCUUUU------GGGUCU UGG-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AGCAAUUGGA GGGCAAGUCU 536 KicIvori 466 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 557 AuePullu 428 CAAUAMUUAC UGAUACAGCG CUCUUUU------GUGUCU UGU-AAUUGG AAUGAAUACA AUUUACCUCC CUUAACGAGG AACAAUUGGA GGGUAUGUCU 519 Hr0Werne 456 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAACCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 547 KirAethi 437 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAACCU CUUAACGAGG AACAAUUGGA GGGCAAGUCU 528 OpbHerpo 448 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAACCU CUUAACGAGG AACAAUUGGA GGGCAAGUCU 539 LehDoli6 417 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAACCU CUUAACGAGG AACAAUUGGA GGGCAAGUCU 508 156Elong 441 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAACCU CUUAACGAGG AACAAUUGGA GGGCAAGUCU 532 PlpBetae 448 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAACCU CUUAACGAGG AACAAUUGGA GGGCAAGUCU 539 KirElate 448 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAACCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 539 42MMycop 416 CAAUAAAUAC UGAUAUAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAACCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 507 21HJunip 448 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUCGG AAUGAGUACA AUUUAAACCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 539 KirMarit 438 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUCGG AAUGAGUACA AUUUAAACCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 529 alongi1189 417 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUCGG AAUGAGUACA AUUUAAACCU CUUAACGAGG AACAAUUGGA GGGCAAGUCU 508 996Bipol 417 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUCGG AAUGAGUACA AUUUAAACCU CCUAACGAGG AACAAUUGGA GGGCAAGUCU 508 996Austr 417 CAAUAAAUAC UGAUACGGGG CUCUUUC------GGGUCU CGU-AAUCGG AAUGAGAACG ACCUAAAUAA CCUAACGAGG AACAAUUGGA GGGCAAGUCU 508 MonPurpu 381 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 472 243Vari2 435 CAAUAAAUAC UGAUACAGGG CUCUUUC------GGGUCU UGU-AAUCGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 526 OngEquin 419 CAAUAAAUAC UGAUACAGG- CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 509 750Supe2 452 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 543 ObaFimic 418 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 509 750Olig2 419 CAAUAAAUAC UGAUACAGGG CUCUUUC------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 510 897Doedy 449 CAAUAAAUAC UGAUACAGGG CUCUUUU------GGGUCU UGU-AAUUGG AAUGAGUACA AUUUAAAUCU CUUAACGAGG NACAAUUGGA GGGCAANUCU 540 ObaDelic 464 CAAUAAAUAA CAAUAUAGGG CUCUUUU------GGGUCU UAU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAAUGAGG AACAAUUGGA GGGCAAGUCU 555 90HRepan 464 CAAUAAAUAA CAAUACAGGG CUCUUUU------GGGUCC UGU-AAUUGG AAUGAGUACA AUUUANNUCC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 555 130Nuda2 470 CAAUAAAUAA CAAUAUAGGG CUCUAUUCUC UAUUGGGUCU UAU-AAUUGG AAUGAGUACA AUUUAAAUCC CUUAACGAGG AACAACUGGA GGGCAAGUCU 568 BulAlbus 469 CAAUAAAUAA CGAUACAGGG CCCAUUC------GGGUCU UGU-AAUUGG AAUGAGUACA AUGUAAAUAC CUUAACGAGG AACAAUUGGA GGGCAAGUCU 560 SayCe108

Figure A36 continued

202 Appendix

Alignment SSU sequences continued

601 611 621 631 641 651 661 671 681 691 700 | | | | | | | | | | | 549 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GUCU GGCUGGCCGG 644 TcdMaxil 549 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GUCU GGCUGGCCGG 644 TcdSetig 548 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAG-UCG UAGUUG-AAC CUUGG-GUCU GGCUGGCCGG 642 TcdFurca 549 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G--UGCAGUU AAAA--- GCUGC UAGUUG A C CUUGG-GUCU GGCUGGCCGG 641 TcdApien 523 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GUCU GGCUGGCCGG 618 Tmarch2639 528 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGUUGGUCGG 623 LemAqu1 532 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGUUGGUCGG 627 LemTerr1 152 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGUUGGUCGG 247 var11783 532 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGUCGG 627 TriAng1020 515 GGUGCCANCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUGAAA- CUUGG-GCCU GGCUGGCCGG 610 Mc8Coral 6 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGUUGGUCGG 101 HVZ81382 485 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGUUGGCCGG 580 Loram 477 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 572 986Defle 477 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 572 OidTenui 493 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 588 Acrassa055 518 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 613 AngFurt 527 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 622 TriSpl1198 522 GGUGCCAGCA GCCGCGG-UA AUACCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 617 472Gutta 490 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 585 7BGrami 556 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-- UUGCAGUU AAAAAGCUCG UAGUUG AAC CUUGA-GCCU GGCUGGCCGG 651 PiaSpec3 509 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGG-CGG 603 164Darwi 498 GGUGCCAGCS GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGUUGGCNGG 593 CudConfu 503 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGUUGGCCGG 598 ShaFlavi 477 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 572 989Roseu 476 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 571 39GPanno 538 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 633 58BInqui 450 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUCGG-GCCU GGCUGGCCGG 545 0LLubri 525 GGUGCCAGCA GCCGCGG- UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG-- AAC CUUGG GCCU GGCUGGCCGG 620 13089Alacu 539 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGUGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 634 NerCinn5 538 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGUGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 633 GsaPutte 521 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGUGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 616 GibPuli5 538 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGUGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGG-CGG 632 HpaLute3 508 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGUGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 603 HymChrys 530 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGUGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 625 Hlug13783 510 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGUGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 605 144Seti3 510 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGUGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 605 PsaBoyd6 539 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGUGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCCUGUCAG 634 98HRetor 508 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGUGGUU AAAAAGCUCG UAGUCG- AAC CUUGG-GCCU GGCCGGCCGG 603 MiaCirro 535 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGUGGUU AAAA-GCUCG UAGUUG- AAC CUUGG--CCU GGCUGGCCGG 628 GmmPeni3 558 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGAGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCUC GGCCG-UCGG 652 NeuCrass 558 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGAGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCC AGCCGGCCGG 653 SorFirmi 508 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGAGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU AGCCGGCCGG 603 ChtElatu 537 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGAGGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 632 KicIvori 558 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 653 AuePullu 520 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUU UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 615 Hr0Werne 548 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 643 KirAethi 529 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAA CUUGG-GUCU GGCUGGCAGG 624 OpbHerpo 540 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAA CUUGG-GUCU GACUGGCGGG 635 LehDoli6 509 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAA CUUGG-GUCU GGCUGGCAGG 604 156Elong 533 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAA CUUGG-GUCU GGCUGGCAGG 628 PlpBetae 540 GGUGCCAGCA GCCGC-G-UA AUU-CAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCAGG 633 KirElate 540 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAA CUUGG-GUCU GGCUAGCGGG 635 42MMycop 508 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAA CUUGG-GUCU GGCUAGCGGG 603 21HJunip 540 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAGUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCUGG 635 KirMarit 530 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 625 alongi1189 509 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAU CUUGG-GCCU GGCUGGCCGG 604 996Bipol 509 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GCCU GGCUGGCCGG 604 996Austr 509 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GUCU GGCUGGCCGG 604 MonPurpu 473 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GUCU GGCUGGCCGG 568 243Vari2 527 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GUCU GGCUGGCCGG 622 OngEquin 510 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAN NUUGG-GUUU GGCUGCUCGG 605 750Supe2 544 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GUUU GGCUGCUCGG 639 ObaFimic 510 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GUUU GGCUGCUCGG 605 750Olig2 511 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC CUUGG-GUCU GGCUGCUCGG 606 897Doedy 541 GGUGCCAACA GCCGCGG-UA AUUCCACCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UANUUGAAAC CUUGG-GCCU GGCUGCUCGG 637 ObaDelic 556 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC UUCAG-CCCU GGCUGGGUGG 651 90HRepan 556 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC UUCAG-GCCU GGCCGGGCGG 651 130Nuda2 569 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAGUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUCG- AAC UUCGG-GACU GGCGGGAUGG 664 BulAlbus 561 GGUGCCAGCA GCCGCGG-UA AUUCCAGCUC CAAUAGCGUA UAUUAAAGUU G-UUGCAGUU AAAAAGCUCG UAGUUG- AAC UUUGG-GCCC GGUUGGCCGG 656 SayCe108

701 711 721 731 741 751 761 771 781 791 800 | | | | | | | | | | | 645 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGACCUU UC-CUUCUGG GG-AAUCG-C AUGCCC-UUC ACUGGGUGUG -U-CGA-GGA UCCAGG-ACU 732 TcdMaxil 645 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGACCUU UC-CUUCUGG GG-AAUCG-C AUGCCC-UUC ACUGGGUGUG -U-CGA-GGA UCCAGG-ACU 732 TcdSetig 643 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGACCUU UC-CUUCUGG GG-AAUCG-C AUGCCC-UUC ACUGGGUGUG -U-CGA-GGA UCCAGG-ACU 730 TcdFurca 642 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGACCUU UC-CUUCUGG GG-AAUCG-C AUGCCC-UUC ACUGGGUGUG -U-CGA-GGA UCCAGG-ACU 729 TcdApien 619 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGACCUU UC-CUUCUGG GG-AAUCG-C AUGCCC-UUC ACUGGGUGUG -U-CGA-GGA UCCAGG-ACU 706 Tmarch2639 624 UCCGCCUCAC -CGCGUGCAC UGA---UCCG AUCGGGCCUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 711 LemAqu1 628 UCCGCCUCAC -CGCGUGCAC UGA---UCCG AUCGGGCCUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 715 LemTerr1 248 UCCGCCUCAC -CGCGUGCAC UGA---UCCG ACCGGGCCUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 335 var11783 628 UCCGCCUCAC -CGCGUGCAC UGA---UCCG GCCGGGUCUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 715 TriAng1020 611 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGUCUU UC-CUCCUGG CU-AGCCC-C AUGCCC-UUC ACUGGGUAUG -U-GGG-NGA ACCAGG-ACU 698 Mc8Coral 102 UCCGCCUCAC -CGCGUGCAC UGG---UCCG ACCGGGUCUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 189 HVZ81382 581 UCCGCCUCAC -CGCGUGCAC UGG---UCCG ACCGGGUCUU UC-CUUCUGA GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA AUCAGG-ACU 668 Loram 573 UCCGCCUCAC -CGCGUGCAC UGG---CCCG GCCGGGCCUU UC-CUCCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 660 986Defle 573 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 660 OidTenui 589 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 676 Acrassa055 614 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 701 AngFurt 623 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 710 TriSpl1198 618 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGUCUU UC-CUCCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 705 472Gutta 586 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGUCUU UC-CUCCUGG GG-AGCCA-C AUGCCC-UUC ACUGGGUGUG -U-UGG-GGA ACCAGG-ACU 673 7BGrami 652 UCCGCCUCAC - CGCGUGCAC UGG---UCCG GCCGGGUUUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 739 PiaSpec3 604 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CUCCUAG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACUAGG-ACU 691 164Darwi 594 UCCGCCUCAC -CGCGUGCAC UGG---UCCG ACCGGGCCUU UC-CUUCUAG GG-AGCCG-C AUGCCC-UUC AUUGGGUGUG -U-UGG-GGA ACUAGG-ACU 681 CudConfu 599 UCCGCCUCAC -CGCGUGCAC UGG---UCCG ACCGGGCCUU UC-CUUCUAG GG-AGCCG-C AUGCCC-UUC AUUGGGUGUG -U-CGG-GGA ACUAGG-ACU 686 ShaFlavi 573 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CUCCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 660 989Roseu 572 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CUCCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 659 39GPanno 634 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 721 58BInqui 546 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CUUCUGG GG-ACCCG-C AUGCAC-UUC A--GUGUGUG -UGCUG-GGG ACCAGG-ACU 632 0LLubri 621 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CUUCUGA GG-AGCCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA AUCAGG-ACU 708 13089Alacu 635 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGGCCUU UC-CCUCUGU GG-AACCC-C AUGCCC-UUC ACUGGGUGUG -G-CGG-GGA AACAGG-ACU 722 NerCinn5 634 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CCUCUGU GG-AACCC-C AUGCCC-UUC ACUGGGUGUG -G-CGG-GGA AACAGG-ACA 721 GsaPutte 617 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGGCCUU UC-CCUCUGU GG-AACCU-C AUGCCC-UUC ACUGGGCGUG -G-CGG-GGA AACAGG-ACU 704 GibPuli5 633 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CCUCUGC GG-AACCC-C AUGCCC-UUC ACUGGGUGUG -G-CGG-GGA AACAGG-ACU 720 HpaLute3 604 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGGCCUU UC-CCUCUGU GG-AACCC-C AUGCCC-UUC ACUGGGUGUG -G-CGG-GGA AACAGG-ACU 691 HymChrys 626 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGGCCUU UC-CCUCUGU GG-AACCC-C AUGCCC-UUC ACUGGGUGUG -G-CGG-GGA AACAGG-ACU 713 Hlug13783 606 UCCCCCUCAC -CGGGUGCAC UGA---UCCA GCCGGGCCUU UC-CCUCUGU GG-AACCC-C AUGGCC-UUC ACUGGCUGUG -G-UGG-GGA AACAGG-ACU 693 144Seti3 606 UCCCCCUCAC -CGGGUGCAC UGA---UCCA GCCGGGCCUU UC-CCUCUGU GG-AACCC-C AUGGCC-UUC ACUGGCCGUG -G-CGG-GGA AACAGG-ACU 693 PsaBoyd6 635 UCCCCCUCAC -CGGGUGCAC UGA---UCUG GCCGGGUCUU UC-CCUCCGC GG-AACCC-C AUGGCC-UUC ACUGGUCGUG -G-CGG-GGA AACGGG-ACU 722 98HRetor 604 UCCCCCUCAC -CGGGUGCAC UGA---UCCG GCCGGGCCUU UC-CCUCUGU GG-AACCC-C AUGGCC-UUC ACUGGCUGUG -- C GGG-GGA AACAGG-ACU 691 MiaCirro 629 UCCCC-UCAC -CGGGUGCAC UGG---UCCG GCCGGGCCUU UC-CCUCUGU GG--ACCG-C AUGCCC-UUC ACUGGGUGUG -C-CGG-GGA AACAGG-ACU 714 GmmPeni3

Figure A36 continued

203 Appendix

Alignment SSU sequences continued

653 UCCGCCUCAC -CGCGUGCAC UGA---CUGG GUCGGGCCUU UU-UUCCUGG AG-AACCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 740 NeuCrass 654 UCCGCCUCAC -CGCGUGCAC UGG---CUCG GUUGGGCCUU UC-CUUCUGG AG-AACCG-C AUGCCC-UUC ACUGGGUGUG -U-CGG-GGA ACCAGG-ACU 741 SorFirmi 604 UCCGCCUCAC -CGCGUGCAC UGG---CUCG GCUGGGUCUU UC-CUUCUGG AG-AACCU-C AUGCCC-UUC ACUGGGUGUG -A-CGG-GGA ACCAGG-ACU 691 ChtElatu 633 UCCGCCUCAC -CGCGUGCAC UGG---UCCG GCCGGGCCUU UC-CCUCUGG GG-AACCG-C AUGCCC-UUC GCUGGGUGUG -C-CGG-GGA ACCAGG-ACU 720 KicIvori 654 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGGCCUU UC-CUUCUGG GG-AGCCG-C AUGCCC-UUC ACUGGGCGUG -U-CGG-GGA ACCAGG-ACU 741 AuePullu 616 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGGCCUU UC-CUUCUGG GG-AGCCG-C AUGGCC-UUC ACUGGCCGUG -U-CGG-GGA ACCAGG-ACU 703 Hr0Werne 644 UCCGCCUCAC -CGCGUGCAC UGG---UUCG GCCGGGCCUU UC-CUUCUGG CG-AGCCA-C AUGCCC-UUC ACUGGGCGUG -C-UGG-GGA ACCAGG-ACU 731 KirAethi 625 UCCGCCUCAC -CGCGUGUAC UUG---UCCG GCCGGGCCUU -C-CUUCUGG AG-AACCU-C AUGCCC-UUC ACUGGGCGUG -U-UGG-GG- ACCAGG-ACU 710 OpbHerpo 636 UCCGCCUCAC -CGCGUGUAC UUG---UCCG GCCGGGCCUU -C-CUUCUGG AG-AACCU-C AUGCCC-UUU ACUGGGCGUG -U-UGG-GG- ACCAGG-ACU 721 LehDoli6 605 UCCGCCUCAC -CGCGUGUAC UUG---UCCG GCCGGGCCUU -C-CUUCUGG AG-AACCU-C AUGCCC-UUC ACUGGGCGUG -U-UGG-GG- ACCAGG-ACU 690 156Elong 629 UCCGCCUCAC -CGCGUGUAC UUG---UCCG GCCGGGCCUU -C-CUUCUGG AG-AACCU-C AUGCCC-UUC ACUGGGCGUG -U-UGG-GG- ACCAGG-ACU 714 PlpBetae 634 UCCGCCUCAC -CGCGUGUAC UUG---ACCG GCCGGGCCUU CU- CUUCUGG AG-AACCU-C AUGCCC-UUU ACUGGGCGUG -U-UGG-GG- ACCAGG-ACU 720 KirElate 636 UCCGCCUCAC -CGCGUGCAC UUG---UCCG GCUGGACCUU UC-CUUCUGG AG-AACCU-C AUGCCC-UUC ACUGGGUGUG -U-UGG-GGA ACCAGG-ACU 723 42MMycop 604 UCCGCCUCAC -CGCGUGCAC UUG---UCCG GCUGGACCUU UC-CUUCUGG AG-AACCU-C AUGCCC-UUC ACUGGGUGUG -U-UGG-GGA ACCAGG-ACU 691 21HJunip 636 UCCGCCUCAC -CGCGUGUAC UGG---UCCG GCCGGGCCUU UC-CUUCUGG AG-AGCCC-C AUGCCC-UUC ACUGGGUGUG -C-GGG-GGA ACCAGG-ACU 723 KirMarit 626 UCCGCCUCAC -CGCGUGUAC UGG---UC-G GCCGGGCCUU UC-CUUCUGG AG-AACCC-C AUGCCC-UUC ACUGGGUGUG -C-GGG-GGA ACCAGG-ACU 712 alongi1189 605 UCCGCCUCAC -CGCGUGUAC UGG---UCUG GCCGGGCCUU UC-CUUCUGG AG-AACCU-C AUGCCC-UUC AUUGGGUGUG -U-UGG-GGA ACCAGG-ACU 692 996Bipol 605 UCCGCCUCAC -CGCGUGUAC UGG---UCUG GCCGGGCCUU UC-CUCCUGG AG-AACCU-C AUGCCC-UUC AGUGGGUGUG -U-UGG-GGA ACCAGG-ACU 692 996Austr 605 UCCGCCUCAU -CGCGAGUAC UGG---UCCG GCCGGACCUU UC-CUUCUGG GG-AACCU-C AUGGCC-UUC ACUGGCUGUG -G--GG-GGA ACCAGG-ACU 691 MonPurpu 569 UCCGCCUCAC -CGCGAGUAC UGG---UCCG GCUGGACCUU UC-CUUCUGG GG-AACCC-C AUGGCC-UUC ACUGGCCGUG -G-CGG-GGA ACCAGG-ACU 656 243Vari2 623 UCCGCUUCGC -GGCGUGCAC UGG---UCCG GCUGGACCUU UC-CUUCUGG GG-AACCC-U AUGGCC-UUC ACUGGCUGUA -G--GG-GGA ACCAGG-ACU 709 OngEquin 606 UCCGCCUAAC -CGCGUGCAC UGA---UGCG GCCGGAUCUU UC-UUUCUGG CC-AACCU-C AUGCCCUUUC AUUGGGUGUG -U-UGG-GGA UCCAGG-ACU 694 750Supe2 640 UCCGCCUAAC -CGCGUGAAC UGA---UGCG GCCGGAUCUU UC-CUUCUGG CC-AACCU-C AUGCCC-UUU AUUGGGUGUG -C-UGG-GGA UCCAGG-ACU 727 ObaFimic 606 UCCGCCUAAC -CGCGUGCAC UGA---UGCG GCCGGAUCUU UC-CUUCUGG CU-AACCU-C AUGCCC-UUU ACUGGGUGUG -C-UGG-GGA UCCAGG-ACU 693 750Olig2 607 UCCGCCUAAC -CGCGUGCAC UGA---UGCG GCCGGAUCUU UC-CUUCUGG CC-AACCC-C AUGCCC-UUU ACUGGGUGUG -G-UGG-GGA UCCAGG-ACU 694 897Doedy 638 UCCGCCUAAC -CGCGUGCAC UGA---UGCG GCCGGGCCUU UC-CUUCUGG CU-AACCU-C AUGCCC-UUC ACUGGGUGUG -C-UGG-GGA UCCAGGAACU 726 ObaDelic 652 UCUGCCUCAC -GGUAUGUAC UGU---C-UG GCUGGGGCUU AC-CUCUUGG UG-AGCUUGC AUGCCG-UUC AUUCGGUGUG -- C AAG-GGA ACCAGG-ACU 739 90HRepan 652 UCCGCCUCAC -GGUGUGUAC UGU---C-UG GCUGGGUCUU AC-CUCUUGG UG-AACCUGC AUGCCC-UUC ACUGGGUGUG -- U AGG-GGA ACCAGG-ACU 739 130Nuda2 665 UCCGCUU-AC -GGUGUGUAC UGU---C-UG GCCGGGCCUU AC-CUCUUGG UGAGGCC-GU AUGCCC-UUU ACUGGGUGUG -C--GGUGGA ACCAGGAA-U 751 BulAlbus 657 UCCGAUUUU- -UUCGUGUAC UGGAUUUCCA ACGGGGCCUU UC-CUUCUGG CU-AACCU-- UGAGUC-CUU G-UGGCUCU- -U--GG-CGA ACCAGG-ACU 742 SayCe108 801 811 821 831 841 851 861 871 881 891 900 | | | | | | | | | | | 733 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA- GGACG UGUG-GUUCU AUUUU-GUUG 826 TcdMaxil 733 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA- GGACG UGUG-GUUCU AUUUU-GUUG 826 TcdSetig 731 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA- GGACG UGUG-GUUCU AUUUU-GUUG 824 TcdFurca 730 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA- GGACG UUUG-GUUCU AUUUU-GUUG 823 TcdApien 707 UUUACU.UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUUGGUUG 801 Tmarch2639 712 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUGA AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 805 LemAqu1 716 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUGA AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 809 LemTerr1 336 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 429 var11783 716 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG- GUUCU AUUUU-GUUG 809 TriAng1020 699 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCAU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 792 Mc8Coral 190 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU UCAUUAGCAU GGAAUAAUAG ARUA-GGACG UGYG-GUCCU AUUUUGGUUG 284 HVZ81382 669 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 762 Loram 661 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 754 986Defle 661 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 754 OidTenui 677 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 770 Acrassa055 702 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 795 AngFurt 711 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 804 TriSpl1198 706 UUUACU-UUG AAAAAAUUAG AGUGUUCCAA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 800 472Gutta 674 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 767 7BGrami 740 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 833 PiaSpec3 692 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG CGCA-GUCUU AUUUU-GUUG 785 164Darwi 682 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 775 CudConfu 687 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 780 ShaFlavi 661 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 754 989Roseu 660 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 753 39GPanno 722 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 815 58BInqui 633 UUUAC--UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 725 0LLubri 709 UUUACU-UUG AAAAAAUUAG AGUGUUC.AA AGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU- GUUG 802 13089Alacu 723 UUUACU-UUG AAAAAAUUAG AGUGCUC-CA GGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAA AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 816 NerCinn5 722 UUUACU-UUG AAAAAAUUAG AGUGCUC-CA GGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAA AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 815 GsaPutte 705 UUUACU-GUG AAAAAAUUAG AGUGCUC-CA GGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUU--GUUG 797 GibPuli5 721 UUUACU-UUG AAAAAAUUAG AGUGCUC-AA GGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 814 HpaLute3 692 UUUACU-UUG AAAAAAUUAG AGUGCUC-AA GGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAA AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 785 HymChrys 714 UUUACU-UUG AAAAAAUUAG AGUGCUC-CA GGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 807 Hlug13783 694 UUUACU-UUG AAAAAAUUAG AGUGCUC-CA GGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 787 144Seti3 694 UUUACU-UUG AAAAAAUUAG AGUGCUC-CA GGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 787 PsaBoyd6 723 UUUACU-UUG AAAAAAUUAG AGUGCUC-CA GGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAA AAUA-GGACG CGUG-GUUCU AUUUU-GUUG 816 98HRetor 692 UUUACU-GUG AAAAAAUUAG AGUGCUC-CA GGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG CGUG-GUUCU AUUUU-GUUG 785 MiaCirro 715 UUUACU-UUG AAAAAAUUAG AGUGCUC-CA GGCAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 808 GmmPeni3 741 UUUACC-GUG AACAAAUCAG AUCGCUC-AA AGAAGGCCU- AUGCUCGAAU GUACUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 834 NeuCrass 742 UUUACU-CUG AACAAAUUAG AUCGCUU-AA AGAAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 835 SorFirmi 692 UUUACU-CUG AACAAAUUAG AUCGCUU-AA AGAAGGCCU- AUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGUG-GUUCU AUUUU-GUUG 785 ChtElatu 721 UUUACU-CUG AACAAAUCAG AUCGCUC-AA AGAAGGCUC- UCGCUCGAAU GCAUUAGCAU GGAAUAAUGG AAUA-GGACG UGCG-GUCCU AUUUU-GUUG 814 KicIvori 742 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUUCU AUUUU-GUUG 835 AuePullu 704 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-AUCCU AUUUG-GUUG 797 Hr0Werne 732 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG -GCG-GUCCU AUUUU-GUUG 824 KirAethi 711 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACGUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUCCU AUUUU-GUUG 804 OpbHerpo 722 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACGUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUCCU AUUUU-GUUG 815 LehDoli6 691 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACGUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUCCU AUUUU-GUUG 784 156Elong 715 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACGUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-UUCCU AUUUU-GUUG 808 PlpBetae 721 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU AUGUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUCCU AUUUU-GUUG 814 KirElate 724 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACGUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUCCU AUUUU-GUUG 817 42MMycop 692 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACGUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUCCU AUUUU-GUUG 785 21HJunip 724 UUUACU-GUG AAAAAAUUAG AGUGUUU-AA AGCAGGCCU- UUGCUCGAAU ACGUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUCCU AUUUU-GUUG 817 KirMarit 713 UUUACU-GUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACGUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-AUCCU AUUUU-GUUG 806 alongi1189 693 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACGUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUCCU AUUUU-GUUG 786 996Bipol 693 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACGUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUCCU AUUUU-GUUG 786 996Austr 692 UUUACU-GUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUUCU AUUUU-GUUG 785 MonPurpu 657 UUUACU-GUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUUCU AUUUU-GUUG 750 243Vari2 710 UUUACU-GUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGGAU ACAUUAGCAU GGAAUAAUAG AAUA-GGAUG UGUG-GUUCU AUUUU-GUUG 803 OngEquin 695 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG -GAG-GUUCU AUUUU-GUUG 787 750Supe2 728 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG -GAG-GUUCU AUUUU-GUUG 820 ObaFimic 694 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG -GAG-GUUCU AUUUU-GUUG 786 750Olig2 695 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- UUGCUCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG -GCG-GUUCU AUUUU-GUUG 787 897Doedy 727 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCCU- AUGCUCGGAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG -GCG-GUUCU AUUUU-GUUG 819 ObaDelic 740 UUUACU-UUG AGAAAAUUAG AGUGUUC-AA AGCAGGCAA- AUGCCCGAAU ACAUUAGCAU GGAAUAAUGG AAUA-NGACG UGCG-GUUCU AUUUU-GUUG 833 90HRepan 740 UUUACC-UUG AGAAAAUUAG AGUGUUC-AA AGCAGGCCU- ACGCCCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUCCU AUUUU-GUUG 833 130Nuda2 752 UUUACC-UUG AGAAAAUUAG AGUGUUC-AA AGCAGGCAU- ACGCCCGAAU ACAUUAGCAU GGAAUAAUAG AAUA-GGACG UGCG-GUUCU AUUUU-GUUG 845 BulAlbus 743 UUUACU-UUG AAAAAAUUAG AGUGUUC-AA AGCAGGCGUA UUGCUCGAAU AUAUUAGCAU GGAAUAAUAG AAUA-GGACG UUUG-GUUCU AUUUU-GUUG 837 SayCe108

Figure A36 continued

204 Appendix

Alignment SSU sequences continued

901 911 921 931 941 951 961 971 981 991 1000 | | | | | | | | | | | 827 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 922 TcdMaxil 827 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 922 TcdSetig 825 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 920 TcdFurca 824 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGUAUGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 919 TcdApien 802 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG .GGGCAUCAG UAUUCAAUUG UC-A-GAGGU CAAAUUCUUG GAUUUAUUGA AGACUAACUA 897 Tmarch2639 806 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUC GAUUUAUUGA AGACUAACUA 901 LemAqu1 810 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUGUUG GAUUUAUUGA AGACUAACUA 905 LemTerr1 430 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 525 var11783 810 GUUUCUAGGA CCGCCGUAAU GAUGAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCCAACG UC-A-GAGGU GAAAUUCUUG GAUUGUUGGA AGACUAACUA 905 TriAng1020 793 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGUCAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 888 Mc8Coral 285 GUUUCUAGGA CCGCCGUAAU GAUUAAUKAG GGMUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUAG GAUKUAKUGA AGACUAMCUA 381 HVZ81382 763 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGAUAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 858 Loram 755 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 850 986Defle 755 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 850 OidTenui 771 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A- GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 866 Acrassa055 796 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A- GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 891 AngFurt 805 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 900 TriSpl1198 801 GUUUCUCGGA CCGCC-UAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 895 472Gutta 768 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 863 7BGrami 834 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 929 PiaSpec3 786 GUUUCUAAGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 881 164Darwi 776 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGUGUCAG UAUUGCGUUG UC-A-GAGGU GAAAUUCUUG GAUUUACGCA AGACUAACUA 871 CudConfu 781 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGUGUCAG UAUUGCGUUG UC-A-GAGGU GAAAUUCUUG GAUUUACGCA AGACUAACUA 876 ShaFlavi 755 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 850 989Roseu 754 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 849 39GPanno 816 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 911 58BInqui 726 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 821 0LLubri 803 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC.A- GAGGU GAAAUUCUUG CAUUUAUUGA AGACUAACUA 898 13089Alacu 817 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCCGUUG UC-A-GAGGU GAAAUUCUUG GAUUUACGGA AGACUAACUA 912 NerCinn5 816 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 911 GsaPutte 798 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 893 GibPuli5 815 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 910 HpaLute3 786 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 881 HymChrys 808 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCAUG GAUUUAUUGA AGACUAACUA 903 Hlug13783 788 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUCUAUUGA AGACUAACUA 883 144Seti3 788 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUCUAUUGA AGACUAACUA 883 PsaBoyd6 817 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUCUAUUGA AGACUAACUA 912 98HRetor 786 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUCUAUUGA AGACUAACUA 881 MiaCirro 809 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUCUAUUGA AGACUAACUA 904 GmmPeni3 835 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 930 NeuCrass 836 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 931 SorFirmi 786 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 881 ChtElatu 815 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCCG UAUUCAGUUG UC-A-GAGGU GAAAUUCUUG GAUCUACUGA AGACGAACUA 910 KicIvori 836 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAYUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 931 AuePullu 798 GUUUUUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 893 Hr0Werne 825 GUUUCUAGAA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCGUCCG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACGAACUA 920 KirAethi 805 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 900 OpbHerpo 816 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 911 LehDoli6 785 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 880 156Elong 809 GUUUCUAGGA ACGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 904 PlpBetae 815 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 910 KirElate 818 GUUUCUAGGA UCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 913 42MMycop 786 GUUUCUAGGA UCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 881 21HJunip 818 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 913 KirMarit 807 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A- GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 902 alongi1189 787 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A- GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 882 996Bipol 787 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A- GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 882 996Austr 786 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCGUCAG UAUUCAGCUG UC-A-GAGGU GAAAUUCUUG GAUUUGCUGA AGACUAACUA 881 MonPurpu 751 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCGUCAG UAUUCAGCUG UC-A-GAGGU GAAAUUCUUG GAUUUGCUGA AGACUAACUA 846 243Vari2 804 GUUUCUAGGA CCGCCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCGUCAG UAUUCGGCUG UC-A-GAGGU GAAAUUCUUG GAUUUGCUGA AGACUAACUA 899 OngEquin 788 GUUUCUAGAG CCACCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 883 750Supe2 821 GUUUCUAGAG CCACCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 916 ObaFimic 787 GUUUCUAGAG CCACCGUAAU GAUUAA-UAG GGACAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 882 750Olig2 788 GUUUCUAGAG CCACCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 883 897Doedy 820 GUUUCUAGAG CCACCGUAAU GAUUAA-UAG GGAUAGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 915 ObaDelic 834 GUUUCUANAN UCNCCGUAAU GAUUAA-UAG GGACAGUUGG -GGGCAUUAG UAUUGAGUUG CU-A-GAGGU GAAAUUCUUG GAUUUACUCA AGACUAACUA 929 90HRepan 834 GUUUCUAGGA UCGCCGUAAU GAUAAA-UAG GGACAGUUGG -GGGCAUUUG UAUUGCGUCG CU-A-GAGGU GAAAUUCUUG GAUUGACGCA AGACAAACUA 929 130Nuda2 846 GUUUCUAGGA UCGCCGUAAU GAUUAA-UAG GGACGGUCGG -GGGCAUUAG UAUUCAGUUG CU-A-GAGGU GAAAUUCUUA GAUUUACUGA AGACUAACUU 941 BulAlbus 838 GUUUCUAGGA CCAUCGUAAU GAUUAA-UAG GGACGGUCGG -GGGCAUCAG UAUUCAAUUG UC-A-GAGGU GAAAUUCUUG GAUUUAUUGA AGACUAACUA 933 SayCe108

