An Abstract of the Thesis of

Francisco 1. Camacho for the degree ofDoctor ofPhilosophy in Botany and Plant

Pathology presented on 22 February 1999. Title: Below Ground Biology ofBotrychium pumicola (Ophioglossaceae). Redacted for Privacy Abstract Approved: __

James M. Trappe

Botrychium pumicola Coville is a rare fern, with extant populations in Klamath,

Lake and Deschutes counties, ofcentral Oregon. It grows on subalpine pwnice ridges and lower montane lodgepole pine forest openings on pwnice-rich soils. The goal ofthis research was to better understand the below ground biology ofB. pumicola. Detailed examination ofthe subterranean structures ofB. pumicola revealed sporophytic gemmae attached to the stem. Developing gemmae are a non-photosynthesizing stage in the life cycle ofthis plant and are presumed to depend on mycorrhzial fungi for their nutrition.

Population genetic analysis ofB. pumicola using inter-simple sequence repeats (ISSR) suggests that the gemmae do not disperse far from the parent plant. Examination ofthe endophytic fungal structures in the roots ofB. pumicola reveal arbuscular mycorrhziae and a high abundance ofseptate hyphae. To better characterize the root fungi, the internal transcribed spacer region offungal ribosomal DNA (ITS) was amplified from root DNA by the polymerase chain reaction (PCR). The ITS amplicon was cloned and sequenced in order to characterize the different fungi in a root segment. Arbuscular mycorrhizal-like Below Ground Biology of Botrych;um pum;cola (Ophioglossaceae)

by

Francisco J. Camacho

A THESIS

submitted to

Oregon State University

in partial fulfilhnent of the requirements for the degree of

Doctor ofPhilosophy

Completed February 22, 1999 Commencement June 1999 Doctor ofPhilosophy thesis ofFrancisco J. Camacho presented on February 22. 1999

APPROVED:

Redacted for Privacy

Major Botany and Plant Pathology

Redacted for Privacy

Chair ofDepartment ofBotany and Plant pathology

Redacted for Privacy

Dean ofGrad+hool

I understand that my thesis will become part ofthe permanent collection ofOregon State University libraries. My signature below authorizes release ofmy thesis to any reader upon request.

Redacted for Privacy

~rancisco J. Camacho, Author CONTRIBUTIONS OF AUTHORS

Chapter three was coauthored with Aaron Liston, who advised in the design and analysis, provided laboratory resources, and reviewed several drafts. TABLE OF CONTENTS

Page CHAPTER 1 Introduction...... 1

1.1 Abstract...... ·························· 1

1.2 Introduction...... 1

1.3 Review ofBotrychium pumicola ...... 3

1.4 Other Species ofBotrychium...... 6

1.5 Conclusion...... 11

CHAPTER 2 Below Ground Structures ofBotrychium pumicola, Roots, Stem, and Gerrunae...... 12

2.1 Abstract...... 12

2.2 Introduction...... 13

2.3 Methods...... 15

2.4 Results...... 15

2.5 Discussion...... 22

2.6 Acknowledgments...... 23

CHAPTER 3 Population Structure and Genetic Diversity ofBotrychium pumicola (Ophioglossaceae) Based on Inter-Simple Sequence Repeats (ISSR) ...... 25

3.1 Abstract...... 25

3.2 Introduction...... 26

3.3 Methods...... 28

3.4 Results...... 30

3.5 Discussion...... 38 TABLE OF CONTENTS (cont.)

3.6 Acknowledgments...... 43

CHAPTER 4 The Mycorrhizae ofBotrychium pumicola (Ophioglossaceae)...... 44

4.1 Abstract...... 44

4.2 Introduction...... 45

4.3 Methods...... 48

4.4 Results...... 50

4.5 Discussion...... 54

4.6 Acknowledgments...... 55

CHAPTER 5 DNA Examination ofthe Root Fungal Community ofPumice Grape Fern, Botrychium pumicola (Ophioglossaceae)...... 57

5.1 Abstract...... 57

5.2 Introduction...... 58

5.3 Methods...... 60

5.4 Results...... 66

5.5 Discussion...... 77

5.6 Acknowledgments...... 80

CHAPTER 6 Conclusion...... 82

6.1 Abstract...... 82

6.2 Introduction...... 83

6.3 Discussion...... 84

BIBLIOGRAPHY...... 87

APPENDICES...... 98 TABLE OF CONTENTS (cont.)

Appendix A ISSR Data Set...... 99

Appendix B DNA Alignment ofthe Complete ITS nrDNA Region ...... 110

Appendix C DNA Alignment ofthe 5.8S nrDNA...... 127 LIST OF FIGURES

Figure Page

2.1 Class 1 Botrychium pumicola plant, stem with emergent leaL ...... l 7

2.2 Class 3 Botrychium pumicola plant...... 17

2.3 Class 3 Botrychium pumicola plant...... 17

2.4 Class 3 Botrychium pumicola plant...... 17

2.5 Class 3 Botrychium pumicola plant...... 17

2.6 Class 3 Botrychium pumicola plant...... 18

2.7 Class 5 germinated gemmae ofBotrychium pumicola ...... 18

2.8 The same class 5 germinated gemmae on Botrychium pumicola ...... 18

2.9 The same class 5 germinated gemmae on Botrychium pumicola ...... 18

2.10 Class 6, gemmae on Botrychium pumicola ...... 19

2.11 Class 6, gemmae on Botrychium pumicola ...... 19

2.12 Class 3, developing plants attached to a mature sporophyte ...... 19

2.13 Class 3, developing plants attached to a mature sporophyte ...... 19

2.14 Several plants ofBotrychium pumicola growing entwined ...... 20

2.15 Horizontally developed underground stem ofBotrychium pumicola ...... 20

2.16 Root scar on the underground stem ofBotrychium pumicola ...... 20

2.17 Leafbud ofBotrychium pumicola...... 20

2.18 Unusually enlarged area ofa Botrychium pumicola root...... 20

3.1 Paulina view population ofsampled specimens of Botrychium pumicola ...... 32

3.2 Broken Top population ofsampled specimens ofBotrychium pumicola ...... 33 LIST OF FIGURES (cont.)

Figure ~

3.3 Katati-2 population of sampled specimens ofBotrychium pumicola ...... 34

4.1 Longitudinal section ofBotrychium pumicola root stained with trypan blue ...... 51

4.2 Longitudinal section ofBotrychium pumicola root stained with trypan blue ...... 51

4.3 Arbuscules and aseptate intercellular hyphae in a root ofBotrychium pumicola.51

4.4 Arbuscules and aseptate intercellular hyphae in a root ofBotrychium pumicola.51

4.5 Vesicles in roots ofBotrychium pumicola ...... 51

4.6 Vesicles in roots of Botrychium pumicola ...... 51

4.7 Regularly septate intracellular hyphae in the roots ofBotrychium pumicola...... 52

4.8 Regularly septate intracellular hyphae in the roots ofBotrychium pumicola ...... 52

4.9 Hyphal coils in cells of Botrychium pumicola roots...... 52

4.10 Dense entangled hyphae in cells ofBotrychium pumicola roots...... 52

4.11 Septate hyphae growing in rectangular cells ofBotrychium pumicola extending in to shorter isodiametric cells and forming amorphus masses of stained material...... 53

4.12 Septate hyphae growing in rectangular cells ofBotrychium pumicola extending in to shorter isodiametric cells and forming amorphus masses of stained material ...... 53

4.13 Septate hyphae growing in rectangular cells ofBotrychium pumicola extending in to shorter isodiametric cells and forming amorphus masses of stained material ...... 53

4.14. Amorphous masses of fungal material in root cells ofBotrychium pumicola ...... 53

5.1 Neighbor-joining analysis ofthe ITS region offungi from the roots of Botrychium pumicola ...... 72 LIST OF FIGURES (cont.)

Figure Page

5.2 Stacked bar graph showing the percentage ofphylotype clones from a root segment...... 74

5.3 Neighbor-joining analysis ofthe 5.8S rDNA...... 76 LIST OF TABLES

2.1 Location and number ofplants sample ...... 16

3.1 Location ofpopulations and the number ofindividuals sampled ...... 29

3.2 Primers used in ISSR analyses ofBotrychium pumicola ...... 29

3.3 The shared genotypes, the distance between individuals, and the probability ofthat genotype occurring a second time ...... 35

3.4 The results ofa Mantel test ofthe comparison ofthe genetic and spatial distance matrices ...... 36

3.5 Measures ofgenetic diversity in each population Botrychium pumicola ...... 36

4.1 Location and general habitat type ofthe plants sampled ...... 49

5.1 Location, habitat type, and date of sample populations ofBotrychium pumicola...... 61

5.2 DNA sequences from EMBL or GenBank used in the 5.8S rDNA analysis .....63

5.3 The number ofclones in a phylotype in root library ofBotrychium pumicola ..67

5.4 The different RFLP-types observed in the fungal internal transcribed spacer (ITS) clones of Botrychium pumicola roots and their respective phylotype .....68

5.5 DNA sequences ofthe ITS region from Botrychium pumicola fungi...... 70

5.6 Phylotypes with mUltiple RFLP-types...... 73

5.7 BLAST results ofBotrychium pumicola fungal phylotypes ...... 73 Below Ground Biology of Botrych;um pum;cola (Ophioglossaceae)

Chapter 1

Introduction

1.1 Abstract

Several species ofthe genus Botrychium, a member ofthe Ophioglossaceae, are rare and ofconcern to land managers. Botrychium pumicola is one such species of concern to the National Forest Service, Bureau ofLand Management, and National Park

Service ofcentral Oregon. The life cycle ofBotrychium is similar in the many species.

This is a review ofour knowledge ofB. pumicola and the reproductive biology and mycorrhizal symbiosis ofBotrychium.

1.2 Introduction

Botrychium pumicola Coville is a member ofthe Ophioglossaceae. It is considered by some to be one ofthe world's rarest ferns (D. Wagner, 1995). This species is listed as a Category 1 species under the Federal Endangered Species Act of 1973

(United States Department ofthe Interior, Fish and Wildlife Service, 1993). Category 1 is designated when the Fish and Wildlife Service has sufficient data to warrant listing as 2 threatened or endangered. It is important to increase our understanding ofthe biology of this species in order to better manage for its conservation. The little knowledge that we have on B. pumicola is primarily from above ground observations (Hopkins and O'Neil,

1993). Many unknown aspects ofthe biology, reproduction, and mycorrhizal symbiosis of