1001 1011 1021 1031 1041 1051 1061 1071 1081 1091 1100 | | | | | | | | | | | 923 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1013 TcdMaxil 923 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1013 TcdSetig 921 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1011 TcdFurca 920 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1010 TcdApien 898 CUGCGAA.A- GCAUU-UGCC AA-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG.AU ACCGUCGUAG U-CUUAACCA 989 Tmarch2639 902 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 992 LemAqu1 906 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 996 LemTerr1 526 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UCAAGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 617 var11783 906 CUGCGAA-A- GCAUU-UGCC -A.AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 996 TriAng1020 889 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 979 Mc8Coral 382 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 472 HVZ81382 859 CUGCGAA-A- GCAUU-UAUC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 949 Loram 851 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 941 986Defle 851 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 941 OidTenui 867 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 957 Acrassa055 892 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 982 AngFurt 901 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 991 TriSpl1198 896 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGCGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 986 472Gutta 864 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 954 7BGrami 930 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1020 PiaSpec3 882 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGGGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 972 164Darwi 872 CUGCGAA-A- GCAUU-CACC -A-AGGAUGN UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 962 CudConfu 877 CUGCGAA-N- NNAUU-CACC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 967 ShaFlavi 851 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 941 989Roseu 850 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 940 39GPanno 912 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1002 58BInqui 822 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 912 0LLubri 899 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU.CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 989 13089Alacu 913 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1002 NerCinn5 912 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1001 GsaPutte 894 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 983 GibPuli5 911 CUGCGAA-A- CGAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1000 HpaLute3 882 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 971 HymChrys 904 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 993 Hlug13783 884 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUGA UA-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACUA 973 144Seti3 884 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUGA UA-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACUA 973 PsaBoyd6 913 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUGA UA-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACUA 1002 98HRetor 882 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUGA UA-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACUA 971 MiaCirro 905 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUGA UA-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACUA 994 GmmPeni3 931 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1020 NeuCrass 932 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1021 SorFirmi

Figure A36 continued

205 Appendix

Alignment SSU sequences continued

882 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 971 ChtElatu 911 CUGCGAA-A- GCAUUUUGCC -A-AGGAUGU UUU-CAUUAA UC-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1001 KicIvori 932 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1022 AuePullu 894 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 984 Hr0Werne 921 CUGCGAA-A- GCAUU-CGCC -A-AGGAUGU UUU-CAUUAA UC-AG-GAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCG 1010 KirAethi 901 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCG 991 OpbHerpo 912 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCG 1002 LehDoli6 881 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCG 971 156Elong 905 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCG 995 PlpBetae 911 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1001 KirElate 914 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCG 1004 42MMycop 882 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCG 972 21HJunip 914 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCG 1004 KirMarit 903 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCG 993 alongi1189 883 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCG 973 996Bipol 883 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCG 973 996Austr 882 CUGCGAA-A- GCAUU-CGCC -A-AGGAUGU UUU-CAUUAA UC-AGGGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 972 MonPurpu 847 CUGCGAA-A- GCAUU-CGCC -A-AGGAUGU UUU-CAUUAA UC-AGGGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 937 243Vari2 900 CUGCGAA-A- GCAUU-CGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 990 OngEquin 884 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 974 750Supe2 917 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1007 ObaFimic 883 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG UGCUUAACCA 974 750Olig2 884 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 974 897Doedy 916 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AGUGAAC GAAAGUUAGG GGAUCGAAGA CGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1006 ObaDelic 930 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AA-GAAC GAAGGUUAGG GGAUCGAAAA CGAUCAG-AU ACCGUUGUAG U-CUUAACAG 1019 90HRepan 930 CUGCGAA-A- GCAUU-UGCC -A-AGGAUGU UUU-CAUUAA UC-AA-GAAC GAAGGUUAGG GGAUCGAAAA CGAUCAG-AU ACCGUUGUAG U-CUUAACAG 1019 130Nuda2 942 CUGCGAA-A- GCAUU-UGCC -A-AGGACGU UUU-CAUUGA UC-AA-GAAC GAAGGUUAGG GGAUCAAAAA CGAUUAG-AU ACCGUUGUAG U-CUUAACAG 1031 BulAlbus 934 CUGCGAA-A- GCAUU-UGCC -A-AGGACGU UUU-CAUUAA UC-AA-GAAC GAAAGUUAGG GGAUCGAAGA UGAUCAG-AU ACCGUCGUAG U-CUUAACCA 1023 SayCe108 1101 1111 1121 1131 1141 1151 1161 1171 1181 1191 1200 | | | | | | | | | | | 1014 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-ACUUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1106 TcdMaxil 1014 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-ACUUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1106 TcdSetig 1012 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-ACUUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1104 TcdFurca 1011 U-AAACUAUG CCGACUAGGG AUCGGGCCAU GU-U-ACUUU UUU-GAC-UC GCUCGCAACC -UUACCAGAA CUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1103 TcdApien 990 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-ACUUU UUU-GAC.UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1082 Tmarch2639 993 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUUUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGGAGU 1085 LemAqu1 997 U-AAA-CAUG CCGACUAGGG AUCGGCCGAU GUAU-AUUUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UUUGGGUUCU GGGGGGAGUA 1090 LemTerr1 618 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUUUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 710 var11783 997 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUUUU UUU-GAC-UC GCUCGGCACC .UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1089 TriAng1020 980 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1072 Mc8Coral 473 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGG...... 558 HVZ81382 950 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU ...... 1032 Loram 942 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-GC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1034 986Defle 942 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1034 OidTenui 958 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1050 Acrassa055 983 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGAG 1075 AngFurt 992 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1084 TriSpl1198 987 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUUUU UUUUGAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1080 472Gutta 955 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUUUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1047 7BGrami 1021 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUUUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1113 PiaSpec3 973 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1065 164Darwi 963 U-AAACUAUG CCGACUAGGG AUCAGGCGAU GU-U-AUCUU UUU-GAC-UC GCUUGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1055 CudConfu 968 U-AAACUAUG CCGACUAGGG AUCAGGCGAU GU-U-AUCUU UUU-GAC-UC GCUUGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1060 ShaFlavi 942 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1034 989Roseu 941 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUUUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1033 39GPanno 1003 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1095 58BInqui 913 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUGCGAGAA AUCAAAGUUU CU-GGGUUCU GGGGGGAGUA 1005 0LLubri 990 U.AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCUU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1082 13089Alacu 1003 U-AAACUAUG CCGACUAGGG AUCGGACGAU GU-U-AUUUA UU--GAC-UC GUUCGGCACC -UUACGAGAA AUCAAAGUGC UU-GGGCUCC AGGGGGAGUA 1094 NerCinn5 1002 U-AAACUAUG CCGACUAGGG AUUGGACGAU GU-U-AUUUU UU--GAC-UC GUUCAGCACC -UUACGAGAA AUCAAAGUGC UU-GGGCUCC AGGGGGAGUA 1093 GsaPutte 984 U-AAACUAUG CCGACUAGGG AUCGGACGGU GU-U-AUUUU UU--GAC-CC GUUCGGCACC -UUACGAGAA AUCAAAGUGC UU-GGGCUCC AGGGGGAGUA 1075 GibPuli5 1001 U-AAACUAUG CCGACUAGGG AUCGGACGAU GU-U-ACAUU UUU-GAC-GC GUUCGGCACC -UUACGAGAA AUCAAAGUGC UU-GGGCUCC AGGGGGAGUA 1093 HpaLute3 972 U-AAACUAUG CCGACUAGGG AUCGGACGAU GU-U-ACAUU UUU-GAC-GC GUUCGGCACC -UUACGAGAA AUCAAAGUGC UU-GGGCUCC AGGGGGAGUA 1064 HymChrys 994 U-AAACUAUG CCGACUAGGG AUCGGACGAU GU-U-AUUUU UU--GAC-UC GUUCGGCACC -UUACGAGAA AUCAAAGUGC UU-GGGCUCC AGGGGGAGUA 1085 Hlug13783 974 U-AAACUAUG CCGACUAGGG AUCGGACGAU GU-U-AUUCU UU--GAC-GC GUUCGGCACC -UUUCGAGAA AUCAAAGUGC UU-GGGCUCC AGGGGGAGUA 1065 144Seti3 974 U-AAACUAUG CCGACUAGGG AUCGGACGAU GU-U-AUUUC UU--GAC-GC GUUCGGCACC -UUUCGAGAA AUCAAAGUGC UU-GGGCUCC AGGGGGAGUA 1065 PsaBoyd6 1003 U-AAACUAUG CCGACUAGGG AUCGGACGAU GU-U-GUUUU UU--GAC-UC GUUCGGCACC -UUUCGAGAA AUCAAAGUGC UU-GGGCUCC AGGGGGAGUA 1094 98HRetor 972 U-AAACUAUG CCGACUAGGG AUCGGACGAU GU-U-AUUUC UU--GAC-GC GUUCGGCACC -UUUCGAGAA AUCAAAGUGC UU-GGGCUCC AGGGGGAGUA 1063 MiaCirro 995 U-AAACUAUG CCGACUAGGG AUCGGACGGU GU-U-UAUAC UU--GAC-CC GUUCGGCACC -UUUCGAGAA AUCAAAGUGC UU-GGGCUCC AGGGGGAGUA 1086 GmmPeni3 1021 U-AAACUAUG CCGAUUAGGG AUCGGACGGU GU-U-AUUUU UU--GAC-CC GUUCGGCACC -UUACGAUAA AUCAAAAUGU UU-GGGCUCC UGGGGGAGUA 1112 NeuCrass 1022 U-AAACUAUG CCGAUUAGGG AUCGGACGAU GU-U-AUUUU UU--GAC-UC GUUCGGCACC -UUACGAUAA AUCAAAAUGU UU-GGGCUCC UGGGGGAGUA 1113 SorFirmi 972 U-AAACUAUG CCGAUUAGGG AUCGGACGGC GU-U-AUUUU UU--GAC-CC GUUCGGCACC -UUACGAUAA AUCAAAAUGU UU-GGGCUCC UGGGGGAGUA 1063 ChtElatu 1002 U-AAACUAUG CCGAUUAGGG AUCGGACGAU GC-U-AUUUU UU--GGC-UC GUUCGGCACC -UUACGACAA AUCAAAAUGU UU-GGGCUCC UGGGGGAGUA 1093 KicIvori 1023 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-AUCAU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1115 AuePullu 985 U-AAACUAUG CCGACUAGGG AUCGGUGGAU GU-U-ACUAU UAU-GAC-UC CAUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1077 Hr0Werne 1011 U-AAACCAUG CCGACUAGGG AUCGGGCGAU GU-U-AUUUG GUU-GAC-UC GCCCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1103 KirAethi 992 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-CUUUU UCU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1084 OpbHerpo 1003 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-CUUUU UCU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1095 LehDoli6 972 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-CUUUU UCU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1064 156Elong 996 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-CUUUU UCU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1088 PlpBetae 1002 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-UCAAU AUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUGU UU-GGGUUCU GGGGGGAGUA 1094 KirElate 1005 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-UCUAU CUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1097 42MMycop 973 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-UCUAU CUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1065 21HJunip 1005 U-AAACUAUG CCGACUAGGG AUUGGGCGAU GU-U-UCUAU UUU-GAC-UC GCUCAGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1097 KirMarit 994 U-AAACUAUG CCGACUGGGG AUCGGGCGAU GU-U-UCUAU CUU-GAC-UC GCUCGGCACC -CUAAGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1086 alongi1189 974 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-UCAAU UUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAG.. 1064 996Bipol 974 U-AAACUAUG CCGACUAGGG AUCGGGCGAU GU-U-UCUAU CUU-GAC-UC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAG.. 1064 996Austr 973 U-AAACUAUG CCGACUAGGG AUCGGACGG. GU-U-UCUAU GAU-GAC-CC GUUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1064 MonPurpu 938 U-AAACUAUG CCGACUAGGG AUCGGACGGU GU-U-UCUAU GAU-GAC-CC GUUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1030 243Vari2 991 U-AAACUAUG CCGACUAGGG AUCGGACGG- GC-AACUUUG AAU-AAC-CC GUUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1083 OngEquin 975 U-AAACUAUG CCGACUAGGG AUCGGGCGGU GU-U-CAACU UAU-GAC-CC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1067 750Supe2 1008 U-AAACUAUG CCGACUAGGG AUCGGGCGGU GU-U-CAACU UAU-GAC-CC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1100 ObaFimic 975 U-AAACUAUG CCGACUAGGG AUCGGGCGGU GU-U-CAACU UAU-GAC-CC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1067 750Olig2 975 U-AAACUAUG CCGACUAGGG AUCGGGCGGU GU-U-CAAUU UAU-GAC-CC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1067 897Doedy 1007 U-AAACUAUG CCGACUAGGG AUCGGGCGGU GU-U-UAACU UAU-GAC-CC GCUCGGCACC -UUACGAGAA AUCAAAGUUU UU-GGGUUCU GGGGGGAGUA 1099 ObaDelic 1020 U-AAACUAUG CCGACUAGGG AUCGGGCGAA CU-C-AAGUU UAU-GUG-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1112 90HRepan 1020 U-AAACUAUG CCGACUAGGG AUCGGGCGAC CU-C-GAAUU CAU-GUG-UC GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1112 130Nuda2 1032 U-AAACUAUG CCGACUAGGG AUCGGGCCAC GU-U--AUUU UUU-GAC-UG GCUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1123 BulAlbus 1024 U-AAACUAUG CCGACUAGGG AUCGGGUGGU GU-U-UUUUU AAU-GAC-CC ACUCGGCACC -UUACGAGAA AUCAAAGUCU UU-GGGUUCU GGGGGGAGUA 1116 SayCe108 1201 1211 1221 1231 1241 1251 1261 1271 1281 1291 1300 | | | | | | | | | | | 1107 UGG-UCGCAA GGCUGA-AAC UUAA-AGGAA UUGACGGAA- GAGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1193 TcdMaxil 1107 UGG-UCGCAA GGCUGA-AAC UUAA-AGGAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1193 TcdSetig 1105 UGG-UCGCAA GGCUGAA-AC UUAA-AGGAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1191 TcdFurca 1104 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GNCUUAAUU UGACUCAAC- AC-GGGG-AA 1190 TcdApien 1083 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1169 Tmarch2639 1086 AUG-GUCGCA AGCUGAA-AC UUAA-AGAAA UUGAC-GAA- GGGCA-C-C- ACCCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGA-AA 1172 LemAqu1

Figure A36 continued

206 Appendix

Alignment SSU sequences continued

1091 UGG-UCGCAA GGCUGA--AC UUAA-AGAAA UUGAC-GAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC GGGCUUAAUU UGACUCAAC- AC-GGGG-AA 1176 LemTerr1 711 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUG...... 740 var11783 1090 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGAU-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1176 TriAng1020 1073 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGNCA-C-C- A-CCAGGA-G UGGNNNNNNN -NNNNNNNNN NNNNNNNNN- NN-NNNN-NN 1159 Mc8Coral 559 ...... 558 HVZ81382 1033 ...... 1032 Loram 1035 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1121 986Defle 1035 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1121 OidTenui 1051 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1137 Acrassa055 1076 UAUGUCGCAA GGCUGAA-AC UUAAAAGAAA UUGACGGAA- GG-CA-C-C- A-CCAAGAAG UGGAGCCUGC -GGGUUUAAU UUACUCAAC- AC-GGGG--A 1163 AngFurt 1085 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1171 TriSpl1198 1081 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU CGACUCAAC- AC-GGGG-AA 1167 472Gutta 1048 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G U-GAGCCUGC --GCUUAAUU UGACUCAAC- AC-GGGG-AA 1132 7BGrami 1114 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1200 PiaSpec3 1066 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1152 164Darwi 1056 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGANNGUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1142 CudConfu 1061 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGANNNUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1147 ShaFlavi 1035 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1121 989Roseu 1034 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGC-UGC -GGCUUAAUU UGACUCAAC- AC-GGG--AA 1118 39GPanno 1096 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1182 58BInqui 1006 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1092 0LLubri 1083 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA. GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1169 13089Alacu 1095 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1181 NerCinn5 1094 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1180 GsaPutte 1076 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1162 GibPuli5 1094 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1180 HpaLute3 1065 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1151 HymChrys 1086 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1172 Hlug13783 1066 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAACCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1152 144Seti3 1066 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAACCUNC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1152 PsaBoyd6 1095 UGG-UCGCAA GGCUGAA-AC UUAA-AGGAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAACCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1181 98HRetor 1064 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAACCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1150 MiaCirro 1087 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G U-GAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1172 GmmPeni3 1113 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1199 NeuCrass 1114 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1200 SorFirmi 1064 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1150 ChtElatu 1094 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGG-G UGG-GACUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1179 KicIvori 1116 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1202 AuePullu 1078 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1164 Hr0Werne 1104 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1190 KirAethi 1085 UGG-UCGCAA GGCUGA--AC UUAA-AGGAA UUGACGGAA- GGGCGAC-C- A-CCAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1171 OpbHerpo 1096 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-CCC- A-CCAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1183 LehDoli6 1065 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1151 156Elong 1089 UGG-UCGCAA GGCUGA--AC UUAA-AGGAA UUGACGGAA- CGGCGAC-CA A-CCAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1176 PlpBetae 1095 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1181 KirElate 1098 UGG-UCGCAA GGCUGAA-AC U-AA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1183 42MMycop 1066 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1152 21HJunip 1098 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGC-G UAGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1184 KirMarit 1087 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1173 alongi1189 1065 ...... 1064 996Bipol 1065 ...... 1064 996Austr 1065 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CAAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1151 MonPurpu 1031 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CAAGGC-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGGGAA 1118 243Vari2 1084 UGG-UCGCAA GGCUGAA-AC UUAA-AGAAA UUGACGGAA- GGGCA-C-C- A-CCAGGC-G UGGUGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1170 OngEquin 1068 UGG-UCGCAA GGCUGAA-AC UUAA-AGGAA UUGACGGAA- GGGCA-C-C- A-CCAGAU-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGA-AA 1154 750Supe2 1101 UGG-UCGCAA GGCUGAA-AC UUAA-AGGAA UUGACGGAA- G-GCA-C-C- A-CCAGAU-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGA-AA 1186 ObaFimic 1068 UGG-UCGCAA GGCUGAA-AC UUAA-AGGAA UUGACGGAA- GGGCA-C-C- A-CCAGAU-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- ACGGGGA-AA 1155 750Olig2 1068 UGG-UCGCAA GGCUGAA-AC UUAA-AGGAA UUGACGGAA- GGGCA-C-C- A-CCAGAU-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1154 897Doedy 1100 UGG-UCGCAA GGCUGAA-AC UUAA-AGGAA UUGACGGAA- GGGCA-C-C- A-CCAGAU-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1186 ObaDelic 1113 UGG-UCGCAA GGCUGAA-AC UUAA-AGGAA UUGACGGAA- GGGCA-C-C- A-CCAGGU-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1199 90HRepan 1113 UGG-UCGCAA GGCUGAA-AC UUAA-AGGAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCCGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1199 130Nuda2 1124 UGG-UCGCAA GGCUGAA-AC UUAA-AGGAA UUGACGGAA- GGGCA-C-C- A-CCAGGU-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1210 BulAlbus 1117 UGG-UCGCAA GGCUGAA-AC UUAA-AGGAA UUGACGGAA- GGGCA-C-C- A-CCAGGA-G UGGAGCCUGC -GGCUUAAUU UGACUCAAC- AC-GGGG-AA 1203 SayCe108

1301 1311 1321 1331 1341 1351 1361 1371 1381 1391 1400 | | | | | | | | | | | 1194 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1285 TcdMaxil 1194 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1285 TcdSetig 1192 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1283 TcdFurca 1191 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1282 TcdApien 1170 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U.UUGUGGGU GGUGGCGCAU GGCC-GUUCU UAGUUGGUGG 1261 Tmarch2639 1173 CUCCACCAGG UCCA-GAGAC ACAAUA-AGG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1267 LemAqu1 1177 ACUCACCAGG UCCA-GACAC C-A-AUAAAG GAUUGACAGA AUUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1271 LemTerr1 741 ...... 740 var11783 1177 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1268 TriAng1020 1160 NNNNNNNNNN NNNN-NNNN- N-N-AU-AAG GAUAGACANA -UUGACANCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UANUUGGUGG 1251 Mc8Coral 559 ...... 558 HVZ81382 1033 ...... 1032 Loram 1122 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1213 986Defle 1122 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1213 OidTenui 1138 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1229 Acrassa055 1164 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GNCC-GUUCU UAGUUGGUGA 1255 AngFurt 1172 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1263 TriSpl1198 1168 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1259 472Gutta 1133 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1224 7BGrami 1201 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1292 PiaSpec3 1153 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1244 164Darwi 1143 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGNNCU CUUUCUUGAU U-UUGUGNGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1234 CudConfu 1148 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1239 ShaFlavi 1122 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1213 989Roseu 1119 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1210 39GPanno 1183 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1274 58BInqui 1093 ACUCACCAGG UCCA-GACA- C-A-AA-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU C-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1184 0LLubri 1170 ACUCACCAGG UCCA- GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC.GUUCU UAGUUGGUGG 1261 13089Alacu 1182 ACUCACCAGG UCCA-GACA- C-A-AU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1273 NerCinn5 1181 ACUCACCAGG UCCA-GACA- C-A-AU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1272 GsaPutte 1163 ACUCACCAGG UCCA-GACA- C-A-AU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1254 GibPuli5 1181 ACUCACCAGG UCCA-GACA- C-A-AU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1272 HpaLute3 1152 ACUCACCAGG UCCA-GACA- C-A-AU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1243 HymChrys 1173 ACUCACCAGG UCCA-GACA- C-A-AU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1264 Hlug13783 1153 ACUCACCAGG UCCA-GACA- C-A-GU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-CUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1244 144Seti3 1153 ACUCACCAGG UCCA-GACA- C-A-GU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-CUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1244 PsaBoyd6 1182 ACUCACCAGG UCCA-GACA- C-A-GU-GAG GAUUGACAGA -UUGAUAGCU CUUUCUUGAU U-CUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1273 98HRetor 1151 ACUCACCAGG UCCA-GACA- C-A-GU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-CUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1242 MiaCirro 1173 ACUCACCAGG UCCA-GACG- C-A-AU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUAGGU GGUGGUGCAU GGCC-GGUCU UAGUUGGUGG 1264 GmmPeni3 1200 ACUCACCAGG UCCA-GACA- C-G-AU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UCGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1291 NeuCrass 1201 ACUCACCAGG UCCA-GACA- C-G-AU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UCGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1292 SorFirmi 1151 ACUCACCAGG UCCA-GACA- C-G-AU-GAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UCGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1242 ChtElatu 1180 ACUCACCAGG UCCA-GACA- C-A-AG-UAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1271 KicIvori

Figure A36 continued

207 Appendix

Alignment SSU sequences continued

1203 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1294 AuePullu 1165 ACUCACCAGG UCCA-GACA- C-A-GU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1256 Hr0Werne 1191 ACUCACCGGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1282 KirAethi 1172 ACUCACCAGG UCCA-GAUG- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUUCAGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1263 OpbHerpo 1184 ACUCACCAGG UCCA-GAUG- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUUCAGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1275 LehDoli6 1152 ACUCACCAGG UCCA-GAUG- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUUCAGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1243 156Elong 1177 ACUCACCAGG UCCA-GAUG- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUUCAGGU GGUGGUGCAU GGCC-GUUCU UAGUUCGUGG 1268 PlpBetae 1182 ACUCACCAGG UCCA-GAUG- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUUCAGGU GGUGGUGCAU GGCC-GUUCU UAGUUCGUGG 1273 KirElate 1184 ACUCACCAGG UCCA-GAUG- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUUCAGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1275 42MMycop 1153 ACUCACCAGG UCCA-GAUG- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUUCAGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1244 21HJunip 1185 ACUCACCAGG UCCA-GAUG- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUUCAGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1276 KirMarit 1174 ACUCACCAGG UCCA-GAUG- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUUCAGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1265 alongi1189 1065 ...... 1064 996Bipol 1065 ...... 1064 996Austr 1152 ACUCACCAGG UCCA-GACA- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU C-UUUUGGAU GGUGGUGCAU GGCC-GUUCC UAGUUGGUGG 1243 MonPurpu 1119 ACUCACCAGG UCCA-GACA- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU C-UUUUGGAU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1210 243Vari2 1171 ACUCACCAGG UCCA-GACA- A-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU C-UUUUGGAU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1262 OngEquin 1155 ACUCACCAGG UCCA-GACA- C-A-UU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-AUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1246 750Supe2 1187 ACUCACCAGG UCCA-GACA- C-A-UU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-AUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1278 ObaFimic 1156 ACUCACCAGG UCCA-GACA- C-A-UU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-AUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1247 750Olig2 1155 ACUCACCAGG UCCA-GACA- C-A-UU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-AUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1246 897Doedy 1187 ACUCACCAGG UCCA-GACA- C-A-UU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-AUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1278 ObaDelic 1200 ACUCACCAGG UCCA-GACA- U-A-GC-UAG GAUUGACAGA -UUGAUAGCU CUUUCUUGAU U-CUAUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1291 90HRepan 1200 ACUCACCAGG UCCA-GACA- U-A-AC-UAG GAUUGACAGA -UUGAUAGCU CUUUCUUGAU U-UUAUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1291 130Nuda2 1211 ACUCACCAGG UCCA-GACA- U-A-GU-AAG GAUUGACAGA -UUGAUAGCU CUUUCUUGAU U-CUAUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1302 BulAlbus 1204 ACUCACCAGG UCCA-GACA- C-A-AU-AAG GAUUGACAGA -UUGAGAGCU CUUUCUUGAU U-UUGUGGGU GGUGGUGCAU GGCC-GUUCU UAGUUGGUGG 1295 SayCe108 1401 1411 1421 1431 1441 1451 1461 1471 1481 1491 1500 | | | | | | | | | | | 1286 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACUUU GACUU-UUAA AUAGCUAGGC UAGC-UUUGG CUGGUCGC-U GGCUUCUUAG AAGG-ACUAU 1381 TcdMaxil 1286 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACUUU GACUU-UUAA AUAGCUAGGC UAGC-UUUGG CUGGUCGC-U GGCUUCUUAG AAGG-ACUAU 1381 TcdSetig 1284 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACUUU GACUU-UUAA AUAGCUAGGC UAGC-UUUGG CUGGUCGC-U GGCUUCUUAG AAGG-ACUAU 1379 TcdFurca 1283 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACUUU GACUU-UUAA AUAGCUAGGC UAGC-UUUGG CUGGUCGC-U GGCUUCUUAG AAGG-ACUAU 1378 TcdApien 1262 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACUUU GACUU-UUAA AUAGCUAGGC UAGC-UUUGG CUGGUCGC- U GGCUUCUUAG AAGG.ACUAU 1357 Tmarch2639 1268 AUUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCAA CGUCU-UUAA AUAGCCAGGC UAGC-UUUGG CUGGUCGC- C GGCUUCUUAA AGAG-ACUUU 1363 LemAqu1 1272 AUUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCAA CGUCU-UUAA AUAGCCAGGC UAGC-UUUGG CUGGUCGC- C GGCUUCUUAA AGAG-ACUUU 1367 LemTerr1 741 ...... 740 var11783 1269 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCC. AGACUAUUAA AUAGCCAGAC UUAC-UUUGG UAGGUC-GCC GGCUUCUUAA UGGG-ACUUU 1364 TriAng1020 1252 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU GACCUGCGGG AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUGU 1348 Mc8Coral 559 ...... 558 HVZ81382 1033 ...... 1032 Loram 1214 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGGC UAGC-UUUGG CUGGUC-GCU GGCUUCUUAG AGGG-ACUAU 1310 986Defle 1214 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1310 OidTenui 1230 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1326 Acrassa055 1256 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC- GCC GGCUUCUUAG AGGG-ACUAU 1352 AngFurt 1264 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1360 TriSpl1198 1260 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-CUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1356 472Gutta 1225 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCUCGAC UAGC-UUUGG CUGGUU-GCU AGCUUCUUAG AGGG-ACUAU 1321 7BGrami 1293 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGCUUUG-G CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1389 PiaSpec3 1245 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGGC UAGC-CUCGG CUGGUC-GCG GGCUUCUUAG AGGG-ACUAU 1341 164Darwi 1235 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAN 1331 CudConfu 1240 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1336 ShaFlavi 1214 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGGC UAGC-UUUGG CUGGUC-GCU GGCUUCUUAG AGGG-ACUAU 1310 989Roseu 1211 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGGC UAGC-UUUGG CUGGUC-GCU GGCUUCUUAG AGGG-ACUAU 1307 39GPanno 1275 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGGC UAGC-UUUGG CUGGUC-GCU GGCUUCUUAG AGGG-ACUAU 1371 58BInqui 1185 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1281 0LLubri 1262 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC- GCC GGCUUCUUAG AGGG-ACUAU 1358 13089Alacu 1274 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGUA UUGC-UUUGG CAGUAC-GCU GGCUUCUUAG AGGG-ACUAU 1370 NerCinn5 1273 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUACUAA CUAGUCCGUA UUGC-UUUGG CAGUAC-GCC GACUUCUUAG AGGG-ACUAU 1369 GsaPutte 1255 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGUA UUGC-UUUGG CAGUAC-GCU GGCUUCUUAG AGGG-ACUAU 1351 GibPuli5 1273 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGUA UUGC-UUUGG CAGUAC-GCC GGCUUCUUAG AGGG-ACUAU 1369 HpaLute3 1244 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGUA UUGC-UUUGG CAGUAC-GCU GGCUUCUUAG AGGG-ACUAU 1340 HymChrys 1265 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGUA UUGC-UUUGG CAGUAC-GCU GGCUUCUUAG AGGG-ACUAU 1361 Hlug13783 1245 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGUA CUGC-UUUGG CAGUUC-GCC GGCUUCUUAG AGGG-ACUAU 1341 144Seti3 1245 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGUA CUGC-UUUGN CAGUUC-GCC GGCUUCUUAG AGGG-ACUAU 1341 PsaBoyd6 1274 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCAAU CUGC-UCCGG CAGUUU-GCU GGCUUCUUAG AGGG-ACUAU 1370 98HRetor 1243 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGUA CUGC-UCUGG CAGUUC-GCC GGCUUCUUAG AGGG-ACUAU 1339 MiaCirro 1265 AGUGAUUUGU CAGCUUAAUU GCGAUAACGA ACGAGACCUU CUUCUGCUAA AUAGCCCGAA CUGC-UUUGG CAGUCC-GCC GGCUUCUUAG AGAG-ACUAU 1361 GmmPeni3 1292 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGUA UUGC-UUUGG CAGUAC-GCU GGCUUCUUAG AGGG-ACUAU 1388 NeuCrass 1293 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGUA UUGC-UUUGG CAGUAC-GCU GGCUUCUUAG AGGG-ACUAU 1389 SorFirmi 1243 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGUA UUGC-UUUGG CAGUAC-GCU GGCUUCUUAG AGGG-ACUAU 1339 ChtElatu 1272 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGCG UCGC-UCCGG CGGCGC-GCC GGCUUCUUAG AGGG-ACUAU 1368 KicIvori 1295 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGGC CCGC-UUUGG CGGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1391 AuePullu 1257 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC CCGC-UUUGG CGGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1353 Hr0Werne 1283 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUACUAA AUAGCCCGGC CCGC-CUUGG CGGGUC-GCU GGCUUCUUAG AGGG-ACUAU 1379 KirAethi 1264 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1360 OpbHerpo 1276 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GUC GGCUUCUUAG AGGG-ACUAU 1372 LehDoli6 1244 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1340 156Elong 1269 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1365 PlpBetae 1274 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1370 KirElate 1276 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1372 42MMycop 1245 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1341 21HJunip 1277 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCA GGCUUCUUAG AGGG-ACUAU 1373 KirMarit 1266 AUUGAUCUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGC-UUUGG CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1362 alongi1189 1065 ...... 1064 996Bipol 1065 ...... 1064 996Austr 1244 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUC GGCCC-UUAA AUAGCCCGGU CCGCAUUU-G C-GGCC-GCU GGCUUCUUAA GGGG-ACUAU 1338 MonPurpu 1211 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUC GGCCC-UUAA AUAGCCCGGU CCGCGUUU-G CGGGCC-GCU GGCUUCUUAG GGGG-ACUAU 1306 243Vari2 1263 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCCGAC CCACGUUU-G UGGGCC-GCU GGCUUCUUAG AGGG-ACUAU 1359 OngEquin 1247 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAAGC UAGCUUUU-G CUGGCU-GCU GGCUUCUUAG AGGG-ACUAU 1343 750Supe2 1279 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAAGC UAGCUUUU-G CUGGUU-GCU GGCUUCUUAG AGGG-ACUAU 1375 ObaFimic 1248 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGCUUUU-G CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1344 750Olig2 1247 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC UAGCUUUU-G CUGGUC-GCC GGCUUCUUAG AGGG-ACUAU 1343 897Doedy 1279 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGCCAGGC GAGCGUUU-G CUCGUC-GCC GGCUUCUUAG AGGG-ACUAU 1375 ObaDelic 1292 AGUGAUUUGU CUGGUUAAUU CCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGUCCGGC UGUCUUUU-G AUGGCU-GCU GACUUCUUAG AGGG-ACUGU 1388 90HRepan 1292 AGUGAUUUGU CUGGUUAAUU CCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGUCAGGC CGGCUUUU-G CUGGUC-GCG GACUUCUUAG AGGG-ACUGU 1388 130Nuda2 1303 AGUGAUUUGU CUGGUUAAUU CCGAUAACGA ACGAGACCUU AACCUGCUAA AUAGACAGGC CGGC-UUCGG CUGGUC- GUC GUCUUCUUAG AGGG-ACUGU 1399 BulAlbus 1296 AGUGAUUUGU CUGCUUAAUU GCGAUAACGA ACGAGACCUU AACCUACUAA AUAGUGGUGC UAGCAUUU-G CUGGUU- AUC CACUUCUUAG AGGG-ACUAU 1392 SayCe108 1501 1511 1521 1531 1541 1551 1561 1571 1581 1591 1600 | | | | | | | | | | | 1382 UUGC-UCAA- GCAAAU-GG- AAGUG-CGAG GCAAUAACAG GUCU-GUGAU G-CACACCUU AG-A-GUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1468 TcdMaxil 1382 UUGC-UCAA- GCAAAU-GG- AAGUG-CGAG GCAAUAACAG GUCU-GUGAU G-CACACCUU AG-AUGUUUU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1469 TcdSetig 1380 UUGC-UCAA- GCAAAU-GG- AAGUG-CGAG GCAAUAACAG GUCU-GUGAU G-CAC-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1465 TcdFurca 1379 UUGC-UCAA- GCAAAU-GG- AAGUG-CGAG GCAAUAACAG GUCU-GUGAU G-- ACACCUU AG-A-GUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1464 TcdApien 1358 UUGC-UCAA- GCAAAU-GG- AAGUG-CGAA GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU .GGGC-CGCA CGCG-CG-CU ACACUGACAG 1442 Tmarch2639 1364 CUGC-UCAA- GCAGAA-GG- AAGUA-GUUG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1448 LemAqu1 1368 CUGC-UCAA- GCAGAA-GG- AAGUA-GUUG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1452 LemTerr1 741 ...... 740 var11783