B. pumicola occur underground.

The goals ofthis research were to better understand the reproductive biology and mycorrhizal symbiosis ofBotrychium pumicola. In Chapter 2, detailed observations ofits below ground structures are reported. A means ofasexual reproduction was revealed on the underground stems. These asexual propagules (gemmae) are small globular structures attached to the stem by a small conduit ofcells. Gemmae have previously been reported in a few other species ofBotrychium (Farrar and Johnson-Groh, 1990), but were unknown in B. pumicola. Gemmae can detach from the parent plant and develop into a new plant. The first stages ofthis development are nonphotosynthetic. The plant is thus

presumed to acquire its nourishment from mycorrhizal fungi. Chapter 3 deals with the population genetics ofB. pumicola. In this study the highly variable DNA markers, inter­

simple sequence repeats (ISSR), were used to examine the distribution ofgemmae clones,

produced within popUlations. The disjunct shared ISSR phenotypes do not appear to be

from the dispersal ofgemmae but from the self-fertilization ofgametophytes. Chapters 4

and 5 deal with the fungal endophytes in the roots ofB. pumicola. In chapter 4, roots

stained for the presence offungi showed abundant fungi present. Several different types

ofhyphae were present, probably representing different species offungi. Typical

arbuscular mycorrhizae were present in limited amounts. One common type ofhyphae 3 was septate, which would represent an ascomycete or basidiomycete. Chapter 5 examines the root fungal community with DNA techniques. The internal transcribed spacers (ITS) ofthe rDNA were used to group the fungi. These groups, phylotypes, are distinct enough to suggest that they are different species. The PCR amplified fungal ITS represents a mixed community and was cloned to segregate the different ITS amplicons. The cloned

ITS gave a measure offrequency and abundance ofthe fungi in the roots. The root fungal communities are diverse even within a location. AMF fungi never dominated the fungal community.

This research greatly adds to the knowledge ofBotrychium pumicola biology.

These findings are likely to be applicable to other species ofBotrychium. In the following sections I review knowledge ofB. pumicola prior to my studies and ofthe genus

Botrychium in general to piece together a more complete picture ofB. pumicola.

1.3 Review of Botrychium pumico/a

Botrychium pumicola was first discovered in 1896 by Coville. It was growing on the high open slopes ofLlao Rock, the North rim ofCrater Lake National Park, Oregon.

It was found at Cloud Cap, another locality at Crater Lake, in 1902. The next population was found sixty miles away in Deschutes County, Oregon, at Paulina Peak above

Newberry Crater, in 1928. In 1951 two populations were discovered on Broken Top and nearby Tumalo Mountain (Rogers, 1951). The latter population consisted oftwo

individuals which were both collected, apparently extirpating the population (D. Wagner, 4

1995}. It is now known that most populations ofthis species occur within this Crater

Lake, Broken Top, and Newberry Crater triangle (Hopkins and O'Neil, 1993). Additional populations occur just outside this area, and a disjunct population was discovered in 1941 in Siskiyou Co., but has not been relocated (Hopkins and O'Neil, 1993). Although the

California specimens are not typical ofB. pumicola (Skinner and Pavlik, 1994), W.

Wagner (pers. comm.) has identified them as this species. Extant populations of

Botrychium pumicola are known only from Oregon (Wagner and Wagner, 1993).

The distribution appears closely linked to pumice substrates deposited by eruptions from Mt. Mazama, Newberry Crater, the South Sister, and possibly Mt. Shasta.

As of 1997, 118 sites had been documented in seven central Oregon locales (Joslin,

1997). These comprise four subalpine areas (Crater Lake National Park, Mt. Bachelor,

Broken Top, and Newberry Crater), and three montane lodgepole pine sites (the Chemult area, Katati basin, and China Hat) (Hopkins and O'Neil, 1993). The subalpine sites are over 7,200 feet in elevation on open or sparsely tree covered ridges with 6,700 year old pumice soils. The montane sites are typically pale pumice soils older than the subalpine sites. The plants occur in level openings in lodgepole pine (Pinus contorta Douglas ex

Loudon) forests. These openings are believed to be maintained by frost heaving that limits establishment oflodgepole pine (Hopkins and O'Neil, 1993).

Botrychium pumicola populations have a scattered and patchy distribution over their range. Populations range in size from 1 to 1,500 individuals. Ninety ofthe 118 populations contain fewer that 100 individuals (Joslin, 1997). The total number of individuals is estimated at 13,000 plants. Botrychium pumicola is apparently the only 5 species ofthe genus that occurs on high elevation pumice (Hopkins and O'Neil, 1993). In contrast, many other species ofBotrychium often occur in genus communities, which are comprised ofseveral species ofBotrychium growing together (Wagner and Wagner,

1983). Only at one known site ofB. pumicola in Newberry Caldera, not the typical

pumice habitat, does it occur with two other species, B. simplex E. Hitchcock and B.

multifidum (S.G. Gmelin) Ruprecht (Hopkins and O'Neil, 1993).

Botrychium pumicola is a perennial eusporangiate fern. The leaves can emerge

mid-May in the montane habitats and after the snow pack recedes in subalpine habitats.

Peak emergence ofleaves is mid-July to mid-August, one leafto a stem. The leafis

divided in two parts, the trophophore, a blade like structure with overlapping pairs of

pinnae, and the sporophore, pinnate branches with terminal fertile sporangia in grape-like

clusters. The sporophore and trophophore are joined at ground level and the petiole

reaches down two to several cm to the below ground stem. The leaf portion ofthe plant

dies back at the end ofthe growing season. When sporangia mature, July to August, they

often open by forming a slit, and the yellow spores are blown away in the wind (Hopkins

and O'Neil, 1993). The spores persist in tetrads after maturity (W.H. Wagner, pers.

comm.), unlike other species ofBotrychium. Plants ofB. pumicola have been observed to

produce a leafone year, not in the subsequent year, and then again in the third year

(Joslin, 1997). Presumably the plant is dormant during the year without a leaf. Plants are

often clustered like other species ofBotrychium (Farrar and Johnson-Groh, 1990), with

several leaves arising together. As many as 7 plants may fonn a cluster (Joslin, 1997). 6

1.4 Other Species of Botrychium

Ofthe 50 to 60 species ofBotrychium worldwide, 30 occur in North America north ofMexico (Wagner and Wagner, 1993). The genus typically grows at higher latitudes and usually occurs at montane to alpine elevations. The Pacific Northwest appears to be a center ofdiversity for the genus (W. Wagner, 1984) with 16 species reported from Oregon (D. Wagner, 1992). The genus is divided into three subgenera:

Osmundopteris, Sceptridium, and Botrychium (Wagner and Wagner, 1993).