Figure A36 continued

208 Appendix

Alignment SSU sequences continued

1365 CCGU-CUAA- GCGGAA-GG- AAGUACU-GG UCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG- CG-CU ACACUGACAG 1449 TriAng1020 1349 CGGC-UCAA- GCCGAA-GG- AAGUU-CGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1433 Mc8Coral 559 ...... 558 HVZ81382 1033 ...... 1032 Loram 1311 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUCCU -GGGC-CGCA CGCG-CG-UU ACACUGACAG 1395 986Defle 1311 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1395 OidTenui 1327 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CGCAU ACACUGACCG 1412 Acrassa055 1353 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACCG 1437 AngFurt 1361 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACCG 1445 TriSpl1198 1357 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1441 472Gutta 1322 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1406 7BGrami 1390 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1474 PiaSpec3 1342 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1426 164Darwi 1332 NNGC-UCAA- GN--NU-GG- AAGUU-UGAG GCAAUAACAG ------UGACAG 1370 CudConfu 1337 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1421 ShaFlavi 1311 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGCC-GCA CGCG-CG-CU ACACUGACAG 1395 989Roseu 1308 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGCC-GCA CGCG-CG-CU ACACUGACAG 1392 39GPanno 1372 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1456 58BInqui 1282 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGCC-GCA CGCG-CG-CU ACACUGACAG 1366 0LLubri 1359 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG- AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1443 13089Alacu 1371 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACGG 1455 NerCinn5 1370 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1454 GsaPutte 1352 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACGG 1436 GibPuli5 1370 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACGG 1454 HpaLute3 1341 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1425 HymChrys 1362 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACGG 1446 Hlug13783 1342 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUCCU -GGGC-CGCA CGCG-CG-UU ACACUGACAG 1426 144Seti3 1342 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUCCU -GGGC-CGCA CGCG-CG-UU ACACUGACAG 1426 PsaBoyd6 1371 CGGC-UCAAA GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCUUGUGAU G-C-C-C-UU AG-AUGUCCU -GGGC-CGCA CGCG-CGGUU ACACUGACAA 1458 98HRetor 1340 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUCCU -GGGC-CGCA CGCG-CG-UU ACACUGACAG 1424 MiaCirro 1362 CGGC-UCAA- GCCGAUUGG- AGGUU-GGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1447 GmmPeni3 1389 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAC 1473 NeuCrass 1390 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAC 1474 SorFirmi 1340 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1424 ChtElatu 1369 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GGGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1453 KicIvori 1392 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1476 AuePullu 1354 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1438 Hr0Werne 1380 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCC -GGGC-UGCA CGCG-CG-CU ACACUGACAG 1464 KirAethi 1361 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1445 OpbHerpo 1373 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1457 LehDoli6 1341 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1425 156Elong 1366 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1450 PlpBetae 1371 CGGC-UCAA- GCCGAG-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1455 KirElate 1373 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1457 42MMycop 1342 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1426 21HJunip 1374 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1458 KirMarit 1363 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1447 alongi1189 1065 ...... 1064 996Bipol 1065 ...... 1064 996Austr 1339 CGGC-UCAA- GCCGAU-GG- AAGUG-CGCG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1423 MonPurpu 1307 CGGC-UCAA- GCCGAU-GG- AAGUG-CGCG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1391 243Vari2 1360 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1444 OngEquin 1344 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1428 750Supe2 1376 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1460 ObaFimic 1345 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1429 750Olig2 1344 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1428 897Doedy 1376 CGGC-UCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1460 ObaDelic 1389 CAGCGUCUA- GCUGAC-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1474 90HRepan 1389 CGGCGUCUA- NCCGAC-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACAG 1474 130Nuda2 1400 CGGCGUUUA- GCCGAC-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-AUGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACUG 1485 BulAlbus 1393 CGGUUUCAA- GCCGAU-GG- AAGUU-UGAG GCAAUAACAG GUCU-GUGAU G-C-C-C-UU AG-ACGUUCU -GGGC-CGCA CGCG-CG-CU ACACUGACGG 1478 SayCe108 1601 1611 1621 1631 1641 1651 1661 1671 1681 1691 1700 | | | | | | | | | | | 1469 AGCCAACGAG U-UC----UU -CCUUAGCCG AAAGGUUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1561 TcdMaxil 1470 AGCCAACGAG U-UC----UU -CCUUAGCCG AAAGGUUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1562 TcdSetig 1466 AGCCAACGAG U-UC----UU -CCUUAGCCG AAAGGUUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1558 TcdFurca 1465 AGCCAACGAG U-UC----UU -CCUUAGCCG AAAGGUUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1557 TcdApien 1443 AGCCAACGAG U-UC----UU -CCUUAGCCG AAAGGUUUGG GUAA.UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1535 Tmarch2639 1449 AGCCAACGAG U-UU---CUU -CCUUGUCCG AAAGGGCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1542 LemAqu1 1453 AGCCAACGAG U-UU---CUU -CCUUGUCCG AAAGGGCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1546 LemTerr1 741 ...... 740 var11783 1450 AGCCAACGUG U-UC---UUU -CCUUCACCG AAAGGUGUGG GUAA-UCAUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1543 TriAng1020 1434 ANCCAACGAG U-UC---UUU -CCUUAACCG AAAGGUUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1527 Mc8Coral 559 ...... 558 HVZ81382 1033 ...... 1032 Loram 1396 AGCCAACGAG U-UC--AUCU -CCUUGUCCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1490 986Defle 1396 AGCCAACGAG U-UC--AUCA -CCUUGUCCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1490 OidTenui 1413 GGCCAACGAG U-UU---CUU -CCUUGACCG AAAGGUCUGG GUAA-UCUUG UUAAACCCGG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1506 Acrassa055 1438 GGCCAACGAG U-UU---CUU -CCUUGACCG AAAGGUCUGG GUAA-UCUUG UUAAACCCGG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1531 AngFurt 1446 GGCCAACGAG U-UU---CUU -CCUUGACCG AAAGGUCUGG GUAA-UCUUG UUAAACCCGG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1539 TriSpl1198 1442 AGCCAACGAG U-AU---CUU -CCUUGUUCG AGAGAUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1535 472Gutta 1407 AGCCAACGAG U-UU---CCU -CCUUGGUCG AAAGACCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1500 7BGrami 1475 AGCCAACGAG U-UU---CUU -CCUUGUCCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1568 PiaSpec3 1427 AGCCAACGAG U-UC--AUCC -CCUUGGCCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1521 164Darwi 1371 AGCCAACGAG U-UC--AUCA -CCUUAGCCG AGAGNUUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1465 CudConfu 1422 AGCCAACGAG U-UC--AUCA -CCUUAGCCG AAAGGUUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1516 ShaFlavi 1396 AGCCAACGAG U-UC--AUCA -CCUUGACCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1490 989Roseu 1393 AGCCAACGAG U-UC--AUCA -CCUUGGCCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1487 39GPanno 1457 AGCCAACGAG U-UC--AUCA -CCUUGGCCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1551 58BInqui 1367 AGCCAACGAG U-UC--AUCA -CCUUGGCCG AAAGGCCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1461 0LLubri 1444 AGCCAACGAG U-UC--AUCA -CCUUGGCCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1538 13089Alacu 1456 AGCCAGCGAG U-----ACUU -CCUUGUCCG AAAGGUCCGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1548 NerCinn5 1455 AGCCAGCGAG U-----ACUU -CCUUGUCCG AAAGGUCCGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1547 GsaPutte 1437 AGCCAGCGAG U-----ACUU -CCUUGUCCG AAAGGUCCGG GUAA-UCUUG UUAAACUCCG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1529 GibPuli5 1455 AGCCAGCGAG U-----ACUC -CCUUGGCCG GAAGGCCUGG GUAA-UCUUG UUAAACUCCG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1547 HpaLute3 1426 AGCCAGCGAG U-----ACUC -CCUUGGCCG GAAGGCCCGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1518 HymChrys 1447 AGCCAGCGAG U-----ACUC -CCUUGACCG GAAGGUCCGG GUAA-UCUUG UUAAACUCCG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1539 Hlug13783 1427 AGCCAGCGAG U-----AUUU -CCUUGACCG GAAGGUCCGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1519 144Seti3 1427 AGCCAGCGAG U-----AUUU -CCUUGGCCG GAAGGCCCGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1519 PsaBoyd6 1459 AGCCAGCGAG U-----AUUU -CCUUUGCCG GAAGGUCCGG GUAA-UCUUG UUAAACUUUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1551 98HRetor 1425 GGCCAGCGAG U-----ACCU -CCUUGGCCG AAAGGCCCGG GUAA-UCUUG UUAAACCCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1517 MiaCirro 1448 AGCCAGCGAG U-----AUCU -CCUUGGCCG AAAGGUCCGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1540 GmmPeni3 1474 AGCCAGCGAG U-----ACUC -CCUUGGCCG GAAGGUCCGG GUAA-UCUUG UUAAACUGUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1566 NeuCrass 1475 AGCCAGCGAG U-----ACUC -CCUUGGCCG GAAGGUCCGG GUAA-UCUUG UUAAACUGUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1567 SorFirmi 1425 AGCCAGCGAG U-----ACUC -CCUUGGCCG GAAGGCCCGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1517 ChtElatu 1454 GGCCAGCGAG U-----ACUC -CCUUGGCCG AAAGGCCCGG GUAA-UCUUG UUAAACCCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1546 KicIvori 1477 AGCCAACGAG U-UC--AUUU -CCUUGCCCG GAAGGGUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1571 AuePullu 1439 AGCCAACGAG U-UUU--UUU -CCUUGGCCG GAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1533 Hr0Werne

Figure A36 continued

209 Appendix

Alignment SSU sequences continued

1465 GGCCAACGAG U-UC--AACC -CCUUGGCCG AAAGGUCCGG GUAA-UCUGG UUAAACCCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1559 KirAethi 1446 AGCCAACGAG U-UCU--UCA -CCUUGACCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1540 OpbHerpo 1458 AGCCAACGAG U-UCU--UCA -CCUUGACCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1552 LehDoli6 1426 AGCCAACGAG U-UCU--UCA -CCUUGACCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1520 156Elong 1451 AGCCAAUGAG U-UCU--UUC -CCUUGGCCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1545 PlpBetae 1456 AGCCAACGAG U-UCU--UUG -CCUUGGCCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1550 KirElate 1458 AGCCAACGAG U-UCU--UCA -CCUUGACCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1552 42MMycop 1427 AGCCAACGAG U-UCU--UCA -CCUUGACCG AAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1521 21HJunip 1459 AGCCAACGAG U-UC--AUCA -CCUUGGCCG GAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1553 KirMarit 1448 AGCCAACGAG U-UC--AUCA -CCUUGGCCG GAAGGUCUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1542 alongi1189 1065 ...... 1064 996Bipol 1065 ...... 1064 996Austr 1424 GGCCAGCGAG U-AC--AUCA -CCUUGGCCG AGAGGCCUGG GUAA-UCUUG UUAAACCCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1518 MonPurpu 1392 GGCCAGCGAG U-AC--AUCA -CCUUGGCCG AGAGGUCUGG GUAA-UCUUG UUAAACCCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1486 243Vari2 1445 GGCCAGCGAG U-UC--AUCA -CCUUGGCCG AGAGGUCUGG GUAA-UCUUG UUAAACCCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1539 OngEquin 1429 AGCCAACGAG U-AU--A- AA -CCUUGAUCG AGAGAUUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGGGCAU UGCAAUUAUU GCCCUUCAAC 1522 750Supe2 1461 AGCCAACGAG U-----AUAA ACCUUGAUCG AGAGAUUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGGGCAU UGCAAUUAUU GCCCUUCAAC 1554 ObaFimic 1430 AGCCAACGAG U- AU --A- AA -CCUUAACCG AGAGGUUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGGGCAU UGCAAUUAUU GCCCUUCAAC 1523 750Olig2 1429 AGCCAACGAG U-GU--- A AA -CCUUAGCCG AGAGGCUUGG GUAA-UCUUG UUAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1522 897Doedy 1461 GGCCAACNAA U-UC---UAA -CCUUGGCCG AGAGGUCUGG GUAA-UCUUG UUAAACCCUG UCGUGCUGGG GAUANANCAU UGCNAUUAUU GCUCUUCA.. 1552 ObaDelic 1475 GGCCAGCGAG U- UU-- AUCA -CCUUGGCUG AAAGGCCUGG GUAA-UCUUG UGAAACCUUG UCGUGCUGGG GAUAGANCAU UGCAAUUAUU GCUCUUCAAC 1569 90HRepan 1475 AGCCAGCGAG U-UUU--UUU -CCUUGGCCG AAAGGUCUGG GUAA-UCUUG UGAAACUCUG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1569 130Nuda2 1486 AGCCAGCGAG U-CU--AUCA -CCUUGGCCG AGAGGCCUGG GUAA-UCUUG UGAAACUCAG UCGUGCUGGG GAUAGAGCAU UGCAAUUAUU GCUCUUCAAC 1580 BulAlbus 1479 AGCCAGCGAG U--C---UAA -CCUUGGCCG AGAGGUCUUG GUAA-UCUUG UGAAACUCCG UCGUGCUGGG GAUAGAGCAU UGUAAUUAUU GCUCUUCAAC 1571 SayCe108

1701 1711 1721 1731 1741 1751 1761 1771 1781 1791 1800 | | | | | | | | | | | 1562 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGA-UUCA 1657 TcdMaxil 1563 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGA-UCCA 1658 TcdSetig 1559 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGA-UUCA 1654 TcdFurca 1558 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGA-UCCA 1653 TcdApien 1536 GAGGAAUUC. CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGA-UCCA 1631 Tmarch2639 1543 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUAA 1638 LemAqu1 1547 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUAA 1642 LemTerr1 741 ...... 740 var11783 1544 GAGGAAUAU- CUAGUAAGCG CAUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA.UUG AAUGG-CUAA 1639 TriAng1020 1528 GAGGAAUUC- CUAGUAAGCG CAUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUAA 1623 Mc8Coral 559 ...... 558 HVZ81382 1033 ...... 1032 Loram 1491 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1586 986Defle 1491 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AACGG-CCCA 1586 OidTenui 1507 GAGGAAUUC- CUAGUAGGCG CAAGUCAUC- AGCUUGCGCC GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1602 Acrassa055 1532 GAGGAAUUC- CUAGUAGGCG CAAGUCAUC- AGCUUGCGCC GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1627 AngFurt 1540 GAGGAAUUC- CUAGUAGGCG CAAGUCAUC- AGCUUGCGCC GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1635 TriSpl1198 1536 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUAA 1631 472Gutta 1501 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUAA 1596 7BGrami 1569 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUAA 1664 PiaSpec3 1522 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AACGG-CUCA 1617 164Darwi 1466 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGNNNU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUAA 1561 CudConfu 1517 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUAA 1612 ShaFlavi 1491 GAGGAAUUC- CUAGUAGGCG CGAGUCAUC- AGCUCGUGCC GACUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AACGG-CUAA 1586 989Roseu 1488 GAGGAAUUC- CUAGUAGGCG CGAGUCAUC- AGCUCGUGCC GACUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AACGG-CUAA 1583 39GPanno 1552 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGCU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUAA 1647 58BInqui 1462 GAGGAAUUC- CUAGUAGGCG CAAGUCAUC- AGCUUGUGCC GACUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1557 0LLubri 1539 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC. AGCUUGCG-U GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG.CUCA 1633 13089Alacu 1549 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1644 NerCinn5 1548 GAGGAAUCC- CUGGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1643 GsaPutte 1530 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1625 GibPuli5 1548 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1643 HpaLute3 1519 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1614 HymChrys 1540 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1635 Hlug13783 1520 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1615 144Seti3 1520 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1615 PsaBoyd6 1552 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AACGG-CUCA 1647 98HRetor 1518 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1613 MiaCirro 1541 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1636 GmmPeni3 1567 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1662 NeuCrass 1568 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1663 SorFirmi 1518 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1613 ChtElatu 1547 GAGGAAUCC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1642 KicIvori 1572 GAGGAAUGC- CUAGUAAGCG UACGUCAUC- AGCGUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUGA 1667 AuePullu 1534 GAGGAAUGC- CUAGUAAGCG CAUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1629 Hr0Werne 1560 GAGGAAUGC- CUAGUAAGCG UAUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUUA 1655 KirAethi 1541 GAGGAAUGC- CUAGUAAGCG CGUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1636 OpbHerpo 1553 GAGGAAUGC- CUAGUAAGCG CGUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1648 LehDoli6 1521 GAGGAAUGC- CUAGUAAGCG CGUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1616 156Elong 1546 GAGGAAUGC- CUAGUAAGCG CGUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1641 PlpBetae 1551 GAGGAAUGC- CUAGUAAGCG CGUGUCAUC- AGCACGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1646 KirElate 1553 GAGGAAUGC- CUAGUAAGCG CAUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1648 42MMycop 1522 GAGGAAUGC- CUAGUAAGCG CAUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1617 21HJunip 1554 GAGGAAUGC- CUAGUAAGCG CAUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1649 KirMarit 1543 GAGGAAUGC- CUAGUAAGCG CAUGUCAUC- AGCAUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1638 alongi1189 1065 ...... 1064 996Bipol 1065 ...... 1064 996Austr 1519 GAGGAAUGC- CUAGUAGGCA CGAGUCAUC- AGCUCGUGCC GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1614 MonPurpu 1487 GAGGAAUGC- CUAGUAGGCA CGAGUCAUC- AGCUCGUGCC GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1582 243Vari2 1540 GAGGAAUGC- CUAGUAGGCA CAAGUCAUC- AGCUUGUGCC GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1635 OngEquin 1523 GAGGAAUAU- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1618 750Supe2 1555 GAGGAAUAU- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1650 ObaFimic 1524 GAGGAAUAU- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1619 750Olig2 1523 GAGGAAUAU- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUCA 1618 897Doedy 1553 ...... 1552 ObaDelic 1570 GAGGAAUAC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUUA 1665 90HRepan 1570 GAGGAAUUC- CUAGUAAGCG UGAGUCAUC- AGCUCGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUUA 1665 130Nuda2 1581 GAGGAAUAC- CUAGUAAGCG UGAGUCACC- AACUCGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAC UACCGA-UUG AAUGG-CUUG 1676 BulAlbus 1572 GAGGAAUUC- CUAGUAAGCG CAAGUCAUC- AGCUUGCGUU GAUUACGUCC CUGCCCUUUG UACACACCGC CCGUCGCUAG UACCGA-UUG AAUGG-CUUA 1667 SayCe108 1801 1811 1821 1831 1841 1851 1861 1871 1881 1891 1900 | | | | | | | | | | | 1658 GUGAGGCUUU CGGACUGGC- CCAGGAAGAG -UGGCAACA- CUCAUCUA-G GGCCGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1752 TcdMaxil 1659 GUGAGGCUUU CGGACUGGC- CCAGGAAGAG -UGGCAACA- CUCAUCUA-G GGCCGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1753 TcdSetig 1655 GUGAGGCUUU CGGACUGGC- CCAGGAAGAG -UGGCAACA- CUCAUCUA-G GGCCGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1749 TcdFurca 1654 GUGAGGCUUU CGGACUGGC- CCAGGAAGAG -UGGCAACA- CUCAUCUA-G GGCCGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1748 TcdApien 1632 GUGAGGCUUU CGGACUGGC- CCAGGAAGAG -UGGCAACA- CUCAUCUA-G GGCCGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1726 Tmarch2639 1639 GUGAGGCUUU CAGACUGGC- CCAGGGACGG -CGGCAACG- CCGACCCA-G GGCUGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1733 LemAqu1 1643 GUGAGGCUUU CAGACUGGC- CCAGGGACGG -CGGCAACG- CCGACCCA-G GGCUGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1737 LemTerr1 741 ...... 740 var11783 1640 GUGAGGCUUU CGGACUGGC- CUAGGAAGAG -UGGCAACA- CUCAUCUC-G GGCCGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1734 TriAng1020 1624 GUGAGGCUUU CGGACUGGCC U-AGGAAGAG -UGGCAACA- CUCAUCUC-G GGCCGGAAAG UUAUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1718 Mc8Coral

Figure A36 continued

210 Appendix

Alignment SSU sequences continued

559 ...... 558 HVZ81382 1033 ...... 1032 Loram 1587 GUGAGGCUUU CGGACUGGC- CUAGGGAGAG -UGGCAACA- CUCACCCA-G GGCCGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1681 986Defle 1587 GUGAGGCUUU CGGACUGGCC CUAGGGAGAG -UGGCAACA- CUCACCCA-G GGCCGGAAAG UUGUCCAAAC UUGGUCGUUU AG-AGGAAGU AAAAGUCGUA 1682 OidTenui 1603 GUGAGGCUUU CGGACUGGC- CCAGAGGGAG -UGGCAACG- CUCGCUCA-G GGCCGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGA...... 1684 Acrassa055 1628 GUGAGGCUUU CGGACUGGC- CCAGAGGGAG -UGGCAACG- CUCGCUCA-G GGCCGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1722 AngFurt 1636 GUGAGGCUUU CGGACUGGC- CCAGGGAGAG -UGGCAACG- CUCGCCCA-G GGCCGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1730 TriSpl1198 1632 GUGAGGCUU- CGGACUGGC- C-AGGGAGAG -UGGCGACA- CUC-CCCA-G GGCCGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1723 472Gutta 1597 GUGAGGCUUU CGGACUGGC- CCGGGGAGAG -UGGUAACA- CUCACCC--- --UUGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1687 7BGrami 1665 GUGAGGCUU- UGGACCGGC- CCAGAGAGGG -UGGCAACA- CCUCCUCA-G GGCUGGAAAA UCAUACAAAC UUGGUCAUUU AG- AGGAAGU AAAAGUCGUA 1758 PiaSpec3 1618 GUGAGGCUUU CGGACUGGC- CUAAGAAGAG -UG-CGACG- CUCGUCUA-G GGCCGGAAAG UUGUUCAAAC UUGGUCGUUU AG-AGGAAGU AAAAGUCGUA 1711 164Darwi 1562 GUGAGGCUUU CGGACUGGC- UCAAGCAGAU -UGGCAACG- AUCAGCCC-G AGCUGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1656 CudConfu 1613 GUGAGGCUUU CAGACUGGC- UUAAGCAGAU -UGGCAACG- AUCAGCCC-G AGCUGGAAAG UUGUCCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1707 ShaFlavi 1587 GUGAGGCUUC CGGACUGGC- UCAGGGAGGU -CGGCAACG- ACCACCCA-G AGCUGGAAAG UUGUCCAAAC UUGGUCGUUU AG-AGGAAGU AAAAGUCGUA 1681 989Roseu 1584 GUGAGGCUUU CGGACUGGC- UCAGGGAGGU -CGGCAACG- ACCACCCA-G AGCUGGAAAG UUGUCCAAAC UUGGUCGUUU AG-AGGAAGU AAAAGUCGUA 1678 39GPanno 1648 GUGAGACUUU CGGACUGGC- UCAGAAAGGU -CGGCAACG- ACCAUUCA-G AGCUGGAAAG UUGUUCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1742 58BInqui 1558 GUGAGGCUU- CGGACCUGG- CCAGGGAGGG -CGGCAACA- AUA-C...... 1597 0LLubri 1634 GUGAGGCUUU CGGACCUAC- GUA-AGGAGG UCGGCAACGA CCACCU-A-G CGCGGGAAAG UUGUUCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1728 13089Alacu 1645 GUGAGGCGUC CGGACUGGC- CCAGAGAGGU -GGGCAACU- ACCACUCA-G GGCCGGAAAG UUCUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1739 NerCinn5 1644 GUGAGGCGUU GGGACUGGC- CCAGGAAGGU -GGGAAACU- ACCAUCCA-G GGCCGGAAAC UUCUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1738 GsaPutte 1626 GUGAGGCGUC CGGACUGGC- CCAGAGUGGU -GGGCAACU- ACCGCUCA-G GGCCGGAAAG CUCUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1720 GibPuli5 1644 GUGAGGCGUC CGGACUGGC- CCAGAGAGGU -GGGCAACU- ACCACUCA-G GGCCGGAAAG CUCUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1738 HpaLute3 1615 GUGAGGCGUC CGGACUGGC- CCAGAGUGGU -GGGCAACU- ACCGCUCA-G GGCCGGAAAG CUCUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1709 HymChrys 1636 GUGAGGCGUC CGGACUGGC- CCAGAGAGGU -GGGCAACC- ACCACUCA-G GGCCGGAAAG CUCUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1730 Hlug13783 1616 GUGAGACCUC CGGACUGGC- CCAGAGAGGU -GGGCAACU- ACCACUCA-G GGCCGGAAAG UUGUUCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1710 144Seti3 1616 GUGAGGCCUC CGGACUGGC- CCAGAGAGGU -GGGCAACU- ACCACUCA-G GGCCGGAAAG UUGUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1710 PsaBoyd6 1648 GUGAGGUUUU CGGACUGGC- CCAGGGAGGC -GGGCAACU- GCCACCCC-G GGCCGGAAAG UUGCCCAAAC UCGGUCGUUU AG-AGGAAGU AAAAGUCGUA 1742 98HRetor 1614 GUGAGGCCUU CGGACUGGC- CCAGAGAGGC -GGGCAACU- GCCACUCA-G GGCCGGAAAG UUGUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1708 MiaCirro 1637 GUGAGGCCUC CGGACUGGC- CCAGAGAGGU -GGGCGACU- ACCACUCA-G GGCCGGAAAG CUGUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1731 GmmPeni3 1663 GUGAGGCUUC CGGACUGGC- CCAGGGAGGU -CGGCAACG- ACCACCCA-G GGCCGGAAAG CUAUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1757 NeuCrass 1664 GUGAGGCUUC CGGACUGGC- CCAGGGAGGU -CGGCAACG- ACCACCCA-G GGCCGGAAAG CUAUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1758 SorFirmi 1614 GUGAGGCUUC CGGACUGGC- CCAGAGAGGU -CGGCAACG- ACCACUCA-G GGCCGGAAAG CUAUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1708 ChtElatu 1643 GUGAGGCUUC CGGACUGGC- CCAGGGAGGU -CGGCAACG- ACCACCCA-G GGCCGGAAAG CUAUCCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1737 KicIvori 1668 GUGAGGCCUU CGGACUGGC- CCAGGGAGGU -CGGCAACG- ACCACCCA-G GGCCGGAAAG UUGGUCAAAC UCCGUCAUUU AG-AGGAAGU AAAAGUCGUA 1762 AuePullu 1630 GUGAGGUGUU CGGACUGGC- CCAGGGAGGU -CGGCAACG- ACCACCCA-G GGCCGGAAAG UUCAUCAAAC UGAGUCAUUU AG-AGGAAGU AAAAGUCGUA 1724 Hr0Werne 1656 GUGAGGCUCU CGGACUGGC- CUAGGGAGGU -UGGCAACG- ACCACCCA-G GGCCGGAAAG UUUGUCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1750 KirAethi 1637 GUGAGGCCUU CGGACUGGC- UUGGGGAGGU -UGGCAACG- ACCACCCU-A AGCCGGAAAG UUCGUCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1731 OpbHerpo 1649 GUGAGGCCUC CGGACUGGC- UUGGAGAGGU -UGGCAACG- ACCACUCU-G AGCCGGAAAG UUCGUCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1743 LehDoli6 1617 GUGAGGCCUU CGGACUGGC- UCGGGGAGGU -UGGCAACG- ACCACCCN-G AGCCGGAAAG UUCGUCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1711 156Elong 1642 GUGAGGCCUU CGGACUGGC- UCGAGGAGGU -UGGCAACG- ACCACCCC-G AGCCGGAAAG UUCGUCAAAC UCGGUCAUUU AG-A...... 1720 PlpBetae 1647 GUGAGGCCUU CGGACUGGC- UCGGGGAGGU -UGGCAACG- ACCACCCC-A AGCCGGAAAG UUCGUCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1741 KirElate 1649 GUGAGGCCUU CGGACUGGC- UCAGGGAGGU -UGGCAACG- ACCACCCC-G AGCCGGAAAG UUCGUCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1743 42MMycop 1618 GUGAGGCCUC CGGACUGGC- UUAGGGAGGU -UGGCAACG- ACCACCCC-G AGCCGGAAAG UUCGUCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1712 21HJunip 1650 GUGAGGCCUC CGGACUGGC- UCAGGGAGGU -UGGCAACG- ACCACCCU-G AGCCGGAAAG UUCGUCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1744 KirMarit 1639 GUGAGGCCUC CGGACUGGC- UCAGGGAGGU -UGGCAACGA CCACCCC--G AGCCGGAAAG UUCGUCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1733 alongi1189 1065 ...... 1064 996Bipol 1065 ...... 1064 996Austr 1615 GUGAGGCCUC CGGACUGGC- CCAGGGAGGU -UGGCAACG- ACCCCCCC-G GGCCGGAAAG CUGGUCAAAC UCGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1709 MonPurpu 1583 GUGAGGCCUU CGGACUGGC- UCAGGGGGGU -UGGCAACG- ACCGCCCA-G AGCCGGAAAG UUGGUCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1677 243Vari2 1636 GUGAGGCCUU CGGACUGGC- UUAGGGAGGU -UGGCAACG- ACCACCCA-G AGCCGGAAAG UUGGUCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1730 OngEquin 1619 GUGAGGCCUU CGGAUGACU- CCA-GUUGAU -CGGCAACG- UUCUUC-A-G AGCCGAGAA...... 1671 750Supe2 1651 GUGAGGCCUU CGGAUUGAC- UCCAGUUGAU -CGGCAACG- UUCUUC-AGG AGCCGAGAAG UUGGUCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1745 ObaFimic 1620 GUGAGGCNUU CGGACUGGC- UCCAGUGGAU -CGGCAA-G- UUCUUC-AGG AGCCGGAAA...... 1673 750Olig2 1619 GUGAGGCCUU CGGACUGGC- UCCAGGAGGU -CGGCAACG- ACCACCCA-G AGCCGGAAA...... 1673 897Doedy 1553 ...... 1552 ObaDelic 1666 GUGAGGUNUU CGGAUUGAC- UUUGGGGAGC -CGGAAACG- GCACUCUA-U UGUUGAGAAG UUGAUCAAAC UUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1760 90HRepan 1666 GUGAGACCUC CGGAUUGGC- UUCGGGGAGC -CGGCAACG- GCACCCUG-U CGCUGAGAAN CUGGUCAAAC UUGGUCAUNU AG-AGGAAGU AAAAGUCGUA 1760 130Nuda2 1677 GUGAGAUCUC CGGAUUGGC- GUUGAGGAGC -CGGCAACG- --ACCUCU-U GGCCGAGAAG CUGAUCAAAC CUGGUCAUUU AG-AGGAAGU AAAAGUCGUA 1769 BulAlbus 1668 GUGAGGCCUC AGGAUCUGC- UUAGAGAAGG -GGGCAACU- CCAUCUCA-G AGCGGAGAAU UUGGACAAAC UUGGUCAUUU AG-AGGAACU AAAAGUCGUA 1762 SayCe108 1901 1911 1921 1931 1941 1951 1961 1971 1981 1991 | | | | | | | | | | 1753 ACAAGG-UUU -CCGUAGGUG AA-CCUG-C- G...... 1778 TcdMaxil 1754 ACAAGG-UUU -CCGUAGGUG AA-CCUG-C- G...... 1779 TcdSetig 1750 ACAAGG-UUU -CCGUAGGUG AA-CCUG-C- G...... 1775 TcdFurca 1749 ACAAGG-UUU -CCGUAGGUG AA-CCUG-C- G...... 1774 TcdApien 1727 ACAAGG-UUU -C...... 1736 Tmarch2639 1734 ACAAGG-UUU -CCGUAGGUG AA-C-UGCCG GGA...... 1762 LemAqu1 1738 ACAAGG-UUU -CCGUAGGUG AA-CCUG-CG G...... 1764 LemTerr1 741 ...... 740 var11783 1735 ACAAGG-UUU -CCGUAGGUG AA-C...... 1755 TriAng1020 1719 ACGGGG-U...... 1725 Mc8Coral 559 ...... 558 HVZ81382 1033 ...... 1032 Loram 1682 ACAAGG...... 1687 986Defle 1683 ACAAGG...... 1688 OidTenui 1685 ...... 1684 Acrassa055 1723 ACAAGG-UUU -CCGUAGGUG AA-C...... 1743 AngFurt 1731 ACAAGG-UUU -CCGUAGGUG AA...... 1750 TriSpl1198 1724 ACAAGG-UUU -CCGUAGGUG AA...... 1743 472Gutta 1688 ACAAGG-UUU -CCGUA...... 1701 7BGrami 1759 ACAAGG-UUU -CCGUAGGUG AA-CCUG-C- G...... 1784 PiaSpec3 1712 ACA...... 1714 164Darwi 1657 ACAAGG-U...... 1663 CudConfu 1708 ACAAGG-U...... 1714 ShaFlavi 1682 ACAAGG...... 1687 989Roseu 1679 ACAAGG...... 1684 39GPanno 1743 ACAAGG-UUU -CCGUA...... 1756 58BInqui 1598 ...... 1597 0LLubri 1729 ACAAGG-UUU -CCGUAGGUG AA-CUGG-C- GGA-A...... 1757 13089Alacu 1740 ACAAGG-UCU -CCGUUGGUG AA-CCUG-C- AGAAGGAUC...... 1773 NerCinn5 1739 ACAAGG-UCU -CCGUUGGUG AA-CCUG-C- AGA-AGGAUC ...... 1772 GsaPutte 1721 ACA...... 1723 GibPuli5 1739 ACAAGG-UCU -CCGUUGGUG AA-CCUG-C- AGAAGGAUC...... 1772 HpaLute3 1710 ACAAGG-UCU -CCGUUGGUG AA-CCUG-C- GG...... 1736 HymChrys 1731 ACAAGG-UCU -CCGUAGGUG AA-CUGG-C- GGA-GGG...... 1761 Hlug13783 1711 ACAAGG-UCU -C...... 1720 144Seti3 1711 ACAAGG-UCU -CC...... 1721 PsaBoyd6 1743 ACAGAG...... 1748 98HRetor 1709 A...... 1709 MiaCirro 1732 ACAAGG-UCU -CCGUA...... 1745 GmmPeni3 1758 ACAAGG-UCU -CCGUUGGUG AA-CCAG-C- GGA-GGGAUC AUUA...... 1795 NeuCrass 1759 ACAAGG-UCU -CCGUUGGUG AA-CCAG-C- GGA-GGGAUC AUUA...... 1796 SorFirmi 1709 ACAAGG-UCU -CCGUAGGUG AA-CCUG-C- GG...... 1735 ChtElatu 1738 ACAAGG-UCU -CCGUU...... 1751 KicIvori 1763 ACAAGG-UUU -CCGUAGGUG AA-CCUG-C- AGA-AGGAUC AAGC...... 1800 AuePullu 1725 ACAAGG-UCU -CCGUAGGUG AA-CCUG-C- GGA-GGGAUC AUUA...... 1762 Hr0Werne 1751 ACA...... 1753 KirAethi 1732 ACAGAG-UUU -CCGUA---- AA...... 1747 OpbHerpo