The life cycles ofthe 3 subgenera are similar. The diploid sporophyte produces haploid spores in the sporangia. When the sporangia mature the spores are released and blown away by air currents. Spores require prolonged darkness and the formation of mycorrhizae to develop into a gametophyte (Whittier, 1973). The achlorophyllous bisexual subterranean gametophyte produces archegonia (structures with the egg) and antheridia (structures with the sperm). The sperm are flagellated and need water to reach an egg on the same or a nearby gametophyte. After fertilization the sporophyte develops roots, an underground stem (some refer to it as a rhizome), and a single leaf. The leaf develops and the sporangia mature (Bierhorst, 1971). In addition to reproduction by spores, some species ofBotrychium produce subterranean sporophytic gemmae (Farrar and 10hnson-Groh, 1990). The gemmae are achlorophyllous globular structures about 0.5 mm in diameter. They develop into an adult plant presumably like a newly formed sporophyte. 7

Botrychium pumico/a belongs to the subgenus Botrychium. This subgenus contains some ofthe rarest species offerns. Some species, such as B. gallicomontanum

Farrar and Johnson-Groh (Farrar and Johnson-Groh, 1991) and Botrychium watertonense

W.H. Wagner (Lesica and Ahlenslager, 1996) are known only from one locality.

Subgenus Botrychium contains 25 species world-wide, 21 being North American.

Fourteen ofthe North American species have been described since 1981 (Wagner and

Wagner, 1993) and undescribed species still remain (D. Wagner, 1992). In the continental

United States, nine species are Category 1 under the Federal Endangered Species Act of

1973 (USDI-Fish and Wildlife Service, 1993). This number should increase as the newly described species are considered for threatened or endangered status.

Species ofsubgenus Botrychium are small and often difficult to find. When found they can be difficult to identifY. Subtle morphological differences in the trophophore and sporophore separate species, and several specimens are often needed for reliable identification (Wagner and Wagner, 1993). These cryptic species often occur sympatric

(genus) communities (Wagner and Wagner, 1983). Isozyme analyses demonstrate distinct differences in alleles between the different species (Farrar, pers. comm.). Hybrids can be found in these communities, usually in small numbers (W. Wagner, 1991), but can represent as much as 25% ofthe genus community (Lesica and Ahlenslager, 1996).

Sometimes the hybrids are sterile and they can exhibit hybrid vigor (Ahlenslager and

Lesica, 1996).

The demography ofBotrychium spp. has been studied for both subgenus

Botrychium (Johnson-Groh and Farrar, 1993; Muller, 1993; Lesica and Ahlenslager, 8

1996) and subgenus Sceptridium (Montgomery, 1990; Kelly, 1994). These studies show that Botrychium plants can experience prolonged dormancy, where an individual remains alive but does not produce an above ground leaffor one or more consecutive years. As much as 50% ofthe population can be dormant in a given year (Johnson-Groh and Farrar,

1993). The leafabsence may be caused by environmental stress, such as drought

(Johnson-Groh and Farrar, 1993). Plants will also survive after premature loss ofthe above ground leaf due to herbivory (Montgomery, 1990) or wilting (Muller, 1993). In three species ofthe subgenus Botrychium, 78% ofthese dormancy periods lasted one year, 19% lasted two years and 3% lasted 3 to 4 years (Lesica and Ahlenslager, 1996).

The two subgenera appear to differ in the longevity ofthe plants. Members ofsubgenus

Botrychium are short lived perennials with half-lives ranging from 1.3 years in B. matricariifolium (Doll) A. Braun (Muller, 1993) to around 3 years in B. hesperium

(Maxon & R. T. Clausen) W.H. Wagner & Lellinger, B. paradoxum W.H. Wagner. and B. watertonense (Lesica & Ahlenslager, 1996). The subgenus Sceptridium is more long-lived with a half-life of43.2 years in B. dissectum Sprengel (Montgomery, 1990) and 11.2 years in B. australe R. Br. (Kelly, 1994) .

Because ofthe bisexuality offem gametophytes, it has been assumed that self fertilization is common. Cross-fertilization is hindered by the need for two gametophytes to be close enough for the sperm ofone individual to reach the egg ofanother (Tyron and

Tyron, 1982). Intragametophytic self-fertilization produces a homozygous sporophyte, even ifthe parent sporophyte was heterozygous. Intragametophytic selfing is the most extreme type ofinbreeding in plants (Klekowski. 1979). Isozyme studies have 9 demonstrated that many fern and fern allies have levels of heterozygosity expected in outbreeders (Haulfer, 1987; Soltis and Soltis, 1987). In contrast, all species of

Botrychium studied, including representatives ofthe 3 subgenera, have exhibited high levels ofhomozygosity (McCauley et ai., 1985; Soltis and Soltis, 1986; Watano and

Sahashi, 1992; Farrar pers. comm.), except for one population ofBotrychium multifidum var. robustum (Watano and Sahashi, 1992). Botrychium is thus one ofthe few studied fern genera that apparently inbreeds.