Figure A36 continued

211 Appendix

Alignment SSU sequences continued

1744 ACAAGGUUUU CCCGUAGGUG GACCCUG-C- GGA-A...... 1775 LehDoli6 1712 ACAAGG-UUU -CCGUAGGU...... 1728 156Elong 1721 ...... 1720 PlpBetae 1742 ACAAGAGUUU -CCGUAGGUG AA-CCUG-C- GGA...... 1770 KirElate 1744 ACAAGG-UUU UCCGUAGGUG AA-CCUG-C- GGA-A...... 1773 42MMycop 1713 ACAAGG-UUU -CCGUAG...... 1727 21HJunip 1745 AC...... 1746 KirMarit 1734 ACAAGG...... 1739 alongi1189 1065 ...... 1064 996Bipol 1065 ...... 1064 996Austr 1710 ACAAGG-UUU -CCGUAGGUG AA-CCU...... 1732 MonPurpu 1678 A...... 1678 243Vari2 1731 ACAAGG-UUU -CCGUAGGUG AA-CC...... 1752 OngEquin 1672 ...... 1671 750Supe2 1746 ACAAGG-UUU -CCGUAGGUG AA-CCUG-C- GGA-AGGAUC AUUACCNNAN AGUGAGAAUU CACUUUCUAC CUGCUCGGCG GCCCUCGGGU C 1830 ObaFimic 1674 ...... 1673 750Olig2 1674 ...... 1673 897Doedy 1553 ...... 1552 ObaDelic 1761 ACAAGG-UUU -CCGUAGGUG AA-CCUG-C- G...... 1786 90HRepan 1761 ACAAGG-UUU -CCGUAGGUG AA-CCUG-C- GGA-AGGAUC AUUA...... 1798 130Nuda2 1770 ACAAGG-UUU -CCGUAGGUG AA-CCUG-C- GGA-AGGAUC AUUA...... 1807 BulAlbus 1763 ACAAGG-UUU -CCGUAGGUG AA-CCUG-C- GGA-AGGAUC AUUA...... 1800 SayCe108

Figure A36 continued

212 Appendix

Alignment LSU Sequences

1 11 21 31 41 51 61 71 81 91 100 | | | | | | | | | | | 1 CUCAGUAACG GCGAGUGAAG CGGCAAAAGC UCAAAUUUGA AAUCUGGCUC UUUCAGGGUC CGAGUUGUAA UUUGUAGAAG AUGCUUUGGG UGUGGCUCUG 100 AatYeast 1 CUCAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCUC UUUUAGGGUC CGAGUUGUAA UUUGUAGAAG AUGCUUCGAG UGUGGCUCCG 100 Sl8Root 1 CUCAGUAACG GCGAGUGAAG CGGUAACAGC UCAAAUUUGA AAUCUGGCCC UUUCAGGGUC CGAGUUGUAA UUUGUAGAAG AUGCUUCGGG UGUAGCUCCG 100 C72Const 1 CUCAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCUC UUUCAGGGUC CGAGUUGUAA UUUGUAGAAG AUGUUUCGAG UGUGGCUCCG 100 C72Longi 1 CUCAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCUC UUUCAGGGUC CGAGUUGUAA UUUGUAGAAG AUGUUUCGAG UGUGGCUCCG 100 C72Micro 1 ...... AACAGC UCAAAUUUGA AAUCUGGCUU CUUUAGGGUC CGAGUUGUAA UUUGUAGAAG AUGCUUCGGG UGUGGCUCCG 76 RtaAceri.2 1 CUCAGUAACG GCGAGUGAAG CGGCAAAAGC UCAAAUUUGA AAUCUGGCUC UUUUAGGGUC CGAGUUGUAA UUUGUAGAAG AUGUUUCGGG UGUGGCUCCG 100 TetMarch 1 ...... 0 HyyVirg2 1 CUCAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAGCUGGCUC UUUCAGGGCC CGCAUUGUAA UUUGUAGAGG AUGCUUCGGG UGCGGCGCUG 100 CudLute2 1 CUCAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAGCUGGCUC UUGCAGAGCC CGCGUUGUAA UUUGUAGAGG AUGCUUCAGG CGCGGCGCUG 100 ShaFlav4 1 CUCAGUAACG GCGAGUGAAG CGGUAACAGC UCAAAUUUGA AAUCUGGCCU C-AC--GGUC CGAGUUGUAA UUUGUAGAGG AUGCUUCGAG CAUGG-UCUG 96 GmyPann4 1 CUCAGUAACG GCGAGUGAAG CGGUAACAGC UCAAAUUUGA AAUCUGGCCU C-AC--GGUC CGAGUUGUAA UUUGUAGAGG AUGCUUCGAG CAUGG-UCUG 96 PdgRose2 1 CUCAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCCC CUUCGGGGUC CGAGUUGUAA UUUGUAGAAG AUGCUUCGGG UGCGGCCCCG 100 OidTenu2 1 CUCAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCCC UCUCAGGGUC CGAGUUGUAA UUUGGAGAAG AUGCUUCGGG UGUGGCCCCG 100 Mx2Defl2 1 CCUAGUAACG GCGAGUGAAG CGGUAACAGC UCAAAUUUGA AAUCUGGCUC CUGCGGAGUC CGAGUUGUAA UUUGUAGAAG AUCUUUGGUG CUUGGCCCGG 100 Py2Mori3 1 CUCAGUAACG GCGAGUGAAG CGGUAACAGC UCAAAUUUGA AAUCUGGAUC CAUUGGAGCC CGAGUUGUAA UUUGUAGAAG AUGCUUUGGC GACGAGUCCG 100 BmrGram4 1 UUCAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCCC --ACGUGGCC CGAGUUGUAA UUUGUAGAGG AUGCUUCGGG CGUGG-UCCG 97 LtiVisco 1 CCCAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCCC -CCC-GGGUC CGAGUUGUAA UUUGUAGAGG AUGCUUUGGU GAGGU-GCCG 97 HpaLutea 1 CCUAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCCC -CUC-GGGCC CGAGUUGUAA UUUGCAGAGG AUGCUUUGGU GAGGC-GCCG 97 HymChry2 1 CUUAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCCC -CC--AGGCC CGAGUUGUAA UUUGUAGAGG AUGCUUCGGC GACGC-GACU 96 GsaPutt3 1 ...... UGA AAUCUGGCC- -CUU--GGUC CGAGUUGUAA UUUGUAGAGG AUGCUUUGGU GAGGU-ACCU 58 NerCinn5 1 CCUAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCUC -UC--GGGCC CGAGUUGUAA UUUGUAGAGG AUGACUU-GA UGCGG-UGCC 95 GibPuli4 1 ...... 0 Pt1Seti3 1 ...... 0 PsaBoyd7 1 CCCAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGUCC CCCCGGGGCC CGAGUUGUAA UUUGAAGAGG AUGCUUCGGC AAGGU-GCCG 99 MiaCirr2 1 CCUAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCGU UUCCGACGUC CGAGUUGUAA UUUGCAGAGG AUGCUUUGGC GACGU-GCCU 99 HpiReto3 1 CCCAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCCC -CCC-GGGUC CGAGUUGUAA UUUGCAGAGG AUGCUCUGGC GAGGC-ACCG 97 GmmPeni9 1 CCUAGUAACG GCGAGUGAAG CCGCAACAGC UCAAAUUUGA GAUCUGGCU- -UC---GGCC CGAGUUGUAA UUUGUAGAGG AAACUUUGGU GAGGC-ACCU 94 SorFimic 1 CCUAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCU- -UC---GGCC CGAGUUGUAA UUUGCAGAGG AAGCUUUGGC GCGGC-ACCU 94 ChtGlob3 1 CCUAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCU- -UC---GGCC CGAGUUGUAA UUUGUAGAGG AAGCUUUGGU GAGGC-ACCU 94 NeuCra34 1 CCUAGUAACG GCGAGUSAAG CSGCAACAGC UCAAAUUUGA AAUCUGGCUC UUUUAGAGUC CGAGUUGUAA UUUSCAGAGG -GGCUUUGGC UUUGGCAGCG 99 CuvInaeq 1 CCUAGUAACG GCGAGUBAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCUC UUUCAGAGUC CGAGUUGUAA UUUGCAGAGG GCGCUUUGGC UUUGGCAGCG 100 CblLunat.2 1 ...... AGAAG GGGCUUUGGC UUUGGAAGCG 25 BipSpici 1 CCUAGUAACG GCGAGUVAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCUC UUUCAGAGUC CGAGUUGUAA UUUGCAGAGG GCGCUUUGCU UUGGC-GCGG 99 CuvEragr.2 1 CCUAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCUC UUUUAGGGUC CGAGUUGUAA UUUGCAGAGG GUGCUUUGCU UUGGC-GCGG 99 PlpHerb9 1 CCUAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCUC UUUUAGGGUC CGAGUUGUAA UUUGCAGAGG GCGCUUUGCG UUGGC-GCGG 99 LehDoli6 1 CCUAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCUC UUUUAGNGUC CGAGUUGUAA UUUGCAGAGG GCGCUUUGCG UUGGC-GCGG 99 OpbHerp2 1 CCUAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCCC UCUCAGGGUC CGAGUUGUAA UUUGUAGAGG GUGCUUUGGC AUUGGUUGUG 100 WetCyli2 1 CCUGAUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAUCUGGCUC CCUUGGGGUC CGAGUUGUAA UUUGUAGAGG GUGCUUUGUA UUAGC-GUGG 99 alongi 1 CUCAGUAACG GCGAGUGAAG CGGCAAUAGC UCAAAUUUGA AAUCUGGCCC UUUCAGGGUC CGAGUUGUAA UUUGCAGAGG GCGCUUUGAG UCGGC-GCAG 99 MphMyco3 1 CCUAGUAACG GCGAGUGAAG CGGCAACAGC UCAAAUUUGA AAGCUGGCCU --UC-GGGUC CGCAUUGUAA UUUGUAGAGG AUGCUUUGGG UGAAAGCCAG 97 AuePul20 1 CUCAGUAACG GCGAGUGAAG CGGCAAAAGC UCAGAUUUGA AAUCUGGCGU CAUUGGCGUC CGAGUUGUAA UCUGUAGAGG AGUAUUCGAG UGUGGCGCUG 100 PezVesi2 1 CCUAGUAACG GCGAGCGAAC AGGGAAGAGC UCAAAUUUGA AAUCUGGCGU CCUCGGCGUC CGAGUUGUAA UCUAUAGA-G ACGUUUUCGU GCCGG-CCGU 98 BulAlbu2 101 111 121 131 141 151 161 171 181 191 200 | | | | | | | | | | | 101 GUUUAAGUUC UUUGGAAUAU UACAUCAUAG AGGGUGAGAA UCCCGUAUUG A-C- AGUCGC CGCGCCCG-U GUAAAGCUCU UUCAACGAGU CGAGUUGUUU 197 AatYeast 101 GUCUAAGUUC CUUGGAACAG GACGUCAUAG AGGGUGAGAA UCCCGUAUUG A-U- GGUUGC UUCGCUCA-U GUGAAGCUCU UUCGACGAGU CGAGUUGUUU 197 Sl8Root 101 GUCUAAGUUC CUUGGAACAG GACGUCAUAG AGGGUGAGAA UCCCGUAUUG A-U- GGUUGU UUCGCCCA-U GUGAAGCUCU UUCGACGAGU CGAGUUGUUU 197 C72Const 101 GUCUAAGUUC CUUGGAACAG GACGUCAUAG AGGGUGAGAA UCCCGUAUUG A-U- GGUUGC UUCGCUCA-U GUGAAACUCU UUCGACGAGU CGAGUUGUUU 197 C72Longi 101 GUCUAAGUUC CUUGGAACAG GACGUCAUAG AGGGUGAGAA UCCCGUAUUG A-U- GGUUGC UUCGCUCA-U GUGAAACUCU UUCGACGAGU CGAGUUGUUU 197 C72Micro 77 GUCUAAGUUC CUUGGAACAG GACGUCAUAG AGGGUGAGAA UCCCGUAUUG A-U- GGCUGC UCCGCCCA-U GUGAAGCUCU UUCGACGAGU CGAGUUGUUU 173 RtaAceri.2 101 GUUUAAGUUC UUUGGAAUAU UACAUCAUAG AGGGUGAGAA UCCCGUAUGU GACCGGCAGC CUUCGCCUAU GUGAAACUCU UUCGACGAGU CGAGUUGUUC 200 TetMarch 1 ...... 0 HyyVirg2 101 GUCUAAGUUC UUUGGAACAA GACGUCAUAG AGGGUGAGAA UCCCGUAUGU GACUAGCUGC CUGUGCCUGU GUGAAGCUCC UUCGACGAGU CGAGUUGUUU 200 CudLute2 101 GUCUAAGUUC UUUGGAACAA GACGUCAUAG AGGGUGAGAA UCCCGUAUGU GACUAGCUGC CUGUGCCUGU GUGAAGCUCC UUCGACGAGU CGAGUUGUUU 200 ShaFlav4 97 GCCUAAGUUC CUUGGAACAG GACGUCAUAG AGGGUGAGAA UCCCGUAUGC GGUCAGGUGC CUACGCUCAU GUGAAGCUCC UUCGACGAGU CGAGUUGUUU 196 GmyPann4 97 GCCUAAGUUC CUUGGAACAG GACGUCAUAG AGGGUGAGAA UCCCGUAUGC GGCCAGGUGC CUACGCUCAU GUGAAGCUCC UUCGACGAGU CGAGUUGUUU 196 PdgRose2 101 GUCUAAGUUC CUUGGAACAG GACGUCACAG AGGGUGAGAA UCCCGUACGU GCCGGUGGUC GCCCCCA--U GUGAAGCUCU UUCGACGAGU CGAGUUGUUU 198 OidTenu2 101 GUCUAAGUUC CUUGGAACAG GACGUCACAG AGGGUGAGAA UCCCGUACGU GGCCGGUGCC GCGCCCA--U GUGAAGCUCU UUCGACGAGU CGAGUUGUUU 198 Mx2Defl2 101 CCU-AAGUUC CUUGGAACAG GACGUCGUAG AGGGUGAGAA UCCCGUAUGC GGCCAGUGUC GGCGCCCG-U GUAAAGCUCU UUCGACGAGU CGAGUUGUUU 198 Py2Mori3 101 GCCUAAGUUC CUUGGAACAG GACGUCAUAG AGGGUGAGAA CCCCGUAUGC GGCCGGGUCU CGAUGCUA-U GUAAAGCUCU UUCGACGAGU CGAGUUGUUU 199 BmrGram4 98 GCCUAAGUCC CUUGGCACAG GGCGUCAUAG AGGGUGAGAA CCCCGUAUGU GGCCGGGUCU AGGCCCG--U GUGAAGCUCC GUCGACGAGU CGAGUUGUUU 195 LtiVisco 98 CCC-GAGUUC CCUGGAACGG GACGCCACAG AGGGUGAGAG CCCCGUCUGG C-U- GGCC-A CCGAGCCUCU GUAAAGCUCC UUCGACGAGU CGAGUAGUUU 193 HpaLutea 98 CCU-GAGUUC CCUGGAACGG GACGCCAUAG AGGGUGAGAG CCCCGUCUGG C-U- GGCC-G CCGAGCCUCU GUAAAGCUCC UUCGACGAGU CGAGUAGUUU 193 HymChry2 97 UCC-GAGUUC CCUGGAACGG GACGCCAAAG AGGGUGAGAG CCCCGUCCGG C-C- GUUC-G CCUAGCCUAU GUGAAGCUCC UUCGACGAGU CGAGUAGUUU 192 GsaPutt3 59 UCC-GAGUUC CCUGGAACGG GACGCCAUAG AGGGUGAGAG CCCCGUCUGG U-- U GGAU - A CCGAGCCUCU GUAAAGCUCC UUCGACGAGU CGAGUAGUUU 154 NerCinn5 96 UUCCGAGUUC CCUGGAACGG GACGCCAUAG AGGGUGAGAG CCCCGUCUGG U-U- GGAU-G CCAAAUCUCU GUAAGUCUCC UUCGACGAGU CGAGUAGUUU 192 GibPuli4 1 ...... GU CGAGUUGUUU 12 Pt1Seti3 1 ...... GU CGAGUUGUUU 12 PsaBoyd7 100 UCC-GAGUUC CCUGGAACGG GACGCCGCAG AGGGUGAGAG CCCCGUACGG U-C- GGAC-G CCGAGCCUCU GUGAAGCUCC UUCGACGAGU CGAGUAGUUU 195 MiaCirr2 100 UCC-GAGUUC CCUGGGACGG GACGCCACAG AGGGUGAGAG CCCCGUACGG U-CUGGC-GC CGAGCCGC-U GUAAAGCUCC UUCGACGAGU CGAGUAGUUU 195 HpiReto3 98 UCC-GAGUUC CCUGGAACGG GACGCCAUAG AGGGUGAGAG CCCCGUAUGG A-C- GACU-G CCGAGCCGCU GUAGAGCUCC UUCGACGAGU CGAGUAGUUU 193 GmmPeni9 95 UCU-GAGUCC CUUGGAACAG GGCGCCAUAG AGGGUGAGAG CCCCGUAUAG U-C- GGAU-G CCGAUCCAAU GUAAAGUUCC UUCGACGAGU CGAGUAGUUU 190 SorFimic 95 UCU-GAGUCC CCUGGAACGG GGCGCCAUAG AGGGUGAGAG CCCCGUAUAG U-U- GGAU-G CCUAGCCUGU GUAAAGCUCC UUCGACGAGU CGAGUAGUUU 190 ChtGlob3 95 UCU-GAGUCC CCUGGAACGG GGCGCCAUAG AGGGUGAGAG CCCCGUAUAG U-C- GGCU-G CCGAUCCAAU GUAAAGCUCC UUCGACGAGU CGAGUAGUUU 190 NeuCra34 100 GUCCAAGUUC CUUGGAACAG GACGUCACAG AGGGUGAGAA UCCCGUACGU GGUCGCUAGC UAUUGCCG-U GUAAAGCCCC UUCGACGAGU CGAGUUGUUU 198 CuvInaeq 101 GUCCAAGUUC CUUGGAACAG GACGUCACAR AGGGUGAGAA UCCCGUACGU GGUCGCUAGC UAUUGCCG-U GUAAAGCCCC UUCGACGAGU CGAGUUGUUU 199 CblLunat.2 26 GUCCAAGUUC CUUGGAACAG GACGUCACAR AGGGUGAGAA UCCCGUACGU GGUCGCUAGC UAUUGCCG-U GUAAAGCCCC UUCGACGAGU CGAGUUGUUU 124 BipSpici 100 UCC-AAGUUC CUUGGAACAG GACGUCACAG AGGGUGAGAA UCCCGUACGU GGUCGCUAGC UAUUGCCG-U GUAAAGCCCC UUCGACGAGU CGAGUUGUUU 197 CuvEragr.2 100 UCC-AAGUUC CUUGGAACAG GACGUCACAG AGGGUGAGAA UCCCGUACGU GGUCGCUAGC UAUCGCCG-U GUAAAGCCCC UUCGACGAGU CGAGUUGUUU 197 PlpHerb9 100 UCC-AAGUUC CUUGGAACAG GACGUCACAG AGGGUGAGAA UCCCGUACGU GGUCGCUGGC CUUCGCCG-U GUAAAGCCUC UUCGACGAGU CGAGUUGUUU 197 LehDoli6 100 UCC-AAGUUC UUUGGAACAG GACGUCACAG AGGGUGAGAA UCCCGUACGU GGUCGCUAGC CUUCGCCG-U GUAAAGCCCC UUCGACGAGU CGAGUUGUUU 197 OpbHerp2 101 GUCUAAGUUC CUUGGAACAG GACGUCACAG AGGGUGAGAA UCCCGUACGU GGCCGCCAAC CUUCGCCG-U GUAAAGCCCC UUCGACGAGU CGAGUUGUUU 199 WetCyli2 100 UCU-AAGACC CUUGGAACAG CGCGUCACAG AGGGUGAGAA UCCCGUAUGU GGCCWGCAGC UCUUGCCU-U GU-AAGCCCC UUCGACGAGU CGAGUUGUUU 196 alongi 100 CCU-AAGUUC CUUGGAACAG GUCAUCAUAG AGGGUGAGAA UCCCGUAUGU GGCUGCUUGC CUUCGCCG-U GUAAAGCCCC UUCGAUGAGU CGAGUUGUUU 197 MphMyco3 98 UCU-AAGUUC CUUGGAACAG GACGUCAUAG AGGGUGAGAA UCCCGUAUGU GACUGGAAUG UUAACCUA-U GUAAAGCUCC UUCGACGAGU CGAGUUGUUU 195 AuePul20 101 GCUUAAGUUC CUUGGAACAG GACGUCAUAG AGGGUGAGAA CCCCGUUAAC GGCCUUUGCU UAUGCUCA-U GUGAAUCUCC UUCGACGAGU CGAGUUGUUU 199 PezVesi2 99 GUCCAAGUUC CUUGGAACAG GAUAUCAAAG AGGGUGACAA UCCCGUACUU GACACGACCC GGUGCUC--U GUGAUACGUU UUCUACGAGU CGAGUUGUUU 196 BulAlbu2 201 211 221 231 241 251 261 271 281 291 300 | | | | | | | | | | | 198 GGGAAUGCAG CUCAAAAUGG GAGGUAUAUU UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 297 AatYeast 198 GGGAAUGCAG CUCAAAAUGG GUGGUAAAUU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 297 Sl8Root 198 GGGAAUGCAG CUCUAAAUGG GUGGUAAAUU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 297 C72Const 198 GGGAAUGCAG CUCAAAAUGG GUGGUAAAUU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 297 C72Longi 198 GGGAAUGCAG CUCAAAAUGG GUGGUAAAUU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 297 C72Micro 174 GGGAAUGCAG CUCAAAAUGG GUGGUAAAUU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 273 RtaAceri.2 201 GGGAAUGCAG CUCAAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 300 TetMarch 1 ...... AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCAGUU 40 HyyVirg2 201 GGGAAUGCAG CUCAAAAUGG GUGGUGUACU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 300 CudLute2 201 GGGAAUGCAG CUCAAAAUGG GUGGUGUACU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 300 ShaFlav4 197 GGGAAUGCAG CUCAAAAUGG GUGGUAAAUU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 296 GmyPann4 197 GGGAAUGCAG CUCAAAAUGG GUGGUAAAUU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 296 PdgRose2

Figure A37: Alignment of LSU sequences.

213 Appendix

Alignment LSU Sequences continued

199 GGGAAUGCAG CUCAAAAUGG GUGGUAAAUU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 298 OidTenu2 199 GGGAAUGCAG CUCAAAAUGG GUGGUAUAUU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 298 Mx2Defl2 199 GGGAAUGCAG CUCAAAAUGG GUGGUAAAUU UCAUCUAAGC UAAAUAUGGG CCAGAGACCG AUAGCGAACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 298 Py2Mori3 200 GGGAAUGCAG CUCAAAAUGG GUGGUAAAUU UCAUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGAACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 299 BmrGram4 196 GGGAUUGCAG CUCAAAAUGG GUGGUAAAUU UCAUCUAAGC UAAAUACCGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 295 LtiVisco 194 GGGAAUGCUG CUCAAAAUGG GAGGUAUAUG UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACCU 293 HpaLutea 194 GGGAAUGCUG CUCUAAAUGG GAGGUAUAUG UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 293 HymChry2 193 GGGAAUGCUG CUCUAAAUGG GAGGUAUACG UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 292 GsaPutt3 155 GGGAAUGCUG CUCUAAAUGG GAGGUAUAUG UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 254 NerCinn5 193 GGGAAUGCUG CUCUAAAUGG GAGGUAUAUG UCUUCUAAGC UAAAUACCGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 292 GibPuli4 13 GGGAAUGCUG CUCAAAAUGG GAGGUAAACC CCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 112 Pt1Seti3 13 GGGAAUGCUG CUCAAAAUGG GAGGUAAACC CCUUCUAAGC UAAAUACUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 112 PsaBoyd7 196 GGGAAUGCUG CUCAAAAUGG GAGGUAAACC CCUUCUAAGC UAAAUACCGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 295 MiaCirr2 196 GGGAAUGCUG CUCUAAAUGG GAGGUAAACU CCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 295 HpiReto3 194 GGGAAUGCUG CUCUAAACGG GAGGUAAAUC UCUUCUAAGC UAAAUACUGG CCACAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 293 GmmPeni9 191 GGGAAUGCUG CUCAAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUAUAGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 290 SorFimic 191 GGGAAUGCUG CUCAAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUACCGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 290 ChtGlob3 191 GGGAAUGCUG CUCAAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 290 NeuCra34 199 GGGAAUGCAG CUCUAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 298 CuvInaeq 200 GGGAAUGCAG CUCUAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 299 CblLunat.2 125 GGGAAUGCAG CUCUAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA CCGAAAGAUG AAAAGCACUU 224 BipSpici 198 GGGAAUGCAG CUCUAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 297 CuvEragr.2 198 GGGAAUGCAG CUCUAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 297 PlpHerb9 198 GGGAAUGCAG CUCUAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUACUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 297 LehDoli6 198 GGGAAUGCAG CUCUAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUACUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 297 OpbHerp2 200 GGGAAUGCAG CUCUAAAUGG GAGGUAAAUU CCUUCUAAGC UAAAUACUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 299 WetCyli2 197 GGGAAUGCAG CUCUAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUACUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 296 alongi 198 GGGAAUGCAG CUCUAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUAUUGG CCAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 297 MphMyco3 196 GGGAAUGCAG CUCUAAAUGG GAGGUAAAUU UCUUCUAAGC UAAAUAUUGG CGAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGCACUU 295 AuePul20 200 GGGAAUGCAG CUCUAAAUGG GUGGUAAAUU CCAUCUAAGC UAAAUACUGG CAAGAGACCG AUAGCGCACA AGUAGAGUGA UCGAAAGAUG AAAAGAACUC 299 PezVesi2 197 GGGAAUGCAG CUCAAAAUGG GUGGUGAGUU CCAUCUAAGC UAAAUAUUGG CGAGAGACCG AUAGCGAACA AGUACCGUGA GGGAAAGAUG AAAAGCACUU 296 BulAlbu2 301 311 321 331 341 351 361 371 381 391 400 | | | | | | | | | | | 298 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GCACGCAGUU GAUCAUCCGG UGUUCUCACC GGGGCACUCU 397 AatYeast 298 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GCACGCAGUU GAUCAUCCGG UGUUCUCACC GGUGCACUCU 397 Sl8Root 298 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GCACGCAGUU GAUCAUCCGG UGUUCUCACU GGUGCACUCU 397 C72Const 298 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GCGCGUAGUU GAUCAUCCGG GGUUCUCCCC GGUGCACUCG 397 C72Longi 298 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GCGCGUAGUU GAUCAUCCGG GGUUCUCCCC GGUGCACUCG 397 C72Micro 274 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AAUCAGACUU GCACGCUGCC GAUCAUCUGG GGUUCUCUCC AGUGCACUCG 373 RtaAceri.2 301 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGUUUGC AACCAGACUU GCACGCAGUU GAUCAUCCGG UGAUCUCACC GGGGCACUCU 400 TetMarch 41 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GCACGCCGUC GAUCAUCUCA GGUUCUCCUG GGUGCACUCG 140 HyyVirg2 301 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AAUCAGACUU GCGCGGCGUC GAUCAACCUG GGUUCUCCCU GGUGCACUCG 400 CudLute2 301 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AAUCAGACUU GCGCGGCGUC GAUCAACCUG GGUUCUCCCU GGUGCACUCG 400 ShaFlav4 297 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GCGCGCGGCC GAUCAUCCGG UGUUCUCACC GGUGCACUCG 396 GmyPann4 297 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GCGCGCGGCC GAUCAUCCGG UGUUCUCACC GGUGCACUCG 396 PdgRose2 299 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GCGCGUGGCC GAUCAUCCGG UGUUCGCACC GGUGCACUCG 398 OidTenu2 299 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GCGCGCGGCC GAUCAUCCGG UGCUCGCACC GGUGCACUCG 398 Mx2Defl2 299 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GGGCGUCGCU GAUCAUCCAA AGACAUCUUU GGUGCACUUG 398 Py2Mori3 300 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AACCAGACUU GGGCACUGUG GAUCAUCCGA GGUUCUCCUC GGUGCACUCG 399 BmrGram4 296 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC GACCAGACUU GGGCGCGGCU GAUCAUCCGG CGUUCUCGCC GGUGCAUCGG 395 LtiVisco 294 UGAAAAGAGG GUUAAAUAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGU GACCAGACUU GGGCGCGGCG GAUCAUCCGG GGUUCUCUCC GGUGCACUUC 393 HpaLutea 294 UGAAAAGAGG GUUAAAUAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUCAU GACCAGACUU GGGCCUGGCG AAUCAUCCGG CGUUCUCGCC GGUGCACUUC 393 HymChry2 293 UGAAAAGAGG GUUAAAUAGU ACGUGAAAUU GCUGAAAGGG AAGCGCUUAU GACCAGACUU GGGCUAGGUG AAUCAUCCGG CGUUCUCGCC GGUGCACUUU 392 GsaPutt3 255 UGAAAAGAGG GUUAAAUAGU ACGUGAAAUU GUUGAAAGGG AAGCGCUUGU GACCAGACUU GGGCUUGGUU AAUCAUCCAG GGUUCUCCCU GGUGCACUUG 354 NerCinn5 293 UGAAAAGAGA GUUAAAAAGU ACGUGAAAUU GUUGAAAGGG AAGCGUUUAU GACCAGACUU GGGCUUGGUU AAUCAUCUGG GGUUCUCCCC AGUGCACUUU 392 GibPuli4 113 UGAAAAGAGA GUUAAACAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC GACCAGACUU GGCUCGUCGA AUCAGCCGUC GCUCGUCGGC GGCGCAUUUC 212 Pt1Seti3 113 UGAAAAGAGA GUUAAACAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC GACCAGACUU GGCCCGUCGA AUCAGCCGCC GCUCGUCGGC GGCGCACUUC 212 PsaBoyd7 296 UGAAAAGAGA GUUAAAAAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC GACCAGACUC GGCCCGUCGG AUCAGCCGUC GCUCGUCGGC GGCGCACUCC 395 MiaCirr2 296 UGAAAAGAGA GUUAAACAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUAU GACCAGACUU GGGCUUGGUG GUUCAGCCGU CCUUCUGGGC GGUGCACUCC 395 HpiReto3 294 UGAAAAGAGA GUUAAACAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUCAU GACCAGACUU GCGCCUGGCG GAUCAUCCGG CGUUCUCGCC GGUGCACUCC 393 GmmPeni9 291 UGAAAAGAGG GUUAAAUAGC ACGUGAAAUU GUUGAAAGGG AAGCGUUUGU GACCAGACUU GCGCCGUUCC GAUCAUCCGG UGUUCUCACC GGUGCAUCGG 390 SorFimic 291 UGAAAAGAGG GUUAAAUAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGU GACCAGACUU GCGCCGGGCG GAUCAUCCGG UGUUCUCACC GGUGCAUCCG 390 ChtGlob3 291 UGAAAAGAGA GUCAAAAAGU ACGUGAAAUU GUUGAAAGGG AAGCGUUUGU GACCAGACUU CGCCUUCCAU CAUCAUGUGC UGUUCUCACC GGUGCACUCG 390 NeuCra34 299 UGGAAAGAGA GUCAAACAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AGCCAGACUU GCUUGCAGUU GCUCAUCCGG GCUUUUGCCC GGUGCACUCU 398 CuvInaeq 300 UGGAAAGAGA GUCAAACAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUUC AGCCAGACUU GCUUGCAGUU GCUCAUCCGG GCUUUU-CCC GGUGCACUCC 398 CblLunat.2 225 UGGAAAGAGA GUCAAACAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AGCCAGACUU GCUUGCAGUU GCUCAUCCGG GCUUUUGCCC GGUGCACUCU 324 BipSpici 298 UGGAAAGAGA GUCAAACAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AGCCAGACUU GCUUGCAGUU GCUCAUCCGG GCUUUUGCCC GGUGCACUCU 397 CuvEragr.2 298 UGGAAAGAGA GUCAAACAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AGCCAGACUU GCUUUCAGUU GCUCAUCCGG GCUUUUGCCC GGUGCACUCU 397 PlpHerb9 298 UGGAAAGAGA GUCAAAUAGC ACGUGAAAUU GUUAAAAGGG AAGCGCUUGC AGCCAGACUU GCCCGUAGUU GCUCAUCCAG GCUUUUGCCU -GUGCAUUCU 396 LehDoli6 298 UGGAAAGAGA GUCAAAUAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AGCCAGACUU GCCNGUAGUU GCUUNUCCGG ACUUUUGUCC GGUGCACUCU 397 OpbHerp2 300 UGGAAAGAGA GUCAAAAAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AGCCAGACUU GCCUGUAGUU GCUCACCUCG GCUUCUGCCU UGGGUACUCU 399 WetCyli2 297 UGGAAAGAGA GUCAAAAAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AGCCAGACUU GCCCGUAGUU GUUCAUCUAG GCUCUUGCCU AGUGCACUCU 396 alongi 298 UGGAAAGAGA GUCAAAAAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AGCCAGACGU GCCCGUAGUU GCUCAUCUGG GGUUCUCCCC AGUGCACUCU 397 MphMyco3 296 UGGAAAGAGA GUUAAAAAGC ACGUGAAAUU GUUGAAAGGG AAGCGCUUGC AAUCAGACUU G-UUUAAACU GUUCGGCCGG UCUUCUGACC GGUUUACUCA 394 AuePul20 300 UGAACAGAGA GUUAAAUAGU ACGUGAAAUU GUUGAAAGGG AAGCACUUGC UACCAGACAC GCCUGUAGUU GAUCAACCUC CAUUCUUGGU GGUGCACUCA 399 PezVesi2 297 UGGAAAGAGA GUUAAACAGU ACGUGAAAUU GUUAAAAGGG AAACGAUUGA AGUCAGUCGU G---UCUGGG AGGCUCAGCC GGUU-CUGCC GGUGUAUUCC 392 BulAlbu2 401 411 421 431 441 451 461 471 481 491 500 | | | | | | | | | | | 398 GCUGCGUUCA GGCCAGCAUC CCCUUUGGUG GUUGGAUAAA GGCCUGGGAA UGAGCUUCUU UCGGGGAGUG UUAUAGCGGU UUUGGUGCAA UGCAGCUACC 497 AatYeast 398 GCUGCGUUCA GGCCAGCAUC GGUUUGG-UG GUUGGAUAGA G...... 437 Sl8Root 398 GCUGCGUUCA GGCCAGCAUC GGUUUUGGUG GUUGGAUAAA GGCCUGGGAA UGAGCUUCUU UCGGGGAGUG UUAUAGCCCU CGGUGCAAUG CAGCCACCGG 497 C72Const 398 AUUACGCUCA GGCCAGCAUC GGUUUGGGUG GUUGGAUAAA GGCCUGGGAA UGGGCUUCCU UCGGGGAGUG UUAUAGCCCU CGGUGCAAUG CAGCCAUCCG 497 C72Longi 398 AUUACGCUCA GGCCAGCAUC GGUUUGGGUG GUUGGAUAAA GGCCUGGGAA UGGGCUUCCU UCGGGGAGUG UUAUAGCCCU CGGUGCAAUG CAGCCAUCCG 497 C72Micro 374 GUUGCGUUCA GGCCAGCAUC GGUUUGGGUG GUUGGAUAAA GGCCUGGGAA UGAGCUUCCU UUGGGGAGUG UUAUAGCCCU CGGUGCAAUG CAGUCAUCCG 473 RtaAceri.2 401 GCUGCGUUCA GGCCAGCAUC GGUUUUGGUG GUUGGAUAAA GGCCUGGGAA UGGGCUUCCU UCGGGGAGUG UUAUAGCCCU CGGUGCAAUG CAGCCUCCGG 500 TetMarch 141 GCGGUGUUCA GGCCAGCAUC GGUUUCGGUG GUUGGAUAAA GGCCUGGGAA UGGGCUCCUC UUGGGGAGUG UUAUAGCCCU CGGUGCAAUG CAGCCACCGG 240 HyyVirg2 401 GCGCUGCUCA GGCCAGCAUC AGCUUCAGCG GUUGGAUAAA GGCCUGGGAA CGGGGUUACU UCGGUGACUG UUAUAGCCCU AGGUGCAAUG CAGCCGCUGG 500 CudLute2 401 GCGCUGCUCA GGCCAGCAUC AGUUUCAGCG GUUGGAUAAA GGCCUGGGAA CGGGGUUACU UCGGUGACUG UUAUAGCCCU AGGUGCAAUG CAGCCGUUGG 500 ShaFlav4 397 GCCGUGCUCA GGCCAGCAUC GGUUUCGGCG GUUGGAUAAA GGCCUAGGAA UGGGCUCCUC UCGGGGAGUG UUAUAGCCUG GGGUGCAAUG CAGCCGCUGG 496 GmyPann4 397 GCCGUGCUCA GGCCAGCAUC GGUUUUGGCG GCUGGAUAAA GGCCCAGGAA UGGGCUCCUC UCGGGGAGUG UUAUAGCCUA GGGUGCAAUG CAGCCGCUGG 496 PdgRose2 399 ACCGCGCGCA GGCCAGCAUC GGUUUGGGUG GCUGGAUAAA GGCCUGGGAA UGGGCUCCCC UCGGGGAGUG UUAUAGCCCG AGGUGCAAUG CAGCCACCCG 498 OidTenu2 399 ACCGCGCGCA GGCCAGCAUC GGUUUGGGCG GUUGGAUAAA GGCCCAGGAA UGGGCUCUCU UCGGGGAGUG UUAUAGCCUG GGGCGCAAUG CAGCCGCCCG 498 Mx2Defl2 399 ACGACGCACA GGCCAGCAUC GGUUGGAGUG GUGGGAGAAA GGUUGCGGAA CGGGCUCUUU UCGGAGAGUG UUAUAGCCGG CGACGCAAUA CCGCCACCCC 498 Py2Mori3 400 GCAGUGCGCA GGCCAGCAUC AGUUUGGGUG GUUGGAUAAA GACCUUGGAA UGAGCUCCUU UCGGGGAGUG UUAUAGCCAC AGGUGCCAUG CGACCACCCG 499 BmrGram4 396 CCGCGUGCCA GGCCAGCAUC GGUUCCGGCG GCCAGACAAA GGCCGGCGAA CGGGCUCCCC -CAGGGAGUG UUAUAGCGCC CGGCGCCAUG UGGCCGCUGG 494 LtiVisco 394 GCCGCGUUCA GGCCAGCAUC AGUUCGUCGC GGGGGAUAAA GGCUUGGGAA CGGGCUCUCC ---GGGAGUG UUAUAGCCCG UUGCAUAAUA CCCUGCGGUG 490 HpaLutea 394 GCCAGGCUCA GGCCAGCAUC AGUUCGCCGC GGGGGAUAAA GGUUUGGGAA CGGGCUCCUC -C-GGGAGUG UUAUAGCCCG UUGCGUAAUA -CCUGCGGUG 490 HymChry2 393 GCCUG-UCCA GGCCAGCAUC AGUUCGCUUC GGGGGAUAAA GGCUUGGGAA UGAGCUCCUC -C-GGGAGUG UUAUAGACCG UUGUGUAAUA CCCUGGGGUG 489 GsaPutt3 355 GCCA-GCCCA GGCCAGCAUC AGUUUGUCGC GGGGGAUAAA GGCGUGGGAA UGGGCUCCCC -C-GGGAGUG UUAUAGCCCU UCGUGUAAUA CCCUGCUUUA 451 NerCinn5 393 UCCAG-UCCA GGCCAGCAUC AGUUUUCGCC GGGGGACAAA GACUUGGGAA UGGGCUCCCC UC-GGGAGUG UUAUAGCCCG UUGUGUAAUG CCCUGGCGGG 490 GibPuli4 213 GGCGGGCUCA GGCCAGCAUC AGUUCGCUGA GGGGGAGAAA GGCGGAGGAA UGGGCUCUUC ----GGAGUG UUAUAGCCUA CCGUAUAAUA CCCCUCGGUG 308 Pt1Seti3 213 GGCGGGCUCA GGCCAGCAUC AGUUCGCUGA GGGGGAGAAA GGCGAGGGAA UGGGCUCUUC ----GGAGUG UUAUAGCCCG CCGCGCAAUA CCCCUCGGCG 308 PsaBoyd7 396 GGCGGGCUCG GGCCAGCAUC AGUUCGCCUG GGGGGAGAAA GGCGGGGGAA UGGGCUCCAC ----GGAGUG UUAUAGCCCA CCGCGUAAUA CCCCCGGGCG 491 MiaCirr2 396 GCCGG-UUCA AGGCAGCAUC AGUUGCCGGU UGGGGAGAAA GGUGGGGGAA CGGGCUCCUC ----GGAGUG UUAUAGCCC- --ACACUAUG CCCUCCUGGC 487 HpiReto3

Figure A37 continued

214 Appendix

Alignment LSU Sequences continued

394 GCCUGGCACA GGCCAGCAUC GGUUCGCUGU CGGGGAGAAA GGCGGGGGAA UGGGCUCUUC ----GGAGUG UUAUAGCCCC CCGUGUAAUA CCCUUCGGCG 489 GmmPeni9 391 GGCGG-CUCA GGCCAGCAUC GGUUUUGGUG GGGGGAUAAA GGUCCGGGAA CGAGCUC--C UCCGGGAGUG UUAUAGCCCU GGGCGUAAUG CCCUCGCUGG 487 SorFimic 391 CCCGG-CUCA GGCCAGCAUC GGUUCUCGCG GGGGGAUAAA GGUCCGGGAA CGAGCUCC-- UCCGGGAGUG UUAUAGCCCG GGGCGUAAUG CCCUCGCGGG 487 ChtGlob3 391 GACAGCU-CA GGCCAGCAUC GGUUUUG-GC GGGGGAUAAA GGUCCGGGAA CGAGCUCUCC ---GGGAGUG UUAUAGCCCG GCGU--AAUG CCUCGC-CGG 482 NeuCra34 399 UCUGCAGGCA GGCCAGCAUC AGUUUGGGCG GUGGGAUAAA GGUCUUGACA CGUCCUUCCU UC-GGUUGGC AUAUAGGGGA GACGUC-AUA CCACCGCCUG 496 CuvInaeq 399 UCUGCAGGCA -GCCAGCAUC AGUUU-GGCG GUGGGAUAAA GGUCUUGACA CGUCCUUCCU UC-GGUU-GC AUAUAGGG-A GACGUC-AUA CCACCGCCUG 492 CblLunat.2 325 UCUGCAGGCA GGCCAGCAUC AGUUUGGGCG GUGGGAUAAA GGUCUUGUCA CGACCUUCCU UC-GGUUGGC UUAUAGGGGA GACGCC-AUA CCACCGCCUG 422 BipSpici 398 UCUGCAGGCA GGCCAGCAUC AGUUUGGGCG GUGGGAUAAA GGUCUUGUCA CGACCUCUCU UCGGGGAGGA UUAUAGGGGA GGCGAC-AUA CCACCGCCUG 496 CuvEragr.2 398 UCUGUAGGCA GGCCAGCAUC AGUUUGGGCG GUGGGAUAAA GGCUUUGGAA UGGGCUCUCU UCGGGGAGGC CUUUAGGGGA AGGUGUAAUA CCACCGCUGG 497 PlpHerb9 397 UCUAUGGGCA GGCCAGCAUC AGUUUGGGCG GUUGGAUAAA GGUCUUGUCA ...... 446 LehDoli6 398 UCUNNGGGCA GGCCAGCAUC AGUUUAGGCG GUUGGAUAAA GGUCUUAUCA ...... 447 OpbHerp2 400 UCUACGGGCA GGCCAGCAUC AGUCCGGGCG GUUGGAUAAA UGCCUCUAAA UGACCUCUCC UCGGGGAGGA UUAUAGGGUA GGCGGC-AUA CAACCGCCUG 498 WetCyli2 397 UCUGCGGGCA GGCCAGCAUC AGUCCAGGCG GUCGGAUAAA GGCCUGCGGA A-GUGGCUCU UUCGGGAGUG UUAUAGCCCA GGGUGCCAUG CGGCCGCCU- 494 alongi 398 UCUAUGGGCA GGCCAGCAUC AGUCCGGGCG GUUGGAGAAA GACCUUGUCA ...... 447 MphMyco3 395 GUUU-GGACA GGCCAGCAUC AGUUUCGGCG GCCGGAUAAA GGCUCGGGAA UGGGCCUUCA CUUGAAGGUG UUAUAGCCCA GGGUGUAAUA CGGCCGCCGG 493 AuePul20 400 GCUAUAGGUG GGUCAGCAUC AGUUACGGCG GUGGGAUAAA GACCUGGGAA UGAGCUUCUU UCGGGAAGUG UUAUAGCCCU GGGUGUAAUA CCGCCGCUGU 499 PezVesi2 393 UC-UCAGACG GGUCAACAUC AGUUUUGUCC GACGGAUAAU GGCGGGGGAA AGAGCACCUC -C-GGGUGUG UUAUAGCCCG CUGUCGCAUA CGCCGGAUGA 489 BulAlbu2 501 511 521 531 | | | | 498 GGGACGAGGA CCGCUUCGGC UAGGAUCGGC GUAAGGUUG 536 AatYeast 438 ...... 437 Sl8Root 498 GACCAGG...... 504 C72Const 498 GACCAGG...... 504 C72Longi 498 GACCAGG...... 504 C72Micro 474 GACCAGGACC GCGCUUCGGC UAGGAUCGGC GUAAGGUUG 512 RtaAceri.2 501 GACCAGGACC GCGCUUCGGC UAGGAUCGGC GUAAGGUUG 539 TetMarch 241 GACCAGGACC GCGCUUCGGC UAGGAUCGG...... 269 HyyVirg2 501 GACUAGGACC GCGCUUCGGC UAGGAUCGGC GUAAGGUUG 539 CudLute2 501 GACUAGGACC GCGCUUCGGC UAGGAUCGGC GUAAGGUUG 539 ShaFlav4 497 GACCAGGACC GCGCUUCGGC UAGGAUCGGC GU...... 528 GmyPann4 497 GACCAGGACC GCGCUUCGGC UAGGAUCGGC GU...... 528 PdgRose2 499 GACCAGGACC GCGCUUCGGC UAGGAUCGGC GU...... 530 OidTenu2 499 GACCAGGACC GCGCUUCGGC UAGGAUCGGC GU...... 530 Mx2Defl2 499 GACCAGGACC GCGCUUCGGC UAGGAUCGGC GUAAGGUUG 537 Py2Mori3 500 GACUAGGACC GCGCUUCGGC UAGGAUCGGC GUAAGGUUG 538 BmrGram4 495 GAACAAGACC GCGCCCUGGC UAAGAUCGGC GUAA..... 528 LtiVisco 491 GACUAGGACC GCGCAUCUGC AAGGAUCGGC GUAAGGUCA 529 HpaLutea 491 GACUAGGUCC GCGCA-CUGC AAGGAUCGGC GUAAGGUCA 528 HymChry2 490 GACUAGGUAC GCGU-UCUGC AAGGAUCGGC GUAAGGUCA 527 GsaPutt3 452 GACUAGGUUC GCGCAUCUGC AAGGAU...... 477 NerCinn5 491 GACUAGGUUC GCGCUUCUGC AAGGAU...... 516 GibPuli4 309 GACUAGGACC GCGCAUCUGC AAGGAUCGGC GUAAGGUUG 347 Pt1Seti3 309 GACUAGGACC GCGCAUCUGC AAGGAUCGGC GUAAGGUCG 347 PsaBoyd7 492 GACUAGGACC GCGCGUAUGC AAGGAUCGGC GUAAGG... 527 MiaCirr2 488 GACUAGUU-A GCGUUCUU-C ACGGAUCGGC GUAAGGUUA 524 HpiReto3 490 GACCAGG...... 496 GmmPeni9 488 GACCAGGUUC GCGCAUC-UG CGAUGCUGCG UAAAGGUCA 525 SorFimic 488 GACCAGGUUC GCGCAUC-UG CAGGAUCGGC GUAAGGUCA 525 ChtGlob3 483 GACCAGGUUC GCGCAUCUGC AAGGAUCGGC GUAAGGUCA 521 NeuCra34 497 GACUAGGUCC GCGCAUCUGC UAGGAU...... 522 CuvInaeq 493 GACUAGGUCC GCGCAUCUGC UAGGAUCGGC GUAAGGCUG 531 CblLunat.2 423 GACUAGGUCC GCGCAUCUGC UAGGAU...... 448 BipSpici 497 GACUAGGUCC GCGCAUCUGC UAGGAUCGGC GUAAGGCUG 535 CuvEragr.2 498 GACUAGGUCC GCGCUUCUGC UAGGAUCGGC GUAAGGCUG 536 PlpHerb9 447 ...... 446 LehDoli6 448 ...... 447 OpbHerp2 499 GACUAGGUCC GCGCAUCUGC UAGGAUCGGC GUAAGGCUG 537 WetCyli2 495 GACUAGG...... 501 alongi 448 ...... 447 MphMyco3 494 GACUAGGUCC GCGCUUCGGC UAGGAUCGGC GUAAGGUUG 532 AuePul20 500 GACUAGGCCC GCGCUUCGGC GAGGAUCGGC AUAAGGUCG 538 PezVesi2 490 GACUAGGCAU GCAGCUUAGC UAGGAUUGAC GUAAGGCUU 528 BulAlbu2

Figure A37 continued

215 Appendix

Alignment ITS sequences Tetracladium marchalianum

1 11 21 31 41 51 61 71 81 91 100 | | | | | | | | | | | 1 ...... G CGGAAG-AUC AUUACC-GAG UUCAUGCCCU AUAAACGGG- UAGA-UCUCC CACCCU-UUG UAUACCUUU- 65 Tmarch312B 1 ...... CCGUA GGUGAACCUG CGGAAGGAUC AUUACC-GAG UUCAUGCCCU AUAAACGGG- UAGA-UCUCC CACCCU-UUG UAUACCUUU- 80 Tmarch2629 1 ...... G CGGAAG-AUC AUUACC-GAG UUCAUGCCCU AUAAACGGG- UAGA-UCUCC CACCCU-UUG UAUACCUUU- 65 TmarchL27 1 ...... CAA GGUUUCCGUA GGUGAACCUG CGGAAGGAUC AUUACC-GAG UUCAUGCCCU AUAAACGGG- UAGA-UCUCC CACCCU-UUG UAUACCUUU- 88 Tmarch2639 1 .UCGUAACAA GGUUUCCGUA GGUGAACCUG CGGAAGGAUC AUUACC-GAG UUCAUGCCCU AUAAACGGG- UAGA-UCUCC CACCCU-UUG UAUACCUUU- 94 Tmarch1939 1 ...... AACCUG CGGAAGGAUC AUUACC-GAG UUCAUGCCCU AUAAACGGG- UAGA-UCUCC -ACCCU-UUG UAUACCUUU- 70 Tmarch2619 1 ...... G CGGAAG-AUC AUUACC-GAG UUCAUGCCCU AUAAACGGG- UAGA-UCUCC CACCCU-UUG UAUACCUUU- 65 TmarchEL50 1 ...... G CGGAAG-AUC AUUACC-GAG UUCAUGCCCU -UA--CGGG- NAGA-UCUCC CACCCU-UUG UAUAC--UAU 61 TcdFurc2 1 ...... G CGGAAG-AUC AUUACC-GAG UUCAUGCCCU -U---CGGGG UAGA-UCUCC CACCCU-UUG UAUAC--UAU 61 TcdMaxi2 1 ...... G CGGAAG-AUC AUUACC-GAG UUCAUGCCCU -UA--CGGG- UAGA-UCUCC CACCCU-UUG UAUACCUU-- 61 TcdApie2 1 ...... C AUUACC-GAG UUCAUGCCCU -CA--CGGG- UAGA-CCUCC CACCCU-UUG UAUACCUU-- 52 AxeEctom 1 GUCGUAACAA GGUUUCCGUA GGUGAACCUG CGGAAGGAUC AUUACC-GAG AU-AUGCCCU -U---CGGGG UAGA-CCUCC CACCCU-GUG UAUACCUU-- 90 DyiDimor 1 ...... C AUUAC-AGAG UUCAUGCCCU -U---CGGGG UAGA-CCUCC CACCC--UUG AAUA---UAU 50 Hl1Speci 1 ...... C AUUAC-AGAG UUCAUGCCCU -U---CGGGG UAGA-CCUCC CACCC--UUG AAUA---UAU 50 EczIsola 1 ...... C-AGAG UUCAUGCCCU --- CA CGGG-- UAGA UCUCC CACCC--UUG AAUAUUUUAU 48 EodMycor.2 1 ...... UCCGUA GGUGAACCUG CGGAAGGAUC AUUAC-AGAG UUCAUGCCCU -UA--CGGG- UAGA-UCUCC CACCC--UUG -AAUACU-AU 76 EodMycor.2 1 ...... C AUUAC-AGAG UUCUCGCCCU ----CCGGG- UAGAU UCUCC CACCC-ACUG UGAUUG--AU 51 OidTenu2 101 111 121 131 141 151 161 171 181 191 200 | | | | | | | | | | | 66 ACCU-UUGUU GCUUUGGCGG GCCG-CCUAG ------CUACUGGCU UCNGCUGGUA AGUGCCCGCC AGAGG-A-CC CAAAACCC-- -UG-AAUUAU 146 TcdMarc5 81 ACCU-UUGUU GCUUUGGCGG GCCG-CCUAG ------CUACUGGCU UCGGCUGGUA AGUGCCCGCC AGAGG-A-CC CAAAACCC-- -UG-AAUUAU 161 Tmarch2629 66 ACCU-UUGUU GCUUUGGCGG GCCG-CCUAN ------CUACUGGCU UCUGCUGGUA AGUGCCCGCC AGAGGGA-CC CAAAACCC-- -UG-AAUUAU 147 TcdMarc4 89 ACCU-UUGUU GCUUUGGCGG GCCG-CCUAG ------CUACUGGCU UCGGCUGGUA AGUGCCCGCC AGAGG-A-CC CAAAACCC-- -UG-AAUUAU 169 Tmarch2639 95 ACCU-UUGUU GCUUUGGCGG GCCG-CCUAG ------CUACUGGCU UCGGCUGGUA AGUGCCCGCC AGAGG-A-CC CAAAACCC-- -UG-AAUUAU 175 Tmarch1939 71 ACCU-UUGUU GCUUUGGCGG GCCG-CCUAG ------CUACUGGCU UCGGCUGGUA AGUGCCCGCC AGAGG-A-CC CAAAACCC-- -UG-AAUUAU 151 Tmarch2619 66 ACCU-UUGUU GCUUUGGCGG GCCG-CCUAG ------CUACUGGCU UCGGCUGGUA AGUGCCCGCC AGAGG-A-CC CAAAACCC-- -UG-AAUUAU 146 TcdMarc6 62 ACCU-UUGUU GCUUUGGCGG GCCG-CCUAG ------CUACUGGCU UCGGNUGGUA NGUGCCCGCC AGAGG-A-CC CAAAACCC-- -UG-AAUUAU 142 TcdFurc2 62 ACCU-UUGUU GCUUUGGCGG GCCG-CCUAG ------CUACUGGCU UCGGCUGGUA AGUGCCCGCC AGAGA-A-CC CAAAACCC-- -UG-AAUUAU 142 TcdMaxi2 62 ACCU-UUGUU GCUUUGGCGG GCCG-CCUAG ------CUACUGGCU UCGGCUGGUA NGUGCCCGCC AGAGGGA-CC CAAAACCC-- -UGGAAUUAU 144 TcdApie2 53 ACCUUUUGUU GCUUUGGCGG GCCG-CCUGG ------CUACUGGCU UCGGCUGGUA AGUGCCCGCC AGAGG-A-CC CCAAACCC-A AACCCAUUUA 137 AxeEctom 91 ACCU-CUGUU GCUUUGGCGG GCCG-CCUAG ------CUACCGGCU UCGGCUGGUA AGUGCCCGCC AGAGG-A-CC CCAAAACCC- -UG-AAU-AU 171 DyiDimor 51 ACCU-UUGUU GCUUUGGCGG GCCGCUUCGG ------CUACUGGCU UCGGCUGGUG AGUGCCCGCC AGAGG-A-CC CAAAAUUC-- -UG-AAUUA- 131 Hl1Speci 51 ACCU-UUGUU GCUUUGGCGG GCCGCUUCGG ------CUACUGGCU UCGGCUGGUG AGUGCCCGCC AGAGG-A-CC CAAAAUUC-- -UG-AAUUA- 131 EczIsola 49 ACCU-UUGUU GCUUUGGCGG GCCGCUUCGG ------CUACCGGCU UCGGCUGGUG AGUGCCCGCC AGAGG-ACCC C-AAACUC-- -UG-AAUUA- 129 EodMycor.2 77 ACCU-UUGUU GCUUUGGCGG GCCGCUUCGG ------CUACCGGCU UCGGCUGGUA AGUGCCCGCC AGAGG-A-CC CUAAACUC-- -UG-AAU-GU 157 EodMycor.2 52 A-CU-GUGUU GCUUUGGCGG GCCG-CC-GG GCCCUGCCCG UCCGCCGGCU CCGGCUGGCG CGCGCCCGCC AGAGG-CUCC GCAAACUC-- -UG-AAU-GU 141 OidTenu2 201 211 221 231 241 251 261 271 281 291 300 | | | | | | | | | | | 147 UAGUGUCGNC NGAGUAAAAU AUUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 245 TcdMarc5 162 UAGUGUCGUC UGAGUAAAAU AUUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 260 Tmarch2629 148 UAGUGUCGGC GGAGNAAAAU AUUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC ANCGAAAUGC GAUAAGUAAU 246 TcdMarc4 170 UAGUGUCGUC UGAGUAAAAU AUUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 268 Tmarch2639 176 UAGUGUCGUC UGAGUAAAAU AUUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 274 Tmarch1939 152 UAGUGUCGUC UGAGUAAAAU AUUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 250 Tmarch2619 147 UAGUGUCGUC UGAGUAAAAU AUUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 245 TcdMarc6 143 UAGUGUCGUC UGAGUAAAAU AUUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 241 TcdFurc2 143 UAGUGNCGUC UGAGUAAAAU AUUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 241 TcdMaxi2 145 UAGUGUCGUC UGAGUAAAAU AUUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 243 TcdApie2 138 UAGUGUCGUC UGAGCAAAA- GUUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 235 AxeEctom 172 UAGUGUCGUC UGAGUAAAAU -UUUAAUA-U UUAAAACUUU CAACAACGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 269 DyiDimor 132 UAGUGUCGUC UGAGUACUAU AU--AAUA-G UUAAAACUUU CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 228 Hl1Speci 132 UAGUGUCGUC UGAGUACUAU AU--AAUA-G UUAAAACUUU CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 228 EczIsola 130 UAGUGUCGUC UGAGUACUAU AA--AAUA-G UUAAAACUUU CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 226 EodMycor.2 158 UAAUGUCGUC UGAGUACUAU AU--AAUA-G UUAAAACUUU CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 254 EodMycor.2 142 CAGUGUCGUC UGAGUACUAU AU--AAUA-G UUAAAACUUU CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU 238 OidTenu2 301 311 321 331 341 351 361 371 381 391 400 | | | | | | | | | | | 246 GUGAAUUGCA NAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC GAGCGUCAUU AUCACCCC-U 344 TcdMarc5 261 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC GAGCGUCAUU AUCACCCC-U 359 Tmarch2629 247 GUGAAUUGCA NAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC NAGCGUCAUU AUCACCCC-U 345 TcdMarc4 269 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC GAGCGUCAUU AUCACCCC-U 367 Tmarch2639 275 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC GAGCGUCAUU AUCACCCC-U 373 Tmarch1939 251 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC GAGCGUCAUU AUCACCCC-U 349 Tmarch2619 246 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC GAGCGUCAUU AUCACCCC-U 344 TcdMarc6 242 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC GAGCGUCAUU AUCACCCC-U 340 TcdFurc2 242 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC GAGCGUCAUU AUCACCCC-U 340 TcdMaxi2 244 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC GAGCGUCAUU AUCACCCC-U 342 TcdApie2 236 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC GAGCGUCAUU AUCACCCC-U 334 AxeEctom 270 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUAUUC GAGCGUCAUU AUCACCCC-U 368 DyiDimor 229 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGGGGGGC AUGCCUGUUC GAGCGUCAUU AUAACCCC-U 327 Hl1Speci 229 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGGGGGGC AUGCCUGUUC GAGCGUCAUU AUAACCCC-U 327 EczIsola 227 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUGUUC GAGCGUCAUU AUAACCCC-U 325 EodMycor.2 255 GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC CCCUUGGUAU UCCGAGGGGC AUGCCUGUUC GAGCGUCAUU AUAACCC--U 352 EodMycor.2 239 GCGAAUUGCA GAAUUCAGUG AGUCAUCGAA UCUUUGAACG CACAUUGCGC CCUGUGGUAU UCCGCAGGGC AUGCCUGUUC GAGCGUCAUU UCAACCC--U 336 OidTenu2 401 411 421 431 441 451 461 471 481 491 500 | | | | | | | | | | | 345 CAAGCUC-AG CUUGGUGUUG AGGCCUGCUG UCA-A-GGC- AGCCUCUAAA AGCAGUGGCA GUGCUGUCAG GCUCUAAGCG UAGUAAUCUC UCUCGCUACA 440 TcdMarc5 360 CAAGCUC-AG CUUGGUGUUG AGGCCUGCUG UCA-A-GGC- AGCCUCUAAA AGCAGUGGCA GUGCUGUCAG GCUCUAAGCG UAGUAAUCUC UCUCGCUACA 455 Tmarch2629 346 CAAGCUC-AG CUUGGUGUUG AGGCCUGCUG UCA-A-GGC- AGCCUCUAAA AGCAGUGGCA GUGCUGUCAG GCUCUAAGCG UANUAAUCUC UCUCGCUACA 441 TcdMarc4 368 CAAGCUC-AG CUUGGUGUUG AGGCCUGCUG UCA-A-GGC- AGCCUCUAAA AGCAGUGGCA GUGCUGUCAG GCUCUAAGCG UAGUAAUCUC UCUCGCUACA 463 Tmarch2639 374 CAAGCUC-AG CUUGGUGUUG AGGCCUGCUG UCA-A-GGC- AGCCUCUAAA AGCAGUGGCA GUGCUGUCAG GCUCUAAGCG UAGUAAUCUC UCUCGCUACA 469 Tmarch1939 350 CAAGCUC-AG CUUGGUGUUG AGGCCUGCUG UCA-A-GGC- AGCCUCUAAA AGCAGUGGCA GUGCUGUCAG GCUCUAAGCG UAGUAAUCUC UCUCGCUACA 445 Tmarch2619 345 CAAGCUC-AG CUUGGUGUUG AGGCCUGCUG UCA-A-GGC- AGCCUCUAAA AGCAGUGGCA GUGCUGUCAG GCUCUAAGCG UAGUAAUCUC UCUCGCUACA 440 TcdMarc6 341 CAAGCCU-AG CUUGGUGUUG AGGCCUGCUG UCA-A-GGC- AGCCUCUAAA AUCAGUGGCA GUGCUGUCAG GCUCUAAGCG UAGUAAAAUU CAUCGCUAUA 436 TcdFurc2 341 CAAGCCU-AG CUUGGUGUUG AGACCUGCUG UCA-A-GGC- AGUCUCUAAA AUCAGUGGCA GUGCUGUCAG GCUCUAAGCG UANUAAA-UU CAUCGCUAUA 435 TcdMaxi2 343 CAAGCCU-AG CUUGGUGUUG AGGCCUGCUG UCA-A-GGC- AGCCUCUAAA AUCAGUGGCA GUGCUGUCAG GCUCUAAGCG UAGUAAACUU CAUCGCUAUA 438 TcdApie2 335 CAAGCUCCGG CUUGGUGUUG AGGCCUGCUG UCA-A-GGC- ACCCUCUAAA AUCAGUGGCA GUGCCGUCAG GCUCUAAGCG UAGUAA-AUC CAUCGCUAUA 430 AxeEctom 369 CAAGCUC-AG CUUGGUGUUG GGGCCUGCCA UCA-C-GGC- AGCCCCUAAA AUCAGUGGCG GUGCCCUCAG GCUCUAAGCG CAGUAAUUUC UCUCGCUACA 464 DyiDimor 328 CAAGCUC-AG CUUGGUGUUG GGGCCUGCCG UC--U-GGC- AGCCCUUAAA AUCAGUGGCG GUGCCAUCUG GCUCUAAGCG UAGUAAUUUU UCUCGCUACA 422 Hl1Speci 328 CAAGCUC-AG CUUGGUGUUG GGGCCUGCCG UC--U-GGC- AGCCCUUAAA AUCAGUGGCG GUGCCAUCUG GCUCUAAGCG UAGUAAUUUU UCUCGCUACA 422 EczIsola 326 CAAGCUC-GG CUUGGUGUUG GGGCCUGCCG -CACG-GGC- AGCCCUUAAA AUCAGUGGCG GUGCCAUCUG GCUCUAAGCG UAGUAAUUCU UCUCGCUAUA 421 EodMycor.2 353 CAAGCCU-AG CUUGGUGUUG GGGCCUGCUG UUACC-GGC- AGCCCUUAAA ACUAGUGGCG GUGCCACCUG GCUCUAAGCG UAGUAAUACU UCUCGCUAUA 449 EodMycor.2 337 CAAGCUC-UG CUUGGUGUUG GGCCCUGCCC GCUCGCGGC- CGGCCCUAAA GACAGUGGCG GCGCCGUCUG GCUCUAAGCG UAGUA-CAUC UCUCGCUCUA 433 OidTenu2

501 511 521 531 541 551 561 571 581 591 600 | | | | | | | | | | | 441 GACACCU-GA UGGACACUCG CCAG-AACCC CCC-A-UCUU UUAAUGAUUG ACCUCGGAUU AGGUAGGGAU ACCCGCUGAA CUUAA...... 521 TcdMarc5 456 GACACCU-GA UGGACACUCG CCAG-AACCC CCCCA-UCUU UUAAUGAUUG ACCUCGGAUU AGGUAGGGAU ACCCGCUGAA CUUAAGCAUA UCAAUAAG.. 550 Tmarch2629 442 GACACCU-GA UGGACACUCG CCAG-AACCC CCC-A-UCUU UUAAUGAUUG ACCUCGGAUU AGGUAGGGAU ACCCGCUGAA CUUAA...... 522 TcdMarc4 464 GACACCU-GA UGGACACUCG CCAG-AACCC CCC-A-UCUU UUAAUGAUUG ACCUCGGAUU AGGUAGGGAU ACCCGCUGAA CUUAAGCAUA UCAAUAAGCG 559 Tmarch2639

Figure A38: Alignment of complete ITS1, 5.8S, ITS2 sequences of Tetracladium marchalianum, closest BLAST hits and Oidiodendron tenuissimum.