Isozyme studies of Botrychium show little variation within a species (McCauley et al., 1985; Soltis and Soltis, 1986; Watano and Sahashi, 1992; Farrar pers. comm.) which might be expected from an inbreeder. However, within a species the genetic diversity is distributed among the populations, as demonstrated by low Gst and Fst values. The lack ofinterpopulational genetic differentiation in species of Botrychium is assumed to result from high rates ofgene flow due to the long distance dispersal of spores (Soltis et ai.,

1988).

Species of Botrychium have a subterranean achiorophyllous mycotrophic gametophyte (Wagner and Wagner, 1993). Most pteridophytes that share this character represent early lineages ofland plants. Similar gametophytes are found in the

Lycopodiaceae (Wagner et al., 1985), considered the earliest extant lineage ofvascular plants (Manhart et aI., 1994; Manhart, 1995). All members ofthe Psilotaceae, another fern ally, have mycotrophic gametophytes (Wagner et aI., 1985). Some early fern lineages, Gleicheniaceae and Schizaeaceae, also have similar gametophytes (Wagner et aI., 1985). All ofthe Ophioglossaceae, to which Botrychium belongs, produce a 10 mycotrophic gametophyte. This family is considered remotely allied with the true ferns and may be more closely related to Psilotaceae based on rbcL (Manhart et ai., 1994), chloroplast SSU rDNA (Manhart, 1995), coxIII (Malek, 1996) and combined data sets

(Wolfet ai., 1998). An alternative view hypothesises a relationship with the cycads or the progymnosperm-seed plants (Wagner, 1964; Bierhorst, 1971; Kato, 1988; Wagner and

Wagner, 1993). The latter is supported by SSU rDNA (Wolfet al. 1998).

About two dozen reports ofBotrychium gametophytes have been published since first described by Hofineister (1855). The gametophytes are regularly associated with endophytic fungi (Daigobo, 1979; Wagner et ai., 1985). Nishida (1956) reported an arbuscule in a gametophyte, but fungal morphology is typically described as aseptate hyphae forming intracellular coils and vesicles (Bruchmann, 1906; Schmid and

Oberwinkler, 1993) much like that ofPsi/otum (Peterson et ai., 1981). Harley and Smith

(1993) grouped Botrychium mycorrhizae with V A mycorrhizae, suggesting an epiparasitism on a chlorophyllous host.

Most ofthe attention on Botrychium mycorrhizae has focused on the gametophyte, probably because ofits achlorophyUous nature. However, the roots ofthis genus lack root hairs, are sparsely branched, and have a large diameter (Wagner and

Wagner, 1993). According to the magnolioid root hypothesis (Baylis, 1975), these are classic characteristics ofobligate mycotrophic plants. These roots are probably not efficient at absorbing nutrients directly from soil, suggesting that the plant must have mycorrhizal fungi to supply those nutrients to the sporophyte. Hepden (1960) found most roots ofB. lunaria (L.) Swartz (subgenus Botrychium) colonized with endophytes. Berch 11 and Kendrick (1982) found almost 100% frequency ofarbuscules in root segments ofB. virginianum (L.) Swartz (subgenus Osmundopteris) and B. oneidense (Gilbert) House

(subgenus Sceptridium). Nair (1989) observed intracellular septate hyphae and vesicles in roots ofB. virginianum var. daucifolium. When the hyphae became old, they clumped together and formed dark irregular bodies that he referred to as arbuscules. Nair (1989) isolated Fusarium oxysporum from the roots and claimed to reinoculate plants and get the vesicular and arbuscular morphology, an unlikely result. The other achlorophyllous stage ofthe Botrychium life cycle is the subterranean gemmae. It would be expected that these would also require mycorrhizae to develop. Farrar and 10hnson-Groh (1990) observed fungal hyphae growing through the 6 to 8 cell thick conduit from a parent stem to a gemma. The gemma would thus have the fungus to help supply nutrients necessary for growth. All developing gemmae observed had endophytic fungi (Farrar pers. comm.).

1.5 Conclusion

Much ofthe biology ofBotrychium pumicola (like other species ofBotrychium) occurs below ground. Sexual reproduction occurs in subterranean gametophytes. Asexual reproduction also takes place below ground, where gemmae are produced on the stem and detach from the parent at maturity. In both ofthese stages ofthe life cycle, the plant receives its nourishment via endophytic fungi. The identities ofthe fungi involved and specificity ofthis relationship are unknown. My research aims to understand both the reproductive biology and the mycorrhizal symbiosis ofB. pumicola. 12

Chapter 2

Below Ground Structures of Botrychium pumicoia, Roots, Stem, and Gemmae.

2.1. Abstract

Botrychium pumicola is in the subgenus Botrychium. Several member ofthis subgenus, including B. pumicola, have a unique type ofasexual reproduction. They produce sporophytic gemmae, which develop on the underground stem. The gemmae may stay attached to or detach from the parent plant as they develop into a mature plant. It is presumed that plants developing from a gemma must receive their carbohydrates from mycorrhizal fungi. Species ofBotrychium are believed to exhibit prolonged dormancy during which the plant remains alive but fails to produce an above ground leaf. This dormancy has been observed in B. pumicola. Because ofthe frequency ofdeveloping gemmae with the parent plant, it is unclear ifplants arising after dormancy are the original plant or a clone. Species ofthis subgenus are believed to be short lived. In B. pumicola the gemmae may provide the continued survival ofthe genet even ifindividual plants are short lived. 13

2.2. Introduction

Much ofthe biology ofBotrychium Swartz (Ophioglossaceae) species takes place underground. Demographic studies on a few species show they can survive below ground without photosynthesizing after herbivory and drought (Montgomery, 1990; Muller,

1992). It is common for Botrychium plants to experience prolonged dormancy in which an individual remains alive but does not produce an above ground leaf for one or more consecutive years. This dormancy has been observed in B. dissectum Sprengel

(Montgomery, 1990), B. matricariifolium (Doll) A. Braun (Muller, 1992), B. campestre

W.H. Wagner & Farrar, B. galicomontanum Farrar & Johnson-Groh, B. simplex E.

Hitchcock (Johnson-Groh & Farrar, 1993), B. paradoxum W.H. Wagner, B. hesperium

(Maxon & R.T. Clausen) W.H. Wagner & Lellinger, B. X watertonense W.H. Wagner

(Lesica & Ahlenslager, 1996) and B. pumicola Coville,(Joslin, 1997). Lesica &

Ahlenslager (1996) found that 78% ofthese dormancy periods lasted one year, 19% lasted two years and 3% lasted 3 to 4 years. As much as half ofthe population could be dormant in a year (Johnson-Groh & Farrar, 1993), possibly because ofenvironmental stress such as drought. Few researchers have examined the underground structures to better understand these events.

In one study, four ofthe ten species ofBotrychium observed produced subterranean sporophytic gemmae (Farrar and Johnson-Groh, 1990). These gemmae are achlorophyllous and develop into an adult plant with chlorophyll. The gemmae are

spherical to semispherical structures around 0.5mm in diameter. During development they ~~ ---~~------

14 are connected to the parental stem by a small conduit oftissue about 6 to 8 cells in diameter. Fungal hyphae can colonize the gemma through this conduit. At maturity the cell ofthe conduit may senesce, severing the gemma from the stem The only other similar structures known are the globular gemmae ofPsi/otum Swartz on its sporophyte rhizomes and gametophytes (Bierhorst, 1971). This observation is noteworthy because molecular systematic studies ofPteridophytes often place Psilotaceae as sister group to the

Ophioglossaceae (Manhart et ai., 1994; Manhart, 1995; Malek, 1996; Wolfet ai., 1998).

Gemma-like tubers also occur in Equisetum, another early lineage ofland plants

(Bierhorst, 1971). Gemma-like structures have also been found on fossil arthrophytes

(Farrar & Johnson-Groh, 1990).

Botrychium pumicola grows mostly in Deschutes, Klamath, and Lake counties in central Oregon (Wagner & Wagner, 1993) and possibly near Mt. Shasta, California

(Wagner pers. comm.). In subalpine communities it occurs in open to partially tree covered sites. It also occurs in open frost pockets oflodgepole pine forest at lower elevation montane sites. These habitats are typically sparsely vegetated raw pumice and pumice-rich soils. Botrychium pumicola is a federal and state candidate for threatened or endangered listing. This study presents the first detailed examination ofthe underground structures ofBotrychium pumicola. 15

2.3. Methods

Individuals ofBotrychium pumicola were sampled from 5 populations in central

Oregon (Table 2.1). Specimens were collected by removing roots with a surrounding

block ofsoil approximately 20 cm in diameter and 20 cm deep. The plants and soil were

placed in plastic bags and kept in a cooler with ice, then at 4° C in the cold room in the lab.

Samples were examined within two weeks ofcollection. Plants were gently teased from

the soil with the use ofwater and an ice pick. Specimens were then observed under the

dissecting microscope for gemmae and size measurements ofthe underground structures

were taken.

2.4. Results

Several specimens ofBotrychium pumicola had gemmae attached to the central

stem (or rhizome) (Camacho, 1996). All six developmental classes described by Farrar and

lohnson-Groh (1990) were observed in B. pumicola: 1) stem with emergent leaf(fig. 2.1),

2) stem with non-emergent leaf, 3) shoot apex without leaf (fig. 2.2, 2.3, 2.4, 2.5, 2.6), 4)

stem-root system without shoot apex,S) germinated (elongated) gemmae (fig. 2.7, 2.8,

2.9), and 6) ungerminated (spherical) gemmae (fig. 2.10, 2.11). Gemmae representing

developmental classes 3 to 6 were found attached to stems ofparent plants (fig. 2.12,

2.13,2.14). One collection had two attached stems, each with an emergent leaf(fig. 2.14). 16

Table 2.1. Location and number ofplants sampled. The general habitat type is included.

Site Name Latitude Longitude Habitat Date sampled plants sampled Icy 430 28' 121 0 25' Montane 30 June 1995 4

Katati-3 430 26' 121 0 18' Montane 30 June 1995 4

Paulina 430 42' 121 0 12' Alpine 30 June 1995 7 (5 in one

View collection) Broken 440 04' 121 0 41' Alpine 16 August 4

Top 1995

LloaRock 420 58' 1220 08' Alpine 23 August 4

1995 17

Fig. 2. I Class I BOlrychium pumicola plant, stem with emergent leaf

Fig. 2.2-2.4 the same Class 3 Bolrychium pumicola plant; 2 roots and a shoot apex. Gemmae are clustered near the stem base (arrow); 3 underside; 4 complete view.

Fig. 2.5 Class 3 Botrychium pumicola plants. The upper root is very long. 18

Fig. 2.6 Class 3 Botrychium pumicola plant.

Fig. 2.7 Class 5 germinated gemmae of Botrychium pumicola.

Fig. 2.8-2.9 The same class 5 germinated gemmae on Botrychium pumicola; 8 Closeup of detached germinated gemmae showing attachment point to sporophyte; 9 germinated gemmae still attached to parent plant. 19

Fig. 2.10-2.11 The same class 6, gemmae on Botrychium pumicofa; 10 close up view; 11 View of the stem with attached gemmae.