216 Appendix

Alignment ITS sequences Tetracladium marchalianum

470 GACACCU-GA UGGACACUCG CCAG-AACCC CCC-A-UCUU UUAAUGAUUG ACCUCGGAUU AGGUAGGGAU ACCCGCUGAA CUUAAGCAUA UCAAUAAGCG 565 Tmarch1939 446 GACACCU-GA UGGACACUCG CCAG-AACCC CCCCA-UCUU UUAAUGAUUG ACCUCGGAUU AGGUAGGGAU ACCCGCUGAA CUUAAGCAUA UCAAUAAGCG 542 Tmarch2619 441 GACACCU-GA UGGACACUCG CCAG-AACCC CCC-A-UCUU UUAAUGAUUG ACCUCGGAUU AGGUAGGNAU ACCCGN-GA- CU-A...... 517 TcdMarc6 437 GACACCU-GG UGGACACUCG CCAG-AACCC CCCCA-UUUU UUAAUGAUUG ACCUCGGAUU AGGUAGGGAU ACCCGCUGAA CUUAAU...... 519 TcdFurc2 436 GACACCU-GG NGGACACUCG CCAG-AACCC CCC-A-UUUU UUAAUGAUUG ACCUCGGAUU AGGUAGGGAU ACCCGCUGAA CUUAA...... 516 TcdMaxi2 439 GACACCU-GG UGGACACUCG CCAG-AACCC CCC-A-UUUU UUAAUGAUUG ACCUCGGAUU AAGUAGGGAU ACCCGCUGAA CUUAA...... 519 TcdApie2 431 GACCCG--GG CGGACGCUCG CCAG-AACAC CCCCA--UUU UUAAUGAUUG A...... 476 AxeEctom 465 GACCCCU-GG AGG-CGCCCG CCAGCAACCC CCC-AUUUUU UUAAUGAUUG ACCUCGGAUU AGGUAGGGAU ACCCGCUGAA CUUAA...... 546 DyiDimor 423 GAGUUCU-GG UGGAUGCUUG CCAUCAACCC CC-AA-UUUU CU-AUGGUUG A...... 469 Hl1Speci 423 GAGUUCU-GG UGGAUGCUUG CCAUCAACCC CC-AA-UUUU CU-AUGGUUG A...... 469 EczIsola 422 GAG-UCCCGG UGGAUGCUUG CCAUUAACCC CC-AA---UU UCAAUGGUUG ACCUCGGAUC AGGUAGGGAU ACCCGCUGAA CUUAAGCAUA UCAAUAAG.. 514 EodMycor.2 450 GAGUCCC-GG UGGAUGCUUG CCAUCAACCC CU-AA-UUUU CU-AUGGUUG ACCUCGGAUC AGGUAGGGAU ACCCGCUGAA CUUAAGCAUA UCAAUAAGCG 545 EodMycor.2 434 G-CGUCC-GG CGGUGGCCCG CCAG-AACCC CC-AA-CUCU --G-UGGUUG ACCUCGGAUC AKGUAKGGAU ACCCGCUGAA CUUAAGCAUA UCAAUAAG.. 523 OidTenu2

601 | 522 ...... 521 TcdMarc5 551 ...... 550 Tmarch2629 523 ...... 522 TcdMarc4 560 G...... 560 Tmarch2639 566 G...... 566 Tmarch1939 543 GAGGAAAAG 551 Tmarch2619 518 ...... 517 TcdMarc6 520 ...... 519 TcdFurc2 517 ...... 516 TcdMaxi2 520 ...... 519 TcdApie2 477 ...... 476 AxeEctom 547 ...... 546 DyiDimor 470 ...... 469 Hl1Speci 470 ...... 469 EczIsola 515 ...... 514 EodMycor.2 546 ...... 545 EodMycor.2 524 ...... 523 OidTenu2

Figure A38 continued

Alignment ITS sequences Alatospora acuminata

1 11 21 31 41 51 61 71 81 91 100 | | | | | | | | | | | 1 GGAUCAUUAA AGAGUUUAGA GACUUCGGUC UACUACUCCA CCCUUUGUUU ACAAUACCAU UGUUGCUUUG GCAGGCCUGU CGCAAGACAA CCGGCUUCGG 100 Alacu12186 1 GGAUCAUUAA AGAGUUUAGA GACUUCGGUC UACUACUCCA CCCUUUGUUU ACAAUACCAU UGUUGCUUUG GCAGGCCUGU CGCAAGACAA CCGGCUUCGG 100 Alacu18799 1 GGAUCAUUAA AGAGUUUAGA GACUUCGGUC UACUACUCCA CC-UUUGUUU ACAAUACCAU UGUUGCUUUG GCAGGCCUGU CGCAAGACAA UCGGCUCCGG 99 alacu37194 1 GGAUCAUUAA AGAGUUUAUG GACUUCGGUC UACUACUCCA CCCUUUGUUU ACAAUACCAU UGUUGCUUUG GCAGGCCCGU CGAAAGACAA CCGGCUUCGG 100 Alacu02383 1 GGAUCAUUAA AGAGUUUAUA GACUUCGGUC UACUACUCCA CCCUUUGUUU ACAAUACCAU UGUUGCUUUG GCAGGCCCGU CGAAAGACAA CCGGCUUCGG 100 Alacu13089 1 ...... CAG AGAACUUG-- CCCUUCGG-- -AUCU--CCA CCC-UUGUUU ACAUUACCUU UGUUGCUUUG GCGGGCCCGU CUUA-GACCA CCGGCUUUGG 84 Lo1Speci 1 GGAUCAUUAA UGAGAUCAUG CCCUUCGG-- -ACCU--CCA CCCUUUGUUU ACAAUACCUU UGUUGCUUUG GCGGCCCCGU CGCAAGACAA CCGGCUCCGG 95 GugPhilo 1 GGAUCAUUAA UGAGAUCAUG -CCUUCGG-- -ACCU--CCA CCC-UCGUAU ACAAUACCUU UGUUGCUUUG GCGGCCC-GU CGCAAGACAA CCGGCUCCGG 92 PmkCoelo 1 GGAUCAUUAA AGAAUUUAGG C-UUUCCG-- -AUAUU-CUA CCC-UUGUAU ACGAUACUA- UGUUGCCUUG GCAGGCU-GU UACUA------77 CudLute4 101 111 121 131 141 151 161 171 181 191 200 | | | | | | | | | | | 101 CUGGUCAGCG CCUGCCAGAG AACCUAAAAC UCAUG-UAUA UUAUUGUCUG AGUACUAUAU AAUAGUUAAA ACUUUCAACA ACGGAUCUCU UGGUUCUGGC 199 Alacu12186 101 CUGGUCAGCG CCUGCCAGAG AACCUAAAAC UCAUG-UAUA UUAUUGUCUG AGUACUAUAU AAUAGUUAAA ACUUUCAACA ACGGAUCUCU UGGUUCUGGC 199 Alacu18799 100 CUGGUCAGCG CCUGCCAGAG GACCUAAAAC UCAUG-UAUA UUAUUGUCUG AGUACUAUAU AAUAGUUAAA ACUUUCAACA ACGGAUCUCU UGGUUCUGGC 198 alacu37194 101 CUGGUCAGUG CCUGCCAGAG GACCUAAAAC UCUUGAUUUA UUAUUGUCCG AGUACUAUAU AAUAGUUAAA ACUUUCAACA ACGGAUCUCU UGGUUCUGGC 200 Alacu02383 101 CUGGUCAGUG CCUGCCAGAG GACCUAAAAC UCUUGAUUUA UUAUUGUCUG AGUACUAUAU AAUAGUUAAA ACUUUCAACA ACGGAUCUCU UGGUUCUGGC 200 Alacu13089 85 CUGGUCAGCG CCCGCCAGAG GACCCAAAAC UCU-G-UUAA U-GUCGUCUG AGUACUAUAU AAU-GUUAAA ACUUUCAACA ACGGAUCUCU UGGUUCUGGC 180 Lo1Speci 96 CUGGUCAGCG GCCGCCAGAG GACUCAAAAC UCA-AAUU- A UUGUCGUCUG AGUACUAUAU AAU-GUUAAA ACUUUCAACA ACGGAUCUCU UGGUUCUGGC 192 GugPhilo 93 CUGGUCAGCG GCCGCCAGAG GA-UCAAAAC UCU-AAUU- A UUGUCGUCUG AGUACUAUAU AAU-GUUAAA ACUUUCAACA ACGGAUCUCU UGGUUCUGGC 188 PmkCoelo 78 ------G CCUGCCGGUG GCCUC-- -AAC --U-GAUCU UUGCUGUCUG AGUACUAUAU AAU-GUUAAA ACUUUCAACA ACGGAUCUCU UGGUUCUGGC 161 CudLute4

201 211 221 231 241 251 261 271 281 291 300 | | | | | | | | | | | 200 AUCGAUGAAG AACGCAGCGA AAUGCGAUAA GUAAUGUGAA UUGCACAAUU CAGUGAAUCA UCGAAUCUUU GAACGCACAU UGCGCCCUCU GGUAUUCCGG 299 Alacu12186 200 AUCGAUGAAG AACGCAGCGA AAUGCGAUAA GUAAUGUGAA UUGCAGAAUU CAGUGAAUCA UCGAAUCUUU GAACGCACAU UGCGCCCUCU GGUAUUCCGG 299 Alacu18799 199 AUCGAUGAAG AACGCAGCGA AAUGCGAUAA GUAAUGUGAA UUGCAGAAUU CAGUGAAUCA UCGAAUCUUU GAACGCACAU UGCGCCCUCU GGUAUUCCGG 298 alacu37194 201 AUCGAUGAAG AACGCAGCGA AAUGCGAUAA GUAAUGUGAA UUGCAGAUUU CAGUGAAUCA UCGAAUCUUU GAACGCACAU UGCGCCCUCU GGUAUUCCGG 300 Alacu02383 201 AUCGAUGAAG AACGCAGCGA AAUGCGAUAA GUAAUGUGAA UUGCAGAAUU CAGUGAAUCA UCGAAUCUUU GAACGCACAU UGCGCCCUCU GGUAUUCCGG 300 Alacu13089 181 AUCGAUGAAG AACGCAGCGA AAUGCGAUAA GUAAUGUGAA UUGCAGAAUU CAGUGAAUCA UCGAAUCUUU GAACGCACAU UGCGCCCUCU GGUAUUCCGG 280 Lo1Speci 193 AUCGAUGAAG AACGCAGCGA AAUGCGAUAA GUAAUGUGAA UUGCAGAAUU CAGUGAAUCA UCGAAUCUUU GAACGCACAU UGCGCCCCCU GGUAUUCCGG 292 GugPhilo 189 AUCGAUGAAG AACGCAGCGA AAUGCGAUAA GUAAUGUGAA UUGCAGAAUU CAGUGAAUCA UCGAAUCUUU GAACGCACAU UGCGCCCCCU GGUAUUCCGG 288 PmkCoelo 162 AUCGAUGAAG AACGCAGCGA AAUGCGAUAA GUAAUGUGAA UUGCAGAAUU CAGUGAAUCA UCGAAUCUUU GAACGCACAU UGCGCCCUCU GGUAUUCCAG 261 CudLute4

301 311 321 331 341 351 361 371 381 391 400 | | | | | | | | | | | 300 GGGGCAUGCC UGUUCGAGCG UCAUUACAAC CCUCAAGCUC UGCUUGGUAU UAGGCUUCAC CCUUCGGGGC GGGCCUCAAA AUCAGUGGCG GUGCCAUUCG 399 Alacu12186 300 GGGGCAUGCC UGUUCGAGCG UCAUUACAAC CCUCAAGCUC UGCUUGGUAU UAGGCUUCAC CCUUCGGGGC GGGCCUCAAA AUCAGUGGCG GUGCCAUUCG 399 Alacu18799 299 GGGGCAUGCC UGUUCGAGCG UCAUUACAAC CCUCAAGCUC UGCUUGGUAU UAGGCUUCAC CCCUAGGGGC GGGCUUUAAA AUCAGUGGCG GUGCCAUUCG 398 alacu37194 301 GGGGCAUGCC UGUUCGAGCG UCAUUACAAC CCUCAAGCUC AGCUUGGUAU UAGGCUUCAC CCUUAGGGGC GGGCUUUAAA AUCAGUGGCG GUGCCAUUCG 400 Alacu02383 301 GGGGCAUGCC UGUUCGAGCG UCAUUACAAC CCUCAAGCUC AGCUUGGUAU UAGGCUUCAC CCUUAGGGGC GGGCUUUAAA AUCAGUGGCG GUGCCAUUCG 400 Alacu13089 281 GGGGCAUGCC UGUUGAGCGU CAUU-ACAAC C-UCAAGCUC UGCU-UGGUA UUGGGCGUCA CCGUCUCGGU GCGCCUUAAA AUCAGUGGCG GUGCCAUUAG 377 Lo1Speci 293 GGGGCAUGCC UGUUGAGCGU CAUU-ACAAC CCUCAAGCUC UGC-UGGUAU UGGGCGUCAC CCCCG--GGU GCGCCUUAAA AUCAGUGGCG GUGCCGUCUG 388 GugPhilo 289 GGGGCAUGCC UGUUGAGCGU CAUU-ACAAC CCUCAAGCUC UGCUUGGUAU UGGGCGUCAC CCCCG--GGU GCGCCUUAAA AUCAGUGGCG GUGCCGUCUG 385 PmkCoelo 262 GGGGCAUGCC UGUUGAGCGU CAUU-ACAAC C-UCACGCCU AGCGUGGUCU UGGGC-UCGC CCUGUAGGGC CUGCCUCAAA GUCAGUGGCG GCGUCGUCUG 358 CudLute4 401 411 421 431 441 451 461 471 | | | | | | | | 400 GCUUCAAGCG UAGUAAUUUU CUCGCUUUGG AGACCGGCUG CGUGCUUGCC AACAACCCCA -AUUUUAUAA AGGUUGAC 476 Alacu12186 400 GCUUCAAGCG UAGUAAUUUU CUCGCUUUGG AGACCGGCUG CGUGCUUGCC AACAACCCCA -AUUUUAUAA AGGUUGAC 476 Alacu18799 399 GCUUCAAGCG UAGUAAUUUU CUCGCUUUGG AGACCGGAUG CGUGCUUGCC AACAACCCCA -AUUUUAUAA AGGUUGAC 475 alacu37194 401 GCUUCAAGCG UAGUAAUUUU CUCGCUUUGG AGAUCGGGUG UGUGUUUGCC AACAACCCCA UAUUUUUUAA AGGUUGAC 478 Alacu02383 401 GCUUCAAGCG UAGUAAUUUU CUCGCUUUGG AGACCGGGUG UGUGUUUGCC AACAACCCCA UAUUUUUUAA AGGUUGAC 478 Alacu13089

378 GCUUCAAGCG UAGUAAUCUU CUCGCUCU-G GAGUC-GGUG UGUGCUUGCC AACAACCCCA -AUUUUAUAA ------445 Lo1Speci 389 GCUUCAAGCG UAGUAAACUU CUCGCUUUGG AG-CCGGGCA GCGUCCUGCC AA-AACCCCA UAUUUUUUAA -GGUUGAC 463 GugPhilo 386 GCUUCAAGCG UAGUAAACUU CUCGCUUUGG AG-CCGGGCA GCGUCCUGCC AA-AACCCCA UAUUUUUUCA -GGUUGAC 460 PmkCoelo 359 ACCCUAAGCG UAGUAAACAC CUCGCUUC-U GGCGU-GGAA GAGGCUUGCU UG-AAACCCA -A-CUUU-AC AGGUUGAC 430 CudLute4

Figure A39: Alignment of complete ITS1, 5.8S, ITS2 sequences of Alatospora acuminata and closest BLAST hits.

217 Appendix

1 11 21 31 41 51 61 71 81 91 100 | | | | | | | | | | | 1 GAAUUGAGUG GAUCAUUACC AUUACCUCAA CGGGGGGGAG UUCAGCAGUG UAUUCGGCUG AACUCCCGCC UGAUUGACGU UACCCAUGUC UUUUGCGUAC 100 LehDoli2 1 GAGGUGACUG CGGAAGGAUC AUUAUUGUGA GGG------UUCGGCCAUA AU------CCUUGCC UUUUGUAGUA 62 Ms3Bipol 1 GAGGUGACUG CGGAAGGAUC AUUAUUGUAG GGG------UUCGGCGCAU GA------ACCCUUGCU CUUUAGUAGU 64 Ms3Froni 1 ...... UC AUUAUCGUGG GGG------CCUGGCCGUG AU------ACCCUUGCC CUUUAGCAUC 46 Ms3Armat 1 GAGGUGACUG CGGAAGGAUC AUUAUCGUAG GG------UUUG-UCGAG AU------GUCCUUGCC UAUUGAGCAC 62 Ms3Corti 1 GAGGUGACUG CGGAAGGAUC AUUAUUGUAG GUG------CUCGGC--AA A------ACCCUUGUC UUUUGUGCAC 61 LsaCaul2 1 GAGGUGACUG CGGAAGGAGC AUUAUUGAGG GGGG------CUUGGU-AAA AGCUGCAC-A CA---CUGU- CUAU------UCCCCUUGUC ---UCUAGUA 79 LehConte 1 GAGGUGACUG CGGAAGGAUC AUUACCGUAG GGGG------UUUG-CGAAA AA------U UCCC-UUGUC ---UUGAGCA 62 AnguLongi1 1 ...... ACCGUAG GGGG------UUUG-CGAAA AA------U UCCC-UUGUC ---UUGAGCA 39 Alongi_106 1 GAGGUGACUG CGGAAGGAUC AUUACCGUAG GGGG------UUUG-CGAAA AA------U UCCC-UUGUC ---UUGAGCA 62 A.longi009 1 ...... ACCGUAG GGGG------UUUG-CGAAA AA------U UCCC-UUGUC ---UUGAGCA 39 Along11891 1 GUGGUGACUG CGGAAGGAUC AUUACCGUAG GGGG------UUUG-CGAAA AA------U UCCC-UUGUC ---UUGAGCA 62 AlongiL22 1 GAGGUGACUG CGGAAGGAUC AUUACUACAA AGGUCGG--- UCAAUAUAAA A------UCG CUGCCGACAU UCAUCCUGUG UU-U---AUA 77 Ms3Rubi 1 GAGGUGACUG CGGAAGGAUC AUUACAAAUU AGCG------U--GG------CCUCCG GGUCGCUGCC UCCCCUUGUC ---UUGAGUA 70 Ms3Ebur2 1 GAGGUGACUG CGG-AGGAUC AUUACACAAA AAGCGGG--- U--GGUAGCA GUUGUUGGGC CAGCCUUGCU GAAUUAUU-- -CCCG-UGUC UU-UUGCGUA 89 PlpPapav 1 ...... AUUACACAAA AAAGG----- U--G-CAACC GC----GGGA -AG--CUG-- -AAUUAUUUU UCCCA-UGUC UU-UUGCGCA 60 CuvErag2 1 ...... CC-UA AG------CCCCUUGCA UU-UGAGCAC 24 LsaVagab 1 CGUAGGGACC GCGGAGGAUC AUUACCUUAG GAGGG----- CC-A-UAGGG C------UUCUUCU UCCC-UUGUC UACUGAUGUU 70 Ms3Ramun 1 ...... UC AUUAACGAUU GCGG------UUUA-CUUCU AC------CCAU U--CUACG-C ---GACCACU 45 Ms3Walke 101 111 121 131 141 151 161 171 181 191 200 | | | | | | | | | | | 101 -AGUUUGUUU UCCCAGCAGG GACUUUGUGC CCCUACGGGA UAUCAAUCCA CCCUUGAAUU UGCAGUCCAU AGUCUGAAAA AUAAUAAUAA UUACAACUUU 199 LehDoli2 63 -U-UCUGUUU CCUCGGCAGU UAGUGCAACA GUUGUGAGGA CCCUAAACAA CCUUUGAAUC UAUCAUGU-U AG------A A-AACUC-AA UUACAACUUU 150 Ms3Bipol 65 -A-CCCGUUU CCUCGGCAGU UGCUUAUUCC AGCCGCAGGA -CCCCCAAAA CCC--GAAUC U-CAAUUU-U AG------AA ACAAUUC-AA UUAAAACUUU 150 Ms3Froni 47 -U-UCCGUUU CCUCGGCA-G C-CUC---GC CCU-ACAGGA ---CCUCAGA CCCUUGAAUC UGUACGAAAU GA------AAGCACAA CUAAAACUUU 125 Ms3Armat 63 C--UUCGUUU CCUCAGCA-G G-CUU---GC CCU-CUGGGA --CCCUC--A CUCUUGAAU- -ACAGUAAUU GA----ACAA ACAAA---AA UCAAAACUUU 141 Ms3Corti 62 C--UUUGUUU CCUCGGCA-- C-UUC---GC CCC-ACAGGA --CCCCC-AA CCCUUG---- -UCCAGUA-U AA-CAAUAAA ACAAC---AA UUAAAACUUU 139 LsaCaul2 80 CAC--UGUUU CCUCGGCA-G G-CUU---GC CCC-AUGGGA --CCCAUC-A CCUUU------UAUUGUUU AA-CCGACAA ACAAU---AA UCAAAACUUU 157 LehConte 63 C-C-UUGUUU CCUCGGCA-G G-CUU---GC CCC-AUAGGA --CCAAUC-A CCUUUG---- -GUAGUAGU- AG-CCGUUAA ACAA----AA UCAAAACUUU 140 AnguLongi1 40 C-C-UUGUUU CCUCGGCA-G G-CUU---GC CCC-AUAGGA --CCAAUC-A CCUUUG---- -GUAGUAGU- AG-CCGUUAA ACAA----AA UCAAAACUUU 117 Alongi_106 63 C-C-UUGUUU CCUCGGCA-G G-CUU---GC CCC-AUAGGA --CCAAUC-A CCUUUG---- -GUAGUAGU- AG-CCGUUAA ACAA----AA UCAAAACUUU 140 A.longi009 40 C-C-UUGUUU CCUCGGCA-G G-CUU---GC CCC-AUGGGA --CCAA-CCA CCUUUG---- -GUAGUAGU- AG-CCGUUAA ACAA----AA UCAAAACUUU 117 Along11891 63 C-C-UUGUUU CCUCGGCA-G G-CUU---GC CCC-AUAGGA --CCAAUC-A CCUUUG---- -GUAGUAGU- AG-CCGUUAA ACAA----AA UCAAAACUUU 140 AlongiL22 78 ---UUUGUUU CCUCGGUG-G G-UUUGUUAC UCC-GUAUGA --CCCUUC-A CCUUUGAAUU UGUCUACCUU GAUUCUUAAA A---UU--AU UUACAACUUU 163 Ms3Rubi 71 C-CGUUGUUC CCUCGGCACG G-U-----GC CCG--U-GGA ---ACACCUA CCCUUGAA-- -GCAUUAAAU GAU---A-AA ACAAUCU--A UUACAACUUU 148 Ms3Ebur2 90 CUC-UUGUUU CCUUGGUG-G G--UU--CGC CCC-ACAGGA -CAAACAUUA CCUUUGAAUU -GCAAUCAGU AGU---A-AA -CAAU---AA UUACAACUUU 172 PlpPapav 61 CU-GUUGUUU CCUGGGCG-G G--UUC--GC CCC-ACAGGA CACACAU--A CCUUU---U- --UGCAGUUA AGU--AA-AA AUAAUC---A UUACAACUUU 139 CuvErag2 25 C--UCUGUUU CCUCGGCG-G G-CUC---GC CCC-AUGGGA --CCAA-CCA CCCUCG---- -GUUGCAG-U AA-CGUAAAA ACAA----AA UCAAAACUUU 102 LsaVagab 71 CUGUUUUGCU CCCUGGC--G GCCCCC------GGAGGG --CCCUUUAA CCCGAGAAGC -GCCUAUCUU GA-UUU------AAGCCU-A UUACAACUUU 149 Ms3Ramun 46 -U-UUUCCUC GGGGGGCU-U GCCCC----- CCU---AGGA -CUCUCAAUA CCUUUGAAU- -GCAGUCAGU GA----AU-A ACAAU---UA UUAAAACUUU 123 Ms3Walke 201 211 221 231 241 251 261 271 281 291 300 | | | | | | | | | | | 200 UAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 299 LehDoli2 151 CAACAAUGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 250 Ms3Bipol 151 CAACAAUGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 250 Ms3Froni 126 CACAAUGGAU CUCUU-GGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 224 Ms3Armat 142 CAACAAUGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCAAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 241 Ms3Corti 140 CAACAAUGGA UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 239 LsaCaul2 158 CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 257 LehConte 141 CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 240 AnguLongi1 118 CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 217 Alongi_106 141 CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 240 A.longi009 118 CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 217 Along11891 141 CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 240 AlongiL22 164 CAACAAUGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 263 Ms3Rubi 149 CAACAAUGGA UCUCUUGGUU CUGGCGUCGA UGAAGAACGC AGCGAAAUGC GAAACGUAGU GUGAAUUGCA GAAUUCCGUG AAUCAUCGAA UCUUUGAACG 248 Ms3Ebur2 173 CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 272 PlpPapav 140 CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUACGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 239 CuvErag2 103 CAACAACGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 202 LsaVagab 150 CAACAAUGGA UCUCUUGGCU CUGGCAUCGA UGAAGAA-CG CAGCGAACUG CGAUAAGUAG UGUGAAUUGC AGAAUUCUAG UGAAUCACGA AUCUUUGAAC 248 Ms3Ramun 124 CAACAAUGGA UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAGU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG 223 Ms3Walke 301 311 321 331 341 351 361 371 381 391 400 | | | | | | | | | | | 300 CACAUUGCGC CCCUUGGUAU UCCAUGGGGC AUGCCUGUUC GAGCGUCAUU UGUACCCUCA AGCUUUGCUU GGUGUUGGGU GAUUGUCCGC GCUUUUUGCG 399 LehDoli2 251 CACAUUGCGC CCUUUGGUAU UCCUUAGGGC AUGCCUGUUC GAGCGUCAUU UAAAC-CUCA AGCACGGCUU CGUGGCAGG- ACCCAAGAGG UAGUAGCGCU 348 Ms3Bipol 251 CACAUUGCGC CCUUUGGUAU UCCUUAGGGC AUGCCUGUUC GAGCGUCAUU UAAAA-CUCA AGCGAAGCUU GGUGAUGGGC GUCUGUC-CC GCACGAUGCA 348 Ms3Froni 225 CACAUUGCGC CCUUUGGUAU UCCUUAGGGC AUGCCUGUUC GAGCGUCAUU UCAA-CCUCA AGCUCAGCUU GGUGAUGGGC AGUUGUCUCC AGCGUUGUGG 323 Ms3Armat 242 CACAUUGCGC CCUUUGGUAU UCCUUAGGGC AUGCCUGUUC GAGCGUCAUU UA-ACCCUCA AGCACAGCUU GGUGUUGGGC GUCUGUC-CC GC--UUCGCG 337 Ms3Corti 240 CACAUUGCGC CCUUUGGUAU UCCUUAGGGC AUGCCUGUUC GAGCGUCAUU UACAAU-UCA AGCUCAGCUU GGUGAUGGGU GUCUGUC-CC GCUUUGCGUG 337 LsaCaul2 258 CACAUUGCGC CCUGUGGUAU UCCGCAGGGC AUGCCUGUUC GAGCGUCAUU UAACCCCUCA AGCUCUGCUU GGUGUUGGGC GUUUGUCC-- GCUUUUUGUA 355 LehConte 241 CACAUUGCGC CCUGUGGUAU UCCGCAGGGC AUGCCUGUUC GAGCGUCAUU UACUACCUCA AGCUCUGCUU GGUGUUGGGC GUUUGUC-CU GCUUUUUGCG 339 AnguLongi1 218 GACAUUGCGC CCUGUGGUAU UCCGCAGGGC AUGCCUGUUC GAGCGUCAUU UACUACCUCA AGCUCUGCUU GGUGUUGGGC GUUUGUC-CU GCUUUUUGCG 316 Alongi_106 241 CACAUUGCGC CCUGUGGUAU UCCGCAGGGC AUGCCUGUUC GAGCGUCAUU UACAUCCUCA AGCUCUGCUU GGUGUUGGGC GUUUGUC-CU GCUUUUUGCG 339 A.longi009 218 CACAUUGCGC CCUGUGGUAU UCCGCAGGGC AUGCCUGUUC GAGCGUCAUU UACUACCUCA AGCUCUGCUU GGUGUUGGGC GUUUGUC-CU GCUUUUUGCA 316 Along11891 241 CACAUUGCGC CCUGUGGUAU UCCGCAGGGC AUGCCUGUUC GAGCGUCAUU UACUACCUCA AGCUCUGCUU GGUGUUGGGC GUUUGUC-CU GCUUUUUGCG 339 AlongiL22 264 CACAUUGCGC CCCUUGGUAU UCCAUUGGGC AUGCCUGUUC GAGCGUCAU- AAGAAAUUCA AGCCCUGCUU GGUGUUGAGU GCCUGUC-CU GC--UUGGCG 359 Ms3Rubi 249 CACAUUGCAC UCCCUGGUAU UCCGGGGAGU AUGCCUGUUC GAGCGUCAUU GGAACC-UCA AGCUCUGCUU GGUGUUGGGC GUCUGUCCCU --UU---GGG 342 Ms3Ebur2 273 CACAUUGCGC CCUUUGGUAU UCCAAAGGGC AUGCCUGUUC GAGCGUCAUU UGUACCCUCA AGCUUUGCUU GGUGUUGGGC GUCUGUCUC- AGUUC--CUG 369 PlpPapav 240 CACAUUGCGC CCUUUGGUAU UCCAAAGGGC AUGCCUGUUC GAGCGUCAUU U-GACCCUCA AGCUUUGCUU GGUGUUGGGC GUUUGUCUU- GGUUUU-GCC 336 CuvErag2 203 CACAUUGCGC CCUUUGGCAU UCCUUAGGGC AUGCCUGUUC GAGCGUCAUC UAAAACCUCA AGCACUGCUU GGUGUUGGGC GCCUGUCC-- GCC----GCG 296 LsaVagab 249 GCACAUUGCG CCUCUUGGAA UCUGAGGGGC AUGCCUGUUG AGCGUCAUUG ACACCCCUCA AGCUCUGCUU GGUGUUGGGC GUCUGUC-CC --UC------339 Ms3Ramun 224 CACAUUGCGC CCCUUGGUAU UCCAUGGGGC AUGCCUGUUC GAGCGUCAUU UGA-CCCUCA AGCUCUGCUU GGUGUUGGGU GUUUGUCC-- GCCAUUGCGU 320 Ms3Walke 401 411 421 431 441 451 461 471 481 491 500 | | | | | | | | | | | 400 CAGACUCGCC UUAAAUCAAU UGGCAGCCGG CAUGUUAGCC UGGAGCGCAG CACAUUUUGC GCACCUUGCU GGCGGUGUUG GCCCCCAUCA AGUCCAUAUA 499 LehDoli2 349 UGGACUCGCC UCGAAUGCAG UUGGCAGCC- -ACC---UCC C-AAUCGGAU CCGAAGU-AG AAUU------CGCUGUGGCU CUCCAAGC-A ACAG--CGUC 432 Ms3Bipol 349 CAAACUCGCC UCAAAUGCAU UGGCAGCCC- -ACU---CCU A-AAUCGGAU CCGAAGU-AG AAUU------CG-ACGU--G GCUCUCAA-A GU----AAAC 427 Ms3Froni 324 CGGACUGCCU U-AAA----U UACGGAGGCG AUCGGCUCAA ACGCAGCAGA CUUGCGUCGC CUGCCC-G-- GGAGGG-UUA CUUUCCGUCA AGAGA----- 409 Ms3Armat 338 CGGACUCGCC UCAAAACGAU UGGCAGCCA- -UCG---GCU U-GAGCGCAG CAGAAUU-GC GUCU------CU-GG-U--G GC-AUCAGUA AG----CAUU 415 Ms3Corti 338 UGGACUCGCC UCAAAUGCAU UGGCAGCUU- -UCG---GCU U-AAACGCAG CAGAUUU-GC GUCG------AGCGGC---G GGCUCUCAGU AG--C-AAA- 417 LsaCaul2 356 UGGACUCGCC UUAA-GAUAU UGGCAGCCA- C-UG---GCU C-GAGCGCAG CAC-UUUUGC GCCC------GG-GGUUGUG GUUCUC---A A-GCC-UAUC 435 LehConte 340 UGGACUCGCC UCAAAUAUAU UGGCAGCCG- -AUG---GCU C-GAGCGCAG CACAUUU-GC GCCU------GA-AUU---G GCUCCCAG-A AG----UAUU 417 AnguLongi1 317 UGGACUCGCC UCAAAUAUAU UGGCAGCCG- -AUG---GCU C-GAGCGCAG CACAUUU-GC GCCU------GA-AUU---G GCUCCCAG-A AG----UAUU 394 Alongi_106 340 UGGACUCGCC UCAAAUAUAU UGGCAGCCG- -AUG---GCU C-GAGCGCAG CACAUUU-GC GCCU------GA-AUU---G GCUCCCAG-A AG----UAUU 417 A.longi009 317 UGGACUCGCC UCAAAUAUAU UGGCAGCCG- -AUG---GCU C-GAGCGCAG CACAUUU-GC GCCU------GA-AUU---G GCUCCCAG-A AG----UAUU 394 Along11891 340 UGGACUCGCC UCAAAUAUAU UGGCAGCCG- -AUG---GCU C-GAGCGCAG CACAUUU-GC GCCU------GA-AUU---G GCUCCCAG-A AG----UAUU 417 AlongiL22 360 CGGACUC-UC UCAAACUA-U UGGCGGUCCC CGCUUU-GC- --GCAGCACA UGUA----GC GCUCU----U ---GGGG-CG G-CAUCGAAA AG------432 Ms3Rubi 343 GGGACUCGCC CCAAAGUCAU UGGCAGCGG- UG-A---GCU CU--GCGCAG CACAUUU-GC GCUUCUCG-- AA-GGGA-CA CGCGUCAGCA AG-----CUC 425 Ms3Ebur2 370 GGGACUCGCC UUAAAGUUAU UGGCAGCCGG -AUG---GUU CGGAGCGCAG CACAAGUCGC GCUC-----U AA---G---- GUC---AGCA UCG-----CC 445 PlpPapav 337 CAAACUCGCC UUAAAC-GAU UGGCAGCCGG CAUG---GUU CGGAGCGCAG CACAUUUUGC GCUU---GC- AAC-A----G GCC---AGCA AUA---GACC 417 CuvErag2

Figure A40: Alignment of complete ITS1, 5.8S, ITS2 sequences of Anguillospora longissima, closest BLAST hits and Massarina and Lophiostoma spp..