Fig. 2.12-2.13 The same class 3, developing plants attached to a mature sporophyte of Botrychium pumicola; 12 complete view; 13 Close up of the apical shoot.

13 20

Fig. 2.14 Several plants of Botrychium pumicofa growing entwined. Two of the sporophytes with an emergent leaf are attached. Class 3, developing plants are also attached to a mature sporophyte.

Fig. 2.15 Horizontally developed underground stem of Botrychium pumicofa.

Fig.2.16 Root scar on the underground stem of Botrychium pumicofa, probably from a detached or dead root.

Fig. 2. 17 Leaf bud of Botrychium pumicofa.

Fig. 2. 18 Unusually enlarged area ofa Botrychium pumicofa root. apparently with a root bud.

15

18 ---- - _. ------_.

21

Presumably, this is an example ofa class 1 plant that developed from a gemma still

attached to the parent.

The number ofgemmae on individual plants ranged from zero to ten. All plants

examined from the Brokentop and Icy populations had gemmae. Three ofthe four

individuals from the Lloa Rock and Katati-3 populations contained gemmae. None ofthe

seven plants with an emergent leaf in the Paulina View population had gemmae, but one

free-living class 3 developing plant had gemmae.

All 10 ofthe class 3 plants had gemmae clustered at the base ofthe stem (fig. 2.2,

2.3). Presumably these plants have developed from gemmae. Developing gemmae without

a shoot apex, class 4 and class 5, did not have additional gemmae. Only three plants were

at this stage. On most ofthe emergent plants that had gemmae, the gemmae were also at

the base ofthe stem, but a few plants had only gemmae scattered along the stem (fig. 2.9,

2.10,2.11). One plant had gemma-like "root buds" clustered at the base ofthe stem.

These structures were not considered gemmae, because the base ofthe structure had no

constriction forming a conduit to the stem.

Underground stems ranged from 1 to 6 cm long and 1.5 to 3 mm wide. Only one

stem had developed horizontally, like a true rhizome (Fig. 2.15). Occasional root scars

from detached or dead roots occurred on stems (Fig. 2.16). At the top ofthe stem

adjacent to the petiole was a green leaf bud that represents the preformed leaf for the

following year (Fig. 2.17).

The longest root, 26 cm, varied in width from 0.5 to 1 mm. Most plants examined

had young developing roots at the upper portion ofthe stem. The Paulina View plants, 22 collected early in their growing season, had several smaller roots, the smallest being 3 mm long. The class 3 plants, developing from gemmae, often had long roots near the top of the stem (fig. 2.5), unlike most mature plants. Most roots did not branch. When they did, mUltiple branches often arose from the same area ofroot. One root had an enlarged portion with an apparent root bud forming (fig. 2.18). The roots were thick and crisp and often snapped in two when bent.

2.5. Discussion

Farrar and 10hnson-Groh (1990) hypothesized that dry climatic conditions increase gemma production, specifically in B. campestre W.H. Wagner & Farrar, which grows in dry prairie environments. Botrychium pumicola also grows in dry habitats and may be exhibiting a similar adaptive response. However, the gemmae ofB. pumicola more resemble those ofB. echo W.H. Wagner in that they often remain attached to the parent plant.

Except for the Paulina View population, most plants examined had gemmae attached to the stem. Although the one class 3 plant did have gemmae, all plants with an emergent leaf lacked gemmae. The Paulina View population is a subalpine site; when it was sampled only a few plants had emerged. These were sampled early in the season and may not have had time to develop gemmae. The other populations were sampled when the aboveground stages ofthe plants were well developed. Even with the plants well into their summer development, not all had gemmae. 23

The roots are large in diameter and lack fine roots and root hairs. According to the magnolioid root hypothesis (Baylis, 1975), these are classic characteristics ofobligately mycotrophic plants. These roots are probably not efficient at absorbing nutrients directly

from soil, suggesting that the plant must have mycorrhizal fungi to supply those nutrients.

Species ofBotrychium are noted for their ability to survive for one to several years

without producing an above ground photosynthesizing leaf(Montgomery, 1990; Muller,

1992; Johnson-Groh & Farrar, 1993; Lesica & Ahlenslager, 1996; Joslin, 1997). All

examined plants had developed preformed leafbuds. It is not known ifthe leafbud can

remain dormant for a year or elongates and fails to reach the surface ofthe ground. In the

case ofB. pumicola, the leafthat arises in subsequent years may well be a new plant, i.e.

an asexual clone produced from gemmae.

It appears that members ofsubgenus Botrychium are short-lived perennials. The

estimated half-lives are 1.3 years in B. matricariifolium (Muller, 1993) and around 3 years

in B. hesperium, B. paradoxum, and B. watertonense (Lesica & Ahlenslager, 1996). Even

though B. pumicola may be short lived, a genet may survive a long time in the same place

with the continued development ofits gemmae.

2.6. Acknowledgments

Thanks to Pat Joslin ofthe Deschutes National Forest and Christine Hopkins for

getting this project started, collecting the plant material, and sharing general knowledge of

Botrychium pumicola. Donald Farrar, Randy Molina, Jim Trappe, and Aaron Liston

assisted with manuscript review. Funding was provided by the Deschutes National Forest. 24

Laboratory resources were provided by Randy Molina at the Corvallis Forestry Sciences

Laboratory ofthe Pacific Northwest Research Station, USDA Forest Service. 25

Chapter 3

Population Structure and Genetic Diversity of Botrychium pumicola

(Ophioglossaceae) Based on Inter-Simple Sequence Repeats (ISSR).