218 Appendix

297 CGGACUCGCC UCAAA-GCAU UGGCGGCC-- UA-G---GCU C-GAGCGCAG CAGAAAC-GC GCCUCGC--- AGAGGGGC-G UCCA-UAGAC -G------373 LsaVagab 340 AGGACUUGCC U-AAAGUUAU UGGCAGCCU- -AUUCCCUUC UGGUA-GUAG CGUA----GC GCUU------GAGA-UUU-G G-CAUCAGCA AG----UGUU 418 Ms3Ramun 321 UGGACUCGCC UUAAAGCAAU UGGCAGCCA- UACG---GCU U-GAGCGCAG CACAUU--GC GUAC------UCUGGGU-AG GCC-UCAG-A AG------UU 398 Ms3Walke 501 511 521 | | | 500 UUGGCUCUUG ACCUCGGAUC AGGUAG 525 LehDoli2 433 UC-AA--UCG GAUCCGAAGU AAAAUU 455 Ms3Bipol 428 UC-AA--GCC UCCUCAAUCG GAUGCG 450 Ms3Froni 410 --- AAGUUUG ACCUCGGAUC AGGUAG 432 Ms3Armat 416 UAUC---UUG ACCUCGGAUC ...... 432 Ms3Corti 418 CCAAA--UUG ACCUCGGA...... 433 LsaCaul2 436 AU-AG--UUG ACCUCGGAUC AGGUAG 458 LehConte 418 UUGG---UUG ACCUCGGAUC AGGUAG 440 AnguLongi1 395 UUGG---UUG ACCUCGGAUC AGGUAG 417 Alongi_106 418 UUGG---UUG ACCUCGGAUC AGGUAG 440 A.longi009 395 UUGG---UUG ACCUCGGAUC AGGUAG 417 Along11891 418 UUGG---UUG ACCUCGGAUC AGGUAG 440 AlongiL22 433 -U-AGC-UUG ACCUCGGAAU CAGGUA 455 Ms3Rubi 426 UCGAC--UUG ACCUCGGAUC AGGUAG 449 Ms3Ebur2 446 UU-AACUUUG ACCUCGGAUC AGGUAG 470 PlpPapav 418 UU--ACUUUG ACCUCGGA...... 433 CuvErag2 374 --- AAGUUUG ACCUCGGAUC AGGUAG 396 LsaVagab 419 UUACAU-UUG ACCUCAAAUC AGGUAG 443 Ms3Ramun 399 UUUC---UUG ACCUCGGA...... 413 Ms3Walke

Figure A40 continued

219 Appendix

Alignment ITS sequences Anguillospora crassa

1 11 21 31 41 51 61 71 81 91 100 | | | | | | | | | | | 1 ...... G---U-- -UCCC------UGCCCU U----CGGGG UAGG------AU 24 AnguCrassa 1 ...... G-AAGGAUC AUUAC----- AGUG---U-- -UCCC------UGCCCU U----CGGGG UAGG------AU 40 AnguCrassa 1 ...... C AUUAC----- AGUG---U-- UUCCC------UGCCCU U----CGGGG UAGG------AU 34 ZeiVariu 1 ...... 0 AnguCrassa 1 ...... CC------UGCCCU U----CGGGG UAGG------AU 20 AnguCrassa 1 ...... CA-G---- AGAA------C-U------UGCCCU U----CGGGG UAG------AU 26 Lo1Speci 1 ...... C AUUACACAAA AGGA---U-- ---A------CCCU C---ACCGG- U------AU 30 Mi4Ciner 1 ...... CAU UACACA--AA AGGA---U-- ---A------CCCU C---ACCGG- U------AU 30 Mi4Minut 1 ...... C AUUAC----- AGAG---U-- -UCA------UGCCCU C---AC-GGG UAG------AU 31 P71Infes 1 ...... AANNAUC N--A------AGNGAAAAA- --CAANN------CGC--- GUUUUUNGGA AAAAAGAANU GGGUACU-AU 55 HyySpeci 1 ...... AAGGAUC AUUA-A---- AGAA------UGCCCC GUUUUUUGAA A------U GGGUUCU-AU 43 HyyEric7 1 ...... C AUUAC----- AGAG---U-- -UCA------UGCCCU GCAAA-- GGG UAG------AC 33 FAbTsug 1 CCCUUCCGUA GGUGAACCUG CGGAAGGAUC AUUAC----- AGAA---U-- -GUA------GGCC-U ---AACCGCC CA------AU 59 ShaFlav8 1 ...... A GGUGAACCUG CGGAAGGAUC AUUA-A---- AGAA---U-- -UUA------GGC--U UU--ACCGCC ------AU 48 CudLute4 1 ...... UC AUUAC----- AGAG-CGUGA UGCUAUGCAG AACUUGUUCU ------GUG- UAG------CUGAC 47 BmrGram5 101 111 121 131 141 151 161 171 181 191 200 | | | | | | | | | | | 25 C---GCCACC C--UU--G-A UU-A-UUUAU -GAG-UGUUG CUUUGGCGGG CCUUG---CG GCC----UAG UCAC-GCCCC GG--CUUC-G G-UGGGGGA- 99 AnguCrassa 41 C---GCCACC C--UU--G-A UU-A-UUUAU -GAG-UGUUG CUUUGGCGGG CCUUG---CG GCC----UAG UCAC-GCCCC GG--CUUC-G G-UGGGGGA- 115 AnguCrassa 35 C---GCCACC C--UU--G-A U-UA-UUUAU -GAG-UGUUG CUUUGGCGGG CCUCG---CG GCC-----UG GCCGCGCCCC GG--CUUC-G G-CGGGGGA- 109 ZeiVariu 1 ...... C--UU--G-A UU-A-UUUAU -GAG-UGUUG CUUUGGCGGG CCUUG---CG GCC----UAG UCAC-GCCCC GG--CUUC-G G-UGGGGGA- 68 AnguCrassa 21 C---GCCACC C--UU--G-A UU-A-UUUAU -GAG-UGUUG CUUUGGCGGG CCUUG---CG GCC----UAG UCAC-GCCCC GG--CUUC-G G-UGGGGGA- 95 AnguCrassa 27 CU--CCCACC C-UUU--GUU UACA-U-UAC CU--UUGUUG CUUUGGCGGG CCC-GUCUUA ------CGAC--CACC GG--CUUU-G G-CUGGUCA- 99 Lo1Speci 31 A--CCCCACC C--GU--GUC U--A-UUUAC UC--UUGUUG CUUUGGCAGG CCG-----UG G------U CACCCACUGC A---GGCU-C UGCCUG-CAU 101 Mi4Ciner 31 A--CCCCACC C--GU--G-U CU-A-UUUAC UC--UUGUUG CUUUGGCAGG CCG-----UG GUCUCCC------ACUGC GGG-CUC--U G-CCUG-CAA 101 Mi4Minut 32 CU--CCCACC C--UU--GUA UAUAUAUAAU UAUC-UGUUG CUUUGGCGGG CCG----CGA GCC---- UAG CU------UGC CCG--GGUUCU GCCCGG-C-- 107 P71Infes 56 -U--CCCAUA CC-GU--GUC UAC---UUAC CU--UUGUUG CUUUGGCGGG CCGCCUUCGG G------CGUCGUNGGU GG--CUCC-G G-CUGA-CA- 129 HyySpeci 44 -U--CCCAAA CC-GU--GUA UAUA---UAC CU--UUGUUG CUUUGGCAGG CCGCCUUCGG G------CGUC GG--CUCA-C G-CUGACC-- 111 HyyEric7 34 CU--CCCACC C--GUGUGUA UAUA---UAC C-A- -UGUUG CUUUGGCAGG CC---UGCCG UCA - GGCUGC CGGCU CCCC GG------ARCUGGUGC- 110 FAbTsug 60 -U--CUCACC CC-AU--G-C -AUACCUGUA CUA--UGUUG CCUUGGCAGG CUG----UUG CCA------108 ShaFlav8 49 AUU-CUCACC C-UUU--GUA UACGUAUA-C U-A--UGUUG CCUUGGCAGG CUG----UU------ACUA- 99 CudLute4 48 CCU--CCACC C--GU--GUC -G-AUUAU-C UCA--UGUUG CUUUGGCGGA UCGGGCUU-- GCCCG------CGCCC- A---CUUUGG-U---- -GGGA 121 BmrGram5 201 211 221 231 241 251 261 271 281 291 300 | | | | | | | | | | | 100 GCGCCCGCCA GAGGA-UUCU A-C---AAAC -CU---G--A UUA- UUAGUG UCGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 181 AnguCrassa 116 GCGCCCGCCA GAGGA-UUCU A-C---AAAC -CU---G--A UUA- UUAGUG UCGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 197 AnguCrassa 110 GCGCCCGCCA GAGGA-UUCU A-C---AAAC - CU---G-AU U-A-UUAGUG UCGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 191 ZeiVariu 69 GCGCCCGCCA GAGGA-UUCU A-C---AAAC -CU---G--A UUA- UUAGUG UCGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 150 AnguCrassa 96 GCGCCCGCCA GAGGA-UUCU A-C---AAAC -CU---G--A UUA- UUAGUG UCGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 177 AnguCrassa 100 GCGCCCGCCA GAGG------A-CCCAAAAC UCUUUUG------UUAAUG UCGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 179 Lo1Speci 102 GUGCCUGCCA GAGGA------CC---AAC -CUCU-G-AA U--UUUAGUG AUGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 180 Mi4Ciner 102 GUGCCUGCCA GAGG------A-CC--AAAC UCU---G-AA UU--UUAGUG AUGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 180 Mi4Minut 108 GUGCCCGCCA GAGGA----- AGCCU-AAAC CCU---G-AA U-G- UUAGUG -CGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 188 P71Infes 130 GCGCCCGCCA GAGG------A-CCC-AAAC CC----GUCU --GUUUAGUG AUGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 209 HyySpeci 112 GCGCCUGCCA GAGG------A-CCC-AAAC UC----GUUU --AUUUAGUG AUGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACGACGGAUC 191 HyyEric7 111 GUGCCUGCCA AAGGA------CCCCUAAAC UCU---GU-A U-A- UUAGUG UCGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 192 FAbTsug 109 --GCCUGCCA GUGGA------CCACAAAC CCU--AG--A--A-UCUCUG CUGUCUGAGC ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 186 ShaFlav8 100 --GCCUGCCG GUGGA------CCUC-AAC UCU--AG--A --A- UCUUUG CUGUCUGAGU ----ACUAUA U--AAUAGUU AAAACUUUCA ACAACGGAUC 176 CudLute4 122 UUAUCCGCCA GGGAAG---- ACC - AAAAC- UCUC-- GUU- --A - UCAGUG AUGUCUGAGG ----AUGAUA- UAAAAUCAUG AAAACUUUCA ACAACGGAUC 201 BmrGram5 301 311 321 331 341 351 361 371 381 391 400 | | | | | | | | | | | 182 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 281 AnguCrassa 198 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 297 AnguCrassa 192 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 291 ZeiVariu 151 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 250 AnguCrassa 178 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 277 AnguCrassa 180 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 279 Lo1Speci 181 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 280 Mi4Ciner 181 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 280 Mi4Minut 189 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 288 P71Infes 210 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 309 HyySpeci 192 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 291 HyyEric7 193 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 292 FAbTsug 187 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 286 ShaFlav8 177 UCUUGGUUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUCAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 276 CudLute4 202 UCUUGGCUCU GGCAUCGAUG AAGAACGCAG CGAAAUGCGA UAAGUAAUGU GAAUUGCAGA AUUUAGUGAA UCAUCGAAUC UUUGAACGCA CAUUGCGCCC 301 BmrGram5 401 411 421 431 441 451 461 471 481 491 500 | | | | | | | | | | | 282 CGUGGUAUUC CGCGGGGCAU GCCUGUUCGA GCGUCAUU-A UAACC-AAUC CCGUUUA-CG GGGUCUUGGG ---CACCGCC UCUAGG---- CG-GGCCUC- 369 AnguCrassa 298 CGUGGUAUUC CGCGGGGCAU GCCUGUUCGA GCGUCAUU-A UAACC-AAUC CCGUUUA-CG GGGUCUUGGG ---CACCGCC UCUAGG---- CG-GGCCUC- 385 AnguCrassa 292 CGUGGUAUUC CGCGGGGCAU GCCUGUU-CG AGCGUCAUUA UGACC- AAUC CCGCUC-GCG GGGUCUUGGG ---CACCGCC UCU-GG---- GCGGGCCUC- 379 ZeiVariu 251 CGUGGUAUUC CGCGGGGCAU GCCUGUUCGA GCGUCAUU-A UAACC-AAUC CCGUUUA-CG GGGUCUUGGG ---CACCGCC UCUAGG---- CG-GGCCUC- 338 AnguCrassa 278 CGUGGUAUUC CGCGGGGCAU GCCUGUUCGA GCGUCAUU-A UAACC-AAUC CCGUUUA-CG GGGUCUUGGG ---CACCGCC UCUAGG---- CG-GGCCUC- 365 AnguCrassa 280 UCUGGUAUUC CGGGGGGCAU GCCUGUUCGA GCGUCAUU-A CAACC--CUC AAGCUCUGCU UGGUAUUGGG CGUCACCGUC UCC-GG---- UG-CGCCUU- 369 Lo1Speci 281 UGUGGUAUUC CGCAGGGCAU GCCUGUUCGA GCGUCAUU-A UAGCC-ACUC AAGCCUCGCU UGGUGUUGGG G-C--UCGC- AGACUU---- GC-GGCCUCU 369 Mi4Ciner 281 UGUGGUAUUC CGCAGGGCAU GCCUGUUCGA GCGUCAUU- A UAGCC -ACUC AAGCCUUGCU UGGUGUUGGG G-C--UCGC- A-AUCUU--- GC-GGCCUCU 369 Mi4Minut 289 CUUGGUAUUC CGGGGGGCAU GCCUGUUCGA GCGUCAUU-A UAACC-AAUC CAGCCUGGCU GGGUCUUGGG --CC-UCGC- ----GGUAUA GCGGGCUUU- 377 P71Infes 310 CUUGGUAUUC CGAGGGGCAU GCCUGUU-CG AGCGUCAUUA UGACC- ACUC AAGCCUAGCU UGGUAUUGGG G-CC--CGC- ----GGUCUC GC-GGCCCCU 398 HyySpeci 292 CUUGGUAUUC CGAGGGGCAU GCCUGUU-CG AGCGUCAUUA UGACC- ACUC AAGCCUAGCU UGGUAUUGGG G-UU--CNC- ----GGUCUC GC-GGCCCUU 380 HyyEric7 293 CUUGGUAUUC CGGGGGGCAU GCCUGUUCGA GCGUCAUU-U CAACC--CUC AAGCGUAACU AGGUCUUGGG UCCUGCC------GGCAA- --CGGCAGCC 379 FAbTsug 287 UCUGGUAUUC CAGGGGGCAU GCCUGUUCGA GCGUCAUU-A CAACC--CUC ACGCCCCGCG UGGUCUUGGG ----CUCGC- CCU--GCAG- ---GGCCUGC 372 ShaFlav8 277 UCUGGUAUUC CAGGGGGCAU GCCUGUUCGA GCGUCAUU-A CAACC--CUC ACGCCUAGCG UGGUCUUGGG ---- CUCGC- CCU--GUAG- ---GGCCUGC 362 CudLute4 302 CUGGGAAUUC CUAGGGGCAU GCCUGUUCGA GCGUCCGUAA CAACCU-CUC AAGCCUAGCU UGGUCUUGGG ACAUGCUGCU CUAGUGG--- --CGGCAGUC 395 BmrGram5 501 511 521 531 541 551 561 571 581 591 600 | | | | | | | | | | | 370 ---AAAAUCA GUGGCGGUCC GGCCAGG--- CUCUGAGCGU AGUAA--AUC UUCUCGCUAU AGG-GUCCU- GGGCGGUA-- - CUGGCCAAC AACCCCC-AA 455 AnguCrassa 386 ---AAAAUCA GUGGCGGUCC GGCCAGG--- CUCUGAGCGU AGUAA--AUC UUCUCGCUAU AGG-GUCCU- GGGCGGUA-- - CUGGCCAAC AACCCCC-AA 471 AnguCrassa 380 ---AAAAUCA GUGGCGGUAC GGCCGGG--- CUCUGAGCGU AGUAA--AUC UUCUCGCUAC AGG-GUCCC- GGGCGG--C- ACUGGCCAGC AACCCCC-AA 465 ZeiVariu 339 ---AAAAUCA GUGGCGGUCC GGCCAGG--- CUCUGAGCGU AGUAA--AUC UUCUCGCUAU AGG-GUCCU- GGGCGGUA-- - CUGGCCAAC AACCCCC-AA 424 AnguCrassa 366 ---AAAAUCA GUGGCGGUCC GGCCAGG--- CUCUGAGCGU AGUAA--AUC UUCUCGCUAU AGG-GUCCU- GGGCGGUA-- - CUGGCCAAC AACCCCC... 449 AnguCrassa 370 ---AAAAUCA GUGGCGGUGC CAUUAGG--- CUUCAAGCGU AGUAA--UUC UUCUCGCUCU GGA-GUUCU- -GGU-GUGU- GCUUGCCAAC AACCCCU-AA 455 Lo1Speci 370 ---AAAAUCA GUGGCGGUG- CCGGUAG--G CUCUGAGCGU AGUAC--AUC UCCUCGCUAU AGA-GUCCU- --AUCGGUC- CCCUGCCAA- AACCCCC-AU 454 Mi4Ciner 370 ---AAAAUUA GUGGCGGUGC CGGUAGG--- CUCUGAGCGU AGUAC--AUC UUCUCGCUAU AGAGUCCU-- G-UCGGUCC- CCU-GCCAA- AACCCCC-AU 454 Mi4Minut 378 ---AAAAACA GUGGCGGUGC UCUCAUG--- CUAUACGCGU AGUAA---UU UUCUCGCUAU AGG-GUUCU- --GGGAGAU- GCUUGCCAAC AACCCCC-AA 462 P71Infes 399 ---AAAAUCA GUGGCGGUGC CGUCUGG--- CUCUAAGCGU AGUAA---UU CUCUCGCUAC AGU-AUCCA- -GGUGG-UAA GCUUGCCAAC AACCCC--AA 482 HyySpeci 381 ---AAAAUCA GUGGCGGUGC CAUCUGG--- CUCUAAGCGU AGUAA--UUU AUCUCGCUAU UGG-GUCC-- -GGUGG-UU- GCUUGCCAAU AACCCCC-AA 465 HyyEric7 380 UU-AAAAUUA GUGGCGGUGC CCUGUUGUGG CCCUGAGCGU AAUAA--CUU UUCUCGCUAU -GGGUGCCCC ---AAGAUG- GCUUGCCAAC AACCCC--AA 469 FAbTsug 373 CUCAAAGUCA GUGGCAGCGC UGUCUGA--C CCCUAAGCGU AGUAA--UAC AUCUCGCUUC UGGGCGCUG- -- GAUGGAG - GCCUGCCAUG AACCCCCCA- 463 ShaFlav8 363 CUCAAAGUCA GUGGCGGCGU CGUCUGA--C CCCUAAGCGU AGUAAAAUAC ACCUCGCUUC UGGGCUGUU- - GGAUAGAG - GCUUGCUGUG AAACCCCCAA 457 CudLute4 396 CUCAAAAGAA GUGGCGGGCC CAUGUAA--- CUCUCCGCGU AGUAA--UAC AUCUCGCGAC AGGGAAGCAG CGGG-----A CUUGCCAA-- AACUCCUUAA 483 BmrGram5

Figure A41: Alignment of complete ITS1, 5.8S, ITS2 sequences of Anguillospora crassa, closest BLAST hits and Mollisia spp..

220 Appendix

Alignment ITS sequences Anguillospora crassa continued

601 611 621 631 641 651 661 671 681 691 700 | | | | | | | | | | | 456 --UCUUUUAC AGGUUGACCU CGGAUCAGGU AGGG-AUACC CG...... 494 AnguCrassa 472 --UCUUUUAC AGGUUGACCU CGGAUCAGGU AGGG...... 503 AnguCrassa 466 --UCUUUUAC AGGUUGACCU CGGAUCAGGU AGGG-AUACC CGCUGAACUU AAGCAUAUCA AU...... 524 ZeiVariu 425 --UCUUUUAC AGGUUGACCU CGGAUCAGGU AGGG-AU-C...... 459 AnguCrassa 450 ...... 449 AnguCrassa 456 ----UUUUAU CAA...... 464 Lo1Speci 455 A-UUUUUUAU AGGUUGA...... 470 Mi4Ciner 455 A---UUUUAU AGGUUGA...... 468 Mi4Minut 463 --U-UUAUAU AGGUUGACCU ...... 479 P71Infes 483 --CUCUC-AC -GGUUGACCU CGGAUCAGGU AGGG-AUACC CGCUGAACUU AA...... 529 HyySpeci 466 --CUUCCAA- -GGUUGACCU CGGAUCAGGU AGG-AAUACC CGCUGAACAU AA...... 512 HyyEric7 470 -UUUUUCUAU -GGUUGACCU ...... 487 FAbTsug 464 ---CUUUUAC AGGUUGACCU CGAAUCAGGU AGGG-AUACC CGCUGAACUU AAGCAUAUCA AUAAGNCGGA GGAA...... 533 ShaFlav8 458 ---CUUU-AC AGGUUGACCU CGGAUCAGGU AGGG-AUACC CGCUGAACUU AAGCAUAUCA AUAAGCNGGA GGAAUU...... 528 CudLute4 484 ---UUGCU-C AGGUUGACCU CGAAUCAGGU AGG-GAUACC CGCUGAACUU AAGCAUAUCA AUAAGCGGAG GAAAAGAAAC CAACAGGGAU UACCUCAGUA 578 BmrGram5 701 711 | | 495 ...... 494 AnguCrassa 504 ...... 503 AnguCrassa 525 ...... 524 ZeiVariu 460 ...... 459 AnguCrassa 450 ...... 449 AnguCrassa 465 ...... 464 Lo1Speci 471 ...... 470 Mi4Ciner 469 ...... 468 Mi4Minut 480 ...... 479 P71Infes 530 ...... 529 HyySpeci 513 ...... 512 HyyEric7 488 ...... 487 FAbTsug 534 ...... 533 ShaFlav8 529 ...... 528 CudLute4 579 ACGGCGAGUG AAGCGGC 595 BmrGram5

Figure A41 continued

1 11 21 31 41 51 61 71 81 91 100 | | | | | | | | | | | 1 ...... GUAG GUGAACCUGC GGAAGGAUCA UUACAGUGU- UCCCUGCCCU UCGGGGUAGG AUCGCCACCC UUGAUUAUU UUAUGAGUGU 82 TriSpl1198 1 UCGUAACAAG GUUUCCGUAG GUGAACCUGC GGAAGGAUCA UUACAGUGU- UCCCUGCCCU UCGGGGUAGG AUCGCCACCC UUGAUUAUU UUAUGAGUGU 98 TriSpl1659 1 ...... UCCGUAG GUGAACCUGC GGAAGGAUCA UUACAGwGU- UCCCUGCCCU UCGGGGUAnG AUCGCCACCC UUGAUUAUU UUAUGAGUGU 85 TriSpl1908 1 ...... CGUAG GUGAACCUGC GGAAGGAUCA UUACAGUGU- UCCCUGCCCU UCGGGGUAGG AUCGCCACCC UUGAUUAUU UUAUGAGUGU 83 TriSpl1238 1 ...... CA UUACAGUGUU UCCCUGCCCU UCGGGGUAGG AUCGCCACCC UUGAUUAU- UUAUGAGUGU 60 ZeiVariu 1 ...... C UUGAUUAU- UUAUGAGUGU 19 AnguCrassa 1 ...... G-AAGGAUCA UUACAGUGU- UCCCUGCCCU UCGGGGUAGG AUCGCCACCC UUGAUUAU- UUAUGAGUGU 66 AnguCrassa 1 ...... GU- UCCCUGCCCU UCGGGGUAGG AUCGCCACCC UUGAUUAU- UUAUGAGUGU 50 AnguCrassa 1 ...... CCUGCCCU UCGGGGUAGG AUCGCCACCC UUGAUUAU- UUAUGAGUGU 46 AnguCrassa 101 111 121 131 141 151 161 171 181 191 200 | | | | | | | | | | | 83 UGCUUUGGCG GGCCUCGCGG CCUGGCCGC GCCCCGGCUC CGGCGGGGGA GCGCCCGCCA GAGGCUUCU ACAAACCUGU GUAUUAGUGU CGUCUGAGUA 180 TriSpl1198 99 UGCUUUGGCG GGCCUCGCGG CCUGGCCGC GCCCCGGCUC CGGCGGGGGA GCGCCCGCCA GAGGCUUCU ACAAACCUGU GUAUUAGUGU CGUCUGAGUA 196 TriSpl1659 86 UGCUUUGGCG GGCCUCGCGG CCUGGCCGC GCCCCGGCUC CGGCGGGGGA GCGCCCGCCA GAGGCUUCU ACAAACCUGU GUAUUAGUGU CGUCUGAGUA 183 TriSpl1908 84 UGCUUUGGCG GGCCUCGCGG CCUGGCCGC GCCCCGGCUC CGGCGGGGGA GCGCCCGCCA GAGGCUUCU ACAAACCUGU GUAUUAGUGU CGUCUGAGUA 181 TriSpl1238 61 UGCUUUGGCG GGCCUCGCGG CCUGGCCGC GCCCCGGCUU CGGCGGGGGA GCGCCCGCCA GAGGAUUCU ACAAACCUGA UUAUUAGUGU CGUCUGAGUA 158 ZeiVariu 20 UGCUUUGGCG GGCCUUGCGG CCUAGUCAC GCCCCGGCUU CGGUGGGGGA GCGCCCGCCA GAGGAUUCU ACAAACCUGA UUAUUAGUGU CGUCUGAGUA 117 AnguCrassa 67 UGCUUUGGCG GGCCUUGCGG CCUAGUCAC GCCCCGGCUU CGGUGGGGGA GCGCCCGCCA GAGGAUUCU ACAAACCUGA UUAUUAGUGU CGUCUGAGUA 164 AnguCrassa 51 UGCUUUGGCG GGCCUUGCGG CCUAGUCAC GCCCCGGCUU CGGUGGGGGA GCGCCCGCCA GAGGAUUCU ACAAACCUGA UUAUUAGUGU CGUCUGAGUA 148 AnguCrassa 47 UGCUUUGGCG GGCCUUGCGG CCUAGUCAC GCCCCGGCUU CGGUGGGGGA GCGCCCGCCA GAGGAUUCU ACAAACCUGA UUAUUAGUGU CGUCUGAGUA 144 AnguCrassa 201 211 221 231 241 251 261 271 281 291 300 | | | | | | | | | | | 181 CUAUAUAAUA GUUAAAACU UUCAACAACG GAUCUCUUGG UUCUGGCAUC GAUGAAGAAC GCAGCGAAAU GCGAUAAGUA AUGUGAAUUG CAGAAUUCAG 279 TriSpl1198 197 CUAUAUAAUA GUUAAAACU UUCAACAACG GAUCUCUUGG UUCUGGCAUC GAUGAAGAAC GCAGCGAAAU GCGAUAAGUA AUGUGAAUUG CAGAAUUCAG 295 TriSpl1659 184 CUAUAUAAUA GUUAAAACU UUCAACAACG GAUCUCUUGG UUCUGGCAUC GAUGAAGAAC GCAGCGAAAU GCGAUAAGUA AUGUGAAUUG CAGAAUUCAG 282 TriSpl1908 182 CUAUAUAAUA GUUAAAACU UUCAACAACG GAUCUCUUGG UUCUGGCAUC GAUGAAGAAC GCAGCGAAAU GCGAUAAGUA AUGUGAAUUG CAGAAUUCAG 280 TriSpl1238 159 CUAUAUAAUA GUUAAAACU UUCAACAACG GAUCUCUUGG UUCUGGCAUC GAUGAAGAAC GCAGCGAAAU GCGAUAAGUA AUGUGAAUUG CAGAAUUCAG 257 ZeiVariu 118 CUAUAUAAUA GUUAAAACU UUCAACAACG GAUCUCUUGG UUCUGGCAUC GAUGAAGAAC GCAGCGAAAU GCGAUAAGUA AUGUGAAUUG CAGAAUUCAG 216 AnguCrassa 165 CUAUAUAAUA GUUAAAACU UUCAACAACG GAUCUCUUGG UUCUGGCAUC GAUGAAGAAC GCAGCGAAAU GCGAUAAGUA AUGUGAAUUG CAGAAUUCAG 263 AnguCrassa 149 CUAUAUAAUA GUUAAAACU UUCAACAACG GAUCUCUUGG UUCUGGCAUC GAUGAAGAAC GCAGCGAAAU GCGAUAAGUA AUGUGAAUUG CAGAAUUCAG 247 AnguCrassa 145 CUAUAUAAUA GUUAAAACU UUCAACAACG GAUCUCUUGG UUCUGGCAUC GAUGAAGAAC GCAGCGAAAU GCGAUAAGUA AUGUGAAUUG CAGAAUUCAG 243 AnguCrassa 301 311 321 331 341 351 361 371 381 391 400 | | | | | | | | | | | 280 UGAAUCAUCG AAUCUUUGAA CGCACAUUGC GCCCCGUGGU AUUCCGCGGG GCAUGCCUGU UCGAGCGUCA UUAUAACCAA UCCAGCUC GCUGGGUCUU 377 TriSpl1198 296 UGAAUCAUCG AAUCUUUGAA CGCACAUUGC GCCCCGUGGU AUUCCGCGGG GCAUGCCUGU UCGAGCGUCA UUAUAACCAA UCCAGCUC GCUGGGUCUU 393 TriSpl1659 283 UGAAUCAUCG AAUCUUUGAA CGCACAUUGC GCCCCGUGGU AUUCCGCGGG GCAUGCCUGU UCGAGCGUCA UUAUAACCAA UCCAGCUC GCUGGGUCUU 380 TriSpl1908 281 UGAAUCAUCG AAUCUUUGAA CGCACAUUGC GCCCCGUGGU AUUCCGCGGG GCAUGCCUGU UCGAGCGUCA UUAUAACCAA UCCAGCUC GCUGGGUCUU 378 TriSpl1238 258 UGAAUCAUCG AAUCUUUGAA CGCACAUUGC GCCCCGUGGU AUUCCGCGGG GCAUGCCUGU UCGAGCGUCA UUAUGACCAA UCCCGCUC GCGGGGUCUU 355 ZeiVariu 217 UGAAUCAUCG AAUCUUUGAA CGCACAUUGC GCCCCGUGGU AUUCCGCGGG GCAUGCCUGU UCGAGCGUCA UUAUAACCAA UCCCGUUU ACGGGGUCUU 314 AnguCrassa 264 UGAAUCAUCG AAUCUUUGAA CGCACAUUGC GCCCCGUGGU AUUCCGCGGG GCAUGCCUGU UCGAGCGUCA UUAUAACCAA UCCCGUUU ACGGGGUCUU 361 AnguCrassa 248 UGAAUCAUCG AAUCUUUGAA CGCACAUUGC GCCCCGUGGU AUUCCGCGGG GCAUGCCUGU UCGAGCGUCA UUAUAACCAA UCCCGUUU ACGGGGUCUU 345 AnguCrassa 244 UGAAUCAUCG AAUCUUUGAA CGCACAUUGC GCCCCGUGGU AUUCCGCGGG GCAUGCCUGU UCGAGCGUCA UUAUAACCAA UCCCGUUU ACGGGGUCUU 341 AnguCrassa

401 411 421 431 441 451 461 471 481 491 500 | | | | | | | | | | | 378 GGGCACCGCC GCCUGG-CGG GCCUCAAAAG CAGUGGCGGU ACGGCCGGGC UCUGAGCGUA GUAAAUCUUC UCGCUACAGG GUCCCGGGCG GCACUGGC 474 TriSpl1198 394 GGGCACCGCC GCCUGG-CGG GCCUCAAAAG CAGUGGCGGU ACGGCCGGGC UCUGAGCGUA GUAAAUCUUC UCGCUACAGG GUCCCGGGCG GCACUGGC 490 TriSpl1659 381 GGGCACCGCC GCCUGG-CGG GCCUCAAAAG CAGUGGCGGU ACGGCCGGGC UCUGAGCGUA GUAAAUCUUC UCGCUACAGG GUCCCGGGCG GCACUGGC 477 TriSpl1908 379 GGGCACCGCC GCCUGG-CGG GCCUCAAAAG CAGUGGCGGU ACGGCCGGGC UCUGAGCGUA GUAAAUCUUC UCGCUACAGG GUCCCGGGCG GCACUGGC 475 TriSpl1238 356 GGGCACCGCC -UCUGGGCGG GCCUCAAAAU CAGUGGCGGU ACGGCCGGGC UCUGAGCGUA GUAAAUCUUC UCGCUACAGG GUCCCGGGCG GCACUGGC 452 ZeiVariu 315 GGGCACCGCC -UCUAGGCGG GCCUCAAAAU CAGUGGCGGU CCGGCCAGGC UCUGAGCGUA GUAAAUCUUC UCGCUAUAGG GUCCUGGGCG GUACUGGC 411 AnguCrassa 362 GGGCACCGCC -UCUAGGCGG GCCUCAAAAU CAGUGGCGGU CCGGCCAGGC UCUGAGCGUA GUAAAUCUUC UCGCUAUAGG GUCCUGGGCG GUACUGGC 458 AnguCrassa 346 GGGCACCGCC -UCUAGGCGG GCCUCAAAAU CAGUGGCGGU CCGGCCAGGC UCUGAGCGUA GUAAAUCUUC UCGCUAUAGG GUCCUGGGCG GUACUGGC 442 AnguCrassa 342 GGGCACCGCC -UCUAGGCGG GCCUCAAAAU CAGUGGCGGU CCGGCCAGGC UCUGAGCGUA GUAAAUCUUC UCGCUAUAGG GUCCUGGGCG GUACUGGC 438 AnguCrassa

501 511 521 531 541 551 561 571 | | | | | | | | 475 CAGCAACCCC CAAAUCUUU CACAGGUUGA CCUCGGAUCA GGUAGGGAU ACCCGCUGAA CUUAAGCAUA UAAU...... 546 TriSpl1198 491 CAGCAACCCC CAAAUCUUU CACAGGUUGA CCUCGGAUCA GGUAGGGAU ACCCGCUGAA CUUAAGCAUA UCAAUAAGCG 568 TriSpl1659 478 CAGCAACCCC CAAAUCUUU CACAGGUUGA CCUCGGAUCA GGUAGGGAU ACCCGCUGAA CUUAAGCAUA UCAAUAAGCG 555 TriSpl1908 476 CAGCAACCCC CAAAUCUUU CACAGGUUGA CCUCGGAUCA GGUAGGGAU ACCCGCUGAA CUUAACCAUA ...... 543 TriSpl1238 453 CAGCAACCCC CAA-UCUUU UACAGGUUGA CCUCGGAUCA GGUAGGGAU ACCCGCUGAA CUUAAGCAUA UCAAU..... 524 ZeiVariu 412 CAACAACCCC CAA-UCUUU UACAGGUUGA CCUCGGAUCA GGUAGGGAU -C...... 459 AnguCrassa 459 CAACAACCCC CAA-UCUUU UACAGGUUGA CCUCGGAUCA GGUAGGG...... 503 AnguCrassa 443 CAACAACCCC CAA-UCUUU UACAGGUUGA CCUCGGAUCA GGUAGGGAU ACCCG...... 494 AnguCrassa 439 CAACAACCCC C...... 449 AnguCrassa

Figure A42: Alignment of complete ITS1, 5.8S, ITS2 sequences of Anguillospora crassa, Tricladium splendens and Zalerion varium.

221 Appendix

Alignment ITS sequences Tricladium splendens

1 11 21 31 41 51 61 71 81 91 100 | | | | | | | | | | | 1 ...... GUAG GUGAACCUGC GGAA-GGAUC AUUAC-AGUG ---U---UCC C------UGCCCUU--- -CGGGGUAGG ------53 TriSpl1198 1 UCGUAACAAG GUUUCCGUAG GUGAACCUGC GGAA-GGAUC AUUAC-AGUG ---U---UCC C------UGCCCUU--- -CGGGGUAGG ------69 TriSpl1659 1 ...... UCCGUAG GUGAACCUGC GGAA-GGAUC AUUAC-AGwG ---U---UCC C------UGCCCUU--- -CGGGGUAnG ------56 TriSpl1908 1 ...... CGUAG GUGAACCUGC GGAA-GGAUC AUUAC-AGUG ---U---UCC C------UGCCCUU--- -CGGGGUAGG ------54 TriSpl1238 1 ...... C AUUAC-AGUG ---U--UUCC C------UGCCCUU--- -CGGGGUAGG ------32 ZeiVariu 1 ...... UCCGUAG GUGAACCUGC GGAA-GGAUC AUUAC-AGAG ---U---UCA ------UGCCCUU--- -CGGGGUAG------54 LhnBicol 1 ...... AA-GGAUC AUUA-AAGAA ------UGCCCCGUUU UUUGAAA------UGGGU 38 HyyEric7 1 ...... AA-NNAUC N--A--AGNG AAAAA---CA ANN------CGC---GUUU UUNGGAAAAA AGAANUGGGU 50 HyySpeci 1 ...... AGGGAUC AUUAC-AGAG ---U---UCA ------UGCCCUU--- AU-GGG-UAG ------35 HyyEric6 1 ...... C AUUAC-AGAG ---U---UCC ------UGCCCUC--- AC-GGG-UAG ------29 HyyFruct 1 ...... UC AUUAC-AGAG -CGUGAUGCU AUGCAGAACU UGUUCU------GUG-UAG------42 BmrGram5 101 111 121 131 141 151 161 171 181 191 200 | | | | | | | | | | | 54 ----AUC--- GCCA- CCCUU G-AU-UAUUU UAU-GAG-UG UUGCUUUGGC GGGCCUCG-- -CGGCC---- UGGCCGCGCC CCGG--CUCC GG-CGGGGGA 131 TriSpl1198 70 ----AUC--- GCCA- CCCUU G-AU-UAUUU UAU-GAG-UG UUGCUUUGGC GGGCCUCG-- -CGGCC---- UGGCCGCGCC CCGG--CUCC GG-CGGGGGA 147 TriSpl1659 57 ----AUC--- GCCA- CCCUU G-AU-UAUUU UAU-GAG-UG UUGCUUUGGC GGGCCUCG-- -CGGCC---- UGGCCGCGCC CCGG--CUCC GG-CGGGGGA 134 TriSpl1908 55 ----AUC--- GCCA- CCCUU G-AU-UAUUU UAU-GAG-UG UUGCUUUGGC GGGCCUCG-- -CGGCC---- UGGCCGCGCC CCGG--CUCC GG-CGGGGGA 132 TriSpl1238 33 ----AUC--- GCCA- CCCUU G-AU-UA-UU UAU-GAG-UG UUGCUUUGGC GGGCCUCG-- -CGGCC---- UGGCCGCGCC CCGG--CUUC GG-CGGGGGA 109 ZeiVariu 55 ----AUCU-- GCCAA- CCUU -UAU-CA--U CAUUAAG-UG UUGCUUUGNC GAAUC--G-- A-GGCCU------UCAC--C CCGG------GG-CCCCUCU 122 LhnBicol 39 UCU-AU-U-- CCCAAACCGU GUAUAUA--- UACCU--UUG UUGCUUUGGC AGGCCGCCUU CGGG------CG UCGG--CUCA CG-CUGACC- 111 HyyEric7 51 ACU-AU-U-- CCCAUACCGU GUCUAC---U UACCU--UUG UUGCUUUGGC GGGCCGCCUU CGGG------CGUCGUNG GUGG--CUCC GG-CUGA-CA 129 HyySpeci 36 ----AUCU-- CCCACCCUAU GUUAUCA--U UACCU--UUG UUGCUUUGGC GGGCCGUC-- A-GGCCUCGG -----UCAGG CUACCGGUCC GG- CUGGUAA 116 HyyEric6 30 ----AAA--C CCCA- CCCUU GUAUAUACUA UA-----UUG UUGCUUUGGC AGGCCGCCUC ACGG------CGUU- --GG---CUC ACGCUGACU- 99 HyyFruct 43 -CUGACCCU- -CCA- CCCGU GUC-G-AUUA U-CUCA--UG UUGCUUUGGC GGA-----UC GGGCUUGCC- ----CGCGCC CACUUUGG-U GGGAGU- UA - 120 BmrGram5 201 211 221 231 241 251 261 271 281 291 300 | | | | | | | | | | | 132 -GCGCCCGCC AGAGG--CUU CUA-C---AA ACC- UG-UGU ---A-UUAGU GUCGUCUGAG U----ACUAU AU--AAU-AG UUAAAACUUU CAACAACGGA 211 TriSpl1198 148 -GCGCCCGCC AGAGG--CUU CUA-C---AA ACC- UG-UGU ---A-UUAGU GUCGUCUGAG U----ACUAU AU--AAU-AG UUAAAACUUU CAACAACGGA 227 TriSpl1659 135 -GCGCCCGCC AGAGG--CUU CUA-C---AA ACC- UG-UGU ---A-UUAGU GUCGUCUGAG U----ACUAU AU--AAU-AG UUAAAACUUU CAACAACGGA 214 TriSpl1908 133 -GCGCCCGCC AGAGG--CUU CUA-C---AA ACC- UG-UGU ---A-UUAGU GUCGUCUGAG U----ACUAU AU--AAU-AG UUAAAACUUU CAACAACGGA 212 TriSpl1238 110 -GCGCCCGCC AGAGGA--UU CUA-C---AA ACC- UG---- AUUA-UUAGU GUCGUCUGAG U----ACUAU AU--AAU-AG UUAAAACUUU CAACAACGGA 189 ZeiVariu 123 -GUAUUCUCC AGAGG------AUNNC-AN AUUCUG- C- - AUUAU ----- GUCGUCUGAG U----ACCAU AA--AAU-AG UUAAAACUUU CAACAACNNN 199 LhnBicol 112 -GCGCCUGCC AGAGG------A-CCC-AA ACUC----GU UU-AUUUAGU GAUGUCUGAG U----ACUAU AU--AAU-AG UUAAAACUUU CAACGACGGA 189 HyyEric7 130 -GCGCCCGCC AGAGG------A-CCC-AA ACCC----GU CU-GUUUAGU GAUGUCUGAG U----ACUAU AU--AAU-AG UUAAAACUUU CAACAACGGA 207 HyySpeci 117 -GCGCCCGCC AGAGG------ACCCCCAA ACUCUG--A- AU-A-UUAGU GUCGUCUGAG U----ACUAU AUU-AAU-AG UUAAAACUUU CAACAACGGA 197 HyyEric6 100 -GUGCCUGCC AGAGG------A-CCCUAA ACUCUG-- AA AU-A--CAGU GUCGUCUGAG U----ACUAU -UU-AAU-AG UUAAAACUUU CAACAACGGA 178 HyyFruct 121 U----CCGCC AGGGAAG--- --A--CCAAA ACUCUC--GU U--A-UCAGU GAUGUCUGAG GAUGAUAUAA AAUC-AU--G --AAAACUUU CAACAACGGA 199 BmrGram5 301 311 321 331 341 351 361 371 381 391 400 | | | | | | | | | | | 212 UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC 311 TriSpl1198 228 UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC 327 TriSpl1659 215 UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC 314 TriSpl1908 213 UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC 312 TriSpl1238 190 UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC 289 ZeiVariu 200 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC 299 LhnBicol 190 UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC 289 HyyEric7 208 UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC 307 HyySpeci 198 UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU GUGAAUUGCA GAAUUUAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC 297 HyyEric6 179 UCUCUUGGUU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU GUGAAUUGCA GAAUUCAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC 278 HyyFruct 200 UCUCUUGGCU CUGGCAUCGA UGAAGAACGC AGCGAAAUGC GAUAAGUAAU GUGAAUUGCA GAAUUUAGUG AAUCAUCGAA UCUUUGAACG CACAUUGCGC 299 BmrGram5 401 411 421 431 441 451 461 471 481 491 500 | | | | | | | | | | | 312 CCCGUGGUAU UCCGCGGGGC AUGCCUGUU- CGAGCGUCAU UAUAACCAA- UCCAGC--UC -GCUGGGUCU UGGGCACCGC -CGCC-U--- -GGCG-GGCC 399 TriSpl1198 328 CCCGUGGUAU UCCGCGGGGC AUGCCUGUU- CGAGCGUCAU UAUAACCAA- UCCAGC--UC -GCUGGGUCU UGGGCACCGC -CGCC-U--- -GGCG-GGCC 415 TriSpl1659 315 CCCGUGGUAU UCCGCGGGGC AUGCCUGUU- CGAGCGUCAU UAUAACCAA- UCCAGC--UC -GCUGGGUCU UGGGCACCGC -CGCC-U--- -GGCG-GGCC 402 TriSpl1908 313 CCCGUGGUAU UCCGCGGGGC AUGCCUGUU- CGAGCGUCAU UAUAACCAA- UCCAGC--UC -GCUGGGUCU UGGGCACCGC -CGCC-U--- -GGCG-GGCC 400 TriSpl1238 290 CCCGUGGUAU UCCGCGGGGC AUGCCUGUU- CGAGCGUCAU UAUGACCAA- UCCCGC--UC -GCGGGGUCU UGGGCACCGC -C-UC-U--- -GGGCGGGCC 377 ZeiVariu 300 CCCUUGGUAU UCCGGGGGGC AUGCCUGUU- CGAGCGUCAU UAUAAACAA- UCCAGU--UU A-CUGGGUCU UGGGCCUCGC GAUUC-U--- --GC- GGGCC 387 LhnBicol 290 CCCUUGGUAU UCCGAGGGGC AUGCCUGUU- CGAGCGUCAU UAUGACCA-C UCAAGC--CU AGCUUGGUAU UGGGG-U-UC NCGGUCUC-- --GC--GGCC 377 HyyEric7 308 CCCUUGGUAU UCCGAGGGGC AUGCCUGUU- CGAGCGUCAU UAUGACCA-C UCAAGC--CU AGCUUGGUAU UGGGG-C-CC GCGGUCUC-- --GC--GGCC 395 HyySpeci 298 CCCUUGGUAU UCCGAGGGGC AUGCCUGUU- CGAGCGUCAU UACAACCC-- UCAAGC--AU UGCUUGGUAU UGGGCUCCGC UACUCACCC- -AGC-GGGCC 389 HyyEric6 279 CCCUUGGUAU UCCGAGGGGC AUGCCUGUU- CGAGCGUCAU UUAUACCAAC UCCUACUCUC AGUAGGGUCU UGGGCUUCGC CUCU------GGGCGGGCC 370 HyyFruct 300 CCCUGGGAAU UCCUAGGGGC AUGCCUGUUC GAGCGUCCGU AACAACCU-C UCAAGC--CU AGCUUGGUCU UGGG-ACAUG CUGCUCUA-G UGG-CGGCAG 393 BmrGram5 501 511 521 531 541 551 561 571 581 591 600 | | | | | | | | | | | 400 UC---AAAAG CAGUGGCGGU ACGGCCGGGC UCUGAGCGUA GUAAAUCUUC UCGCUACAGG -GUCCCGGGC GG--CACUGG CC-AG-CAAC CCCC-AAAUC 490 TriSpl1198 416 UC---AAAAG CAGUGGCGGU ACGGCCGGGC UCUGAGCGUA GUAAAUCUUC UCGCUACAGG -GUCCCGGGC GG--CACUGG CC-AG-CAAC CCCC-AAAUC 506 TriSpl1659 403 UC---AAAAG CAGUGGCGGU ACGGCCGGGC UCUGAGCGUA GUAAAUCUUC UCGCUACAGG -GUCCCGGGC GG--CACUGG CC-AG-CAAC CCCC-AAAUC 493 TriSpl1908 401 UC---AAAAG CAGUGGCGGU ACGGCCGGGC UCUGAGCGUA GUAAAUCUUC UCGCUACAGG -GUCCCGGGC GG--CACUGG CC-AG-CAAC CCCC-AAAUC 491 TriSpl1238 378 UC---AAAAU CAGUGGCGGU ACGGCCGGGC UCUGAGCGUA GUAAAUCUUC UCGCUACAGG -GUCCCGGGC GG--CACUGG CC-AG-CAAC CCCC-AA-UC 467 ZeiVariu 388 UU---AAAAC UAGUGGCGGU GCUCUUAGGC UCUACGCGUA GUAACUUUCU CGCUAUAGAG -- UCCUGA-- GGGAUGCUUG CC-AA-CAAC CCCA-AAUUU 477 LhnBicol 378 CUU--AAAAU CAGUGGCGGU GCCAUCUGGC UCUAAGCGUA GUAAUUUAUC UCGCUAUUGG -GUCC-GG-U GG-UUGCUUG CC-AA-UAAC CCCC-AA-CU 467 HyyEric7 396 CCU--AAAAU CAGUGGCGGU GCCGUCUGGC UCUAAGCGUA GUAA-UUCUC UCGCUACAGU -AUCCAGG-U GG-UAGCUUG CC-AA-CAAC CCC--AA-CU 484 HyySpeci 390 UU---AAAAU CAGUGGCGGU GCCGUCGGGC CCUGAGCGCA GUAAAUAUCC UCGCUAUAGG GACCC-GG-U GG-ACGCUAG CC-AUC-AAC CCCC-AA-CU 479 HyyEric6 371 UU---AAAAU UAGUGGCGGU GCCUUGAGGC UCUACGCGUA GUAAUACUCC UCGCGAUAGA UGUCU---UA GGGUGUCUUG CC-AG-CAAC CCCC-AA-CU 460 HyyFruct 394 UCCUCAAAAG AAGUGGCGGG CCCAUGUAAC UCUCCGCGUA GUAAUACAUC UCGCGACAGG GAAGCA--GC GGG--ACUUG CC-AA--AAC UCCUUAA--U 484 BmrGram5 601 611 621 631 641 651 661 671 681 691 700 | | | | | | | | | | | 491 UUUCACAGGU UGACCUCGGA UCAGGUAGG- GAUACCCGCU GAACUUAAGC AUAUAAU...... 546 TriSpl1198 507 UUUCACAGGU UGACCUCGGA UCAGGUAGG- GAUACCCGCU GAACUUAAGC AUAUCAAUAA GCG...... 568 TriSpl1659 494 UUUCACAGGU UGACCUCGGA UCAGGUAGG- GAUACCCGCU GAACUUAAGC AUAUCAAUAA GCG...... 555 TriSpl1908 492 UUUCACAGGU UGACCUCGGA UCAGGUAGG- GAUACCCGCU GAACUUAACC AUA...... 543 TriSpl1238 468 UUUUACAGGU UGACCUCGGA UCAGGUAGG- GAUACCCGCU GAACUUAAGC AUAUCAAU...... 524 ZeiVariu 478 UCUUA--GGU UGACCUCGGA UCAGGUAGG- GAUACCCGCU GAACUUAAGC AUAUCAAUAA GCGGAGG...... 541 LhnBicol 468 UCCAA--GGU UGACCUCGGA UCAGGUAGG- AAUACCCGCU GAACAUAA...... 512 HyyEric7 485 CUC-AC-GGU UGACCUCGGA UCAGGUAGG- GAUACCCGCU GAACUUAA...... 529 HyySpeci 480 UUCUAA-GUU UGACCUCGGA UCAGGUAGG- GAUACCCGCU GAACUUAA...... 525 HyyEric6 461 UUCUAA-GGU UGA...... 472 HyyFruct 485 UGCU-CAGGU UGACCUCGAA UCAGGUAGG- GAUACCCGCU GAACUUAAGC AUAUCAAUAA GCGGAGGAAA AGAAACCAAC AGGGAUUACC UCAGUAACGG 582 BmrGram5

Figure A43: Alignment of complete ITS1, 5.8S, ITS2 sequences of Tricladium splendens and closest BLAST hits.

222 Appendix

Alingnment ITS sequences Tricladium angulatum

1 11 21 31 41 51 61 71 81 91 100 | | | | | | | | | | | 1 ...... UCCG UAGGUGAACC UGCGGAAGGA UCAUUACAGA GUUCAUGCCC UCACGGG-UA GAUCUCCCAC CCUUGUAUAA CCUAUCA--U 81 SopSolen 1 ...... CAGA GUUCAUGCCC UU-CGGGGUA GAUCUCCCAC CCGUGU-UAU C--AU-ACCA 49 AF141161 1 ...... CAGA GUUCAUGCCC UCACGGG-UA GAUCUCCCAC CCUUGAAUAU UUUAU-ACCU 52 EodMycor.2 1 AAGUCGUAAC AAGGUUUCCG UUGGUGAACC AGCGGAAGGA UCAUUACAGA GUUCAUGCCC UUACGGG-UA GAUCUCCCAC CCUUGAAUAA CUUAC-A--- 95 Mc8Cora2 1 ...... UUCCN UAGGUG-ACC UGCGGAAGGA UCAUUACAGA GUUCAUGCCC UCACGGG-UA GAUCUCCCAC CCUUGAAUA- --CAU-ACCU 79 C59Grevi 1 .....GUAAC AAGGUUUCCG UAGGUGAACC UGCGGAAGGA UCAUUACAGA GUUCAUGCCC UCACGGG-UA GAUCUCCCAC CCUUGAAUA- --CAU-ACCU 90 TriAng1418 1 ...... G UAGGUGAACC UGCGGAAGGA UCAUUACAGA GUUCAUGCCC UCACGGG-UA GAUCUCCcAC CCUUGAAUA- --CAU-ACCU 76 TriAng139 1 .....GUAAC AAGGUUUCCG UAGGUGAACC UGCGGAAGGA UCAUUACAGA GUUCAUGCCC UCACGGG-UA GAUCUCCCAC CCUUGAAUA- --CAU-ACCU 90 TriAng1020 1 ...... AAC AAGGUUUCCG UAGGUGAACC UGCGGAAGGA UCAUUACAGA GUUCAUGCnC UCACGGG-UA GAUCUCCCAC CCUUGAAUA- --CAU-ACCU 88 TriAng0138 1 ...... UCCG UAGGUGAACC UGCGGAAGGA UCAUUACAGA GUUCAUGCCC UU-CGGGGUA GAUCUGCCAA CCUU-UAU-- --CAUCAUUA 78 LhnBicol 101 111 121 131 141 151 161 171 181 191 200 | | | | | | | | | | | 82 --AUGUUGCU UUGGCG------GGCUCCGG ------CCCGCCAGAG G-ACCAC-AA ACUCU-GAAU AUU---AGUG 137 SopSolen 50 --UUGUUGCU UUGGCGGGCC CGCCUCGGCC ------A CCGGCUCCGG CUGGUGAGCG CCCGCCAGAG G-ACCCC- AA ACUCU-GAAA UUU---AGUG 132 AF141161 53 --UUGUUGCU UUGGCGGGCC -GCUUCGGCU ------A CCGGCUUCGG CUGGUGAGUG CCCGCCAGAG G-ACCCC-AA ACUCU-GAA- UU-A-UAGUG 134 EodMycor.2 96 --UUGUUGCU UUGGCGGAC- -GCUUCGGC------A-- -G -CCGCCAGAG A-ACCCU AA ACUCUUG--- UUUA-CAGUG 155 Mc8Cora2 80 --UUGUUGCU UUGGCGGGUC -GCUUCGUCC ------A CCGGCUCCGG CUGGUGCGUG CCCGCCAGAG ACA-CCU-AA ACUCU-GAAA U--AACA-UG 161 C59Grevi 91 --UUGUUGCU UUGGCAGGCC -GCUUCGGCC ------CUG --GGCUUCGG CUCGGGCGUG CCUGCCAGAG G-ACCCC-AA ACUCU--AAA UU-A-CAGUG 172 TriAng1418 77 --UUGUUGCU UUGGCAGGCC -GCUUCGGCC ------CUG --GGCUUCGG CUCGGGCGUG CCUGCCAGAG G-ACCCC-AA ACUCU--AAA UU-A-CAGUG 158 TriAng139 91 --UUGUUGCU UUGGCAGGCC -GCUUCGGCC ------CUG --GGCUUCGG CUCGGGCGUG CCUGCCAGAG G-ACCCC-AA ACUCU--AAA UU-A-CAGUG 172 TriAng1020 89 --UUGUUGCU UUGGCAGGCC -GCUUCGGCC ------CUG --GGCUUCGG CUCGGGCGUG CCUGCCAGAG G-ACCCC-AA ACUCU-GAA- UU-A-CAGUG 170 TriAng0138 79 AG-UGUUGCU UUGNCGAAUC -G---AGGCC ----UUCACC CCGG----GG CCCCUCUGUA UUCUCCAGAG G-AUNNC-AN AUUCU-G-CA UU---NA-UG 157 LhnBicol 201 211 221 231 241 251 261 271 281 291 300 | | | | | | | | | | | 138 UCGUCUGAGU ACUAUAAAAU -AGUUAAAAC UUUCAACAAC GGAUCUCUUG GUUCUGGCAU CGAUGAAGAA CGCAGGGAAA UGCGAUAAGU AAUGUGAAUU 236 SopSolen 133 UCGUCUGAGU ACUAUAUAAU -AGUUAAAAC UUUCAACAAC GGAUCUCUUG GUUCUGGCAU CGAUGAAGAA CGCAGCGAAA UGCGAUAAGU AAUGUGAAUU 231 AF141161 135 UCGUCUGAGU ACUAUAAAAU -AGUUAAAAC UUUCAACAAC GGAUCUCUUG GUUCUGGCAU CGAUGAAGAA CGCAGCGAAA UGCGAUAAGU AAUGUGAAUU 233 EodMycor.2 156 UCGUCUGAGU ACUAUAUAAU -AGUUAAAAC UUUCAACAAC GGAUCUCUUG GUUCUGGCAU CGAUGAAGAA CGCAGCGAAA UGCGAUAAGU AAUGUGAAUU 254 Mc8Cora2 162 UCGUCUGAGU ACUAUAUAAU -AGUUAAAAC UUCCAACAAC GGAUCUCUUG GUUCUGGCAU CGAUGAAGAA CGCAGGGAAA UGCGAUAAGU AAUGUGAAUU 260 C59Grevi 173 UCGUCUGAGU ACUAUAUAAU -AGUUAAAAC UUUCAACAAC GGAUCUCUUG GUUCUGGCAU CGAUGAAGAA CGCAGCGAAA UGCGAUAAGU AAUGUGAAUU 271 TriAng1418 159 UCGUCUGAGU ACUAUAUAAU -AGUUAAAAC UUUCAACAAC GGAUCUCUUG GUUCUGGCAU CGAUGAAGAA CGCAGCGAAA UGCGAUAAGU AAUGUGAAUU 257 TriAng139 173 UCGUCUGAGU ACUAUAUAAU -AGUUAAAAC UUUCAACAAC GGAUCUCUUG GUUCUGGCAU CGAUGAAGAA CGCAGCGAAA UGCGAUAAGU AAUGUGAAUU 271 TriAng1020 171 UCGUCUGAGU ACUAUAUAAU -AGUUAAAAC UUUCAACAAC GGAUCUCUUG GUUCUGGCAU CGAUGAAGAA CGCAGCGAAA UGCGAUAAGU AAUGUGAAUU 269 TriAng0138 158 UCGUCUGAGU ACCAUAAAAU -AGUUAAAAC UUUCAACAAC NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNAAUU 256 LhnBicol 301 311 321 331 341 351 361 371 381 391 400 | | | | | | | | | | | 237 GCAGAAUUCA GUGAAUCAUC GAAUCUUUGA ACGCACAUUG CGCCCCUUGG UAUUCCGAGG GGCAUGCCUG UUCGAGCGUC AUUAU-AACC AAUCUAGCCC 335 SopSolen 232 GCAGAAUUCA GUGAAUCAUC GAAUCUUUGA ACGCACAUUG CGCCCCUUGG UAUUCCGGGG GGCAUGCCUG UUCGAGCGUC AUUAC-AACC C-UCAAGCUC 329 AF141161 234 GCAGAAUUCA GUGAAUCAUC GAAUCUUUGA ACGCACAUUG CGCCCCUUGG UAUUCCGAGG GGCAUGCCUG UUCGAGCGUC AUUAU- AACC CCUCAAGCUC 332 EodMycor.2 255 GCAGAAUUCA GUGAAUCAUC GAAUCUUUGA ACGCACAUUG CGCCCCUUGG UAUUCCGGGG GGCAUGCCUG UUCGAGCGUC AUUAU-AACC CCUCAAGCUC 353 Mc8Cora2 261 GCAGAAUUCA GUGAAUCAUC GAAUCUUUGA ACGCACAUUG CGCCCCUUGG UAUUCCGGGG GGCAUGCCUG UU-CGAGCGU CAUUAUAA-C CCUCAAGCCU 358 C59Grevi 272 GCAGAAUUCA GUGAAUCAUC GAAUCUUUGA ACGCACAUUG CGCCCCUUGG UAUUCCGGGG GGCAUGCCUG UU-CGAGCGU CAUCAAAAAC CCUCAAGCCU 370 TriAng1418 258 GCAGAAUUCA GUGAAUCAUC GAAUCUUUGA ACGCACAUUG CGCCCCUUGG UAUUCCGGGG GGCAUGCCUG UU-CGAGCGU CAUCAAAAAC CCUCAAGCCU 356 TriAng139 272 GCAGAAUUCA GUGAAUCAUC GAAUCUUUGA ACGCACAUUG CGCCCCUUGG UAUUCCGGGG GGCAUGCCUG UU-CGAGCGU CAUCAAAAAC CCUCAAGCCU 370 TriAng1020 270 GCAGAAUUCA GUGAAUCAUC GAAUCUUUGA ACGCACAUUG CGCCCCUUGG UAUUCCGGGG GGCAUGCCUG UU-CGAGCGU CAUCAAAAAC CCUCAAGCCU 368 TriAng0138 257 GCAGAAUUCA GUGAAUCAUC GAAUCUUUGA ACGCACAUUG CGCCCCUUGG UAUUCCGGGG GGCAUGCCUG UU-CGAGCGU CAUUAUAAAC AAUCCAGUUU 355 LhnBicol 401 411 421 431 441 451 461 471 481 491 500 | | | | | | | | | | | 336 UGCUAGGUGU UGGGCCUCGC CACC--CGGC GGG-CCUUAA AACCAGUGGC GGUGCUGCCA GGCUCUAAGC GUAGUAAAUC U-CUCGCUAU AGGGUCCU-G 430 SopSolen 330 UGCUUGGUGU UGGGCGUCCC CGGCAACGGG GUG-CCCUAA AAUCAGUGGC GGUGCCGUCU GGCUCUAAGC GUAGUAAAUC U-CUCGCUCU GGAUGCCC-G 426 AF141161 333 GGCUUGGUGU UGGGGCCUGC C- GCACGGGC AG-CCCUUAA AAUCAGUGGC GGUGCCAUCU GGCUCUAAGC GUAGUAAUUC UUCUCGCUAU AGAGUCCC-G 429 EodMycor.2 354 AGCUUGGUGU UGGGGCCUGC CG--ACUGGC AG-CCCUUAA AAUCAGUGGC GGCGCCAUCU GGCUCUAAGC GUAGUAAUUU CUCUCGCUAU AGGGUCCC-G 449 Mc8Cora2 359 AGCUUGGUGU UGGAGCAUGC CUC---UGGC AG-CUCUUAA AAUCAGUGGC AGUGCCUGUC GGCUCUAAGC GUAGUAAAUU CUCUCGCUAU AGGGACC--G 453 C59Grevi 371 AGCUUGGUGU UGGGGCCUGC CGCC-- UGGC AG-CnCUUAA AAUCAGUGGC GGUGCCGGUC GGCUCUAAGC GUAGUAAUUC UUCUCGCUAU AGACGUC--G 465 TriAng1418 357 AGCUUGGUGU UGGGGCCUGC CGCC-- UGGC AG-CCCUUAA AAUCAGUGGC GGUGCCGGUC GGCUCUAAGC GUAGUAAUUC UUCUCGCUAU AGACGUC--G 451 TriAng139 371 AGCUUGGUGU UGGGGCCUGC CGCC-- UGGC AG-CCCUUAA AAUCAGUGGC GGUGCCGGUC GGCUCUAAGC GUAGUAAUUC UUCUCGCUAU AGACGUC--G 465 TriAng1020 369 AGCUUGGUGU UGGGGCCUGC CGCC- -UGGC AG-CmCUUAA AAUCAGUGGC GGUGCCGGUC GGCUCUAAGC GUAGUAAUUC UUCUCGCUAU AGACGUC--G 463 TriAng0138 356 A-CUGGGUCU UGGGCCUCGC GAUU- CU-GC GGG-CCUUAA AACUAGUGGC GGUGCUCUUA GGCUCUACGC GUAGUAACUU UCUCGCUAUA GAGUCCU--G 449 LhnBicol 501 511 521 531 541 551 561 571 581 591 | | | | | | | | | | 431 G-UGG-ACGC UGGCCAACA ACCCCCAA-- UUUUCUAGGU UGACCUCGGA UCAGGUAGGG AUACCCGCUG AACUUAAGCA UAUCAAUAAG CGGAGG... 521 SopSolen 427 G-UGG-AGAC UUGCCAGUA ACCCCCAA-- UUUUUU...... 457 AF141161 430 G-UGG-AUGC UUGCCAUUA ACCCCCAA-- UUUCAAUGGU UGACCUCGGA UCAGGUAGGG AUACCCGCUG AACUUAAGCA UAUCAAUAAG ...... 514 EodMycor.2 450 G-UGG-ACGC UGGCCAGCA ACCCCCA...... 473 Mc8Cora2 454 G-UGGAACAC GCGCCAG-A ACCCCCAA-C UUUCUAUGGU UGACCUCGGA UCAGGUAGGG AUACCCGCUG AACUUAAGCA UAUCAAUAAG CGGGGGAAG 548 C59Grevi 466 GGCGG--CUC UAGCCAACA ACCCCCAA-C UUCUUA-GUU UGACCUCGGA UCAGGUAGGG AUACCCGCUG AACUUAAGCA UAUCAAU...... 547 TriAng1418 452 GGCGG--CUC UAGCCAACA ACCCCCAA-C UUCUUA-GUU UGACCUCGGA UCAGGUAGGG AUACCCGCUG AACUUAAGCA UAUCAAUAAG CGGAG.... 541 TriAng139 466 GGCGG--CUC UAGCCAACA ACCCCCAA-C UUCUUA-GUU UGACCUCGGA UCAGGUAGGG AUACCCGCUG AACUUAAGCA UAUCAAUAAG CG...... 552 TriAng1020 464 GGCGG--CUC UAGCCAACA ACCCCCAA-C UUCUUA-GUU UGACCUCGGA UCAGGUAGGG AUACCCGCUG AACUUAAGCA UAUCAAUAAG CG...... 550 TriAng0138 450 A--GGGAUGC UUGCCAACA ACCCCAAAUU UUCUUA-GGU UGACCUCGGA UCAGGUAGGG AUACCCGCUG AACUUAAGCA UAUCAAUAAG CGGAGG... 541 LhnBicol

Figure A44: Alignment of complete ITS1, 5.8S, ITS2 sequences of Tricladium angulatum, closest BLAST hits and Mycoarthris corralinus.

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