Francisco J. Camacho and Aaron Liston

Department ofBotany and Plant Pathology

Oregon State University, Corvallis, OR 97331

3.1. Abstract

Species ofBotrychium reproduce by spores that form subterranean gametophytes and a few, like B. pumicola, also reproduce asexually with subterranean sporophytic

gemmae. The goal ofthis study was to examine the genetic diversity ofB. pumicola

populations and to better understand the role ofgemmae. Ninety-nine individuals from

three monitored populations were sampled. The technique ofinter-simple sequence

repeats (ISSR) produced fifteen polymorphic loci and identified seventy-one ISSR

genotypes. Sixteen ofthe ISSR genotypes were shared by more that one individual in a

population, representing potential clones. Ten ofthe sixteen shared genotypes were not

limited to clusters ofplants (groups ofplants growing from the same point). It is

determined that there is a high probability that these shared genotypes arose from

independent sexual events suggesting they were not clones. In addition, the ten potential

clones were disjunct and not in patches as might be expected for an underground 26 propagule. Thus we believe that the long distance dispersal ofgemmae is at best a rare event.

3.2. Introduction

The reproduction ofOphioglossaceae, including Botrychium Swartz, occurs underground. Species in this family ofeusporangiate ferns produce a bisexual subterranean gametophyte that requires prolonged darkness and the fonnation ofmycorrhizae to develop (Whittier, 1973). Underground, the flagellated sperm fertilizes an archegonium and a sporophyte develops. Cross-fertilization may be hindered by the need for two gametophytes to be close enough for the sperm ofone individual to reach the egg of another (Tyron and Tyron, 1982). In another plant group with underground gametophytes, the Lycopodiaceae, self-fertilization appears to be rare (Soltis and Soltis,

1988). Wagner et ai. (1985) provided theoretical arguments for possible outcrossing in species ofBotrychium, however isozyme studies suggest inbreeding is the norm

(McCauleyet aI., 1985; Soltis and Soltis, 1986; Watano and Sahashi, 1992; Farrar pers. comm.).

In addition to reproduction by spores, some species ofOphioglossaceae have subterranean asexual reproduction. The genus Ophioglossum can reproduce by means of root buds (Cascio and Thomas 1993) and some species ofBotrychium reproduce asexually by achlorophyllous subterranean sporophytic gemmae (Farrar and

Johnson-Grob, 1990). ~ ------

27

Botrychium pumicola Coville reproduces by gemmae (Camacho, 1996). Of25 observations ofB. pumicola below ground organs, no gametophytes were found, only gemmae. This observation led to the hypothesis that gemmae are an important part ofthe life cycle for this species. The gemmae ofB. pumicola are approximately 0.4 mm wide and develop on the underground stem. The gemmae do not appear capable ofeasily dispersing away from the parent plant.

In Oregon, Botrychium pumicola occurs in Deschutes, Klamath, and Lake counties (Wagner and Wagner, 1993). This species is one ofOregon's rarest ferns. This species is also reported from Mt. Shasta, California (W.H. Wagner, pers. comm.). It occupies specialized habitats in subalpine communities and open frost pockets of lodgepole pine, Pinus contorta, forest at lower elevation montane sites. These habitats are typically sparsely vegetated raw pumice and pumice-rich soils. Population sizes range from one to 1500 or more plants (Hopkins and O'Neil, 1993).

Molecular markers have been widely used to characterize clones in plants

(Sheffield et aI., 1989; Bayer, 1990; Smith et aI., 1992; Parker and Hamrick, 1992; Parks and Werth, 1993; Neuhaus et al., 1993; Hsiao and Rieseberg, 1994; Stiller and Denton,

1995; Waycott, 1995; Ayres and Ryan, 1997; Montalvo et al., 1997). These molecular techniques represent the only reasonable way ofdistinguishing ramets from a genet, in a fragmenting clonal plant (Parks and Werth, 1993). Inter-simple sequence repeats (ISSR) within a species can be a highly variable region ofDNA (Salimath et al. 1995). ISSR have the advantage over randomly amplified polymorphic DNA (RAPD) in that the primers are longer, allowing for more stringent annealing temperatures (Wolfe and Liston, 1998). 28

These higher temperatures apparently provide a higher reproducibility ofbands than in

RAPD (Wolfe et al. 1998; Nagaoka and Ogihara 1997). Tsumura et al. (1996) found that most oftheir ISSR bands (96%) segregated according to Mendelian expectations. Our study used ISSR to examine three populations ofB. pumicola in order to evaluate asexual reproduction in this species.

3.3. Methods

Three mapped populations ofBotrychium pumicola on the Deschutes National

Forest were sampled during the summer of 1997 (Table 3.1). Ninety-nine individuals were sampled. Individuals were sampled by removing a portion ofthe leaf. These leaves were stored on ice and then at -200 C until DNA was extracted. DNA was extracted with a

Qiagen (Chatsworth, CA) DNeasy plant extraction kit. A subset ofthe samples was used to screen ISSR primers for polymorphic loci.

Each ISSR reaction was carried out in a total volume of17 Ill; containing 8.5 III

MasterAmp 2X PCR PreMix D and one unit ofAmpliTherm polymerase (Epicentre,

Madison, WI), 5.5 III double deionized water, 1 III BSA (10 mg/rnl), 1 III ofprimer (10

nmoVrnl), and 10-20 ng ofgenomic DNA (1 III volume). Initial denaturation was carried

out for 1 min at 940 C, followed by 34 cycles of45 seconds at 940 C, 30 seconds at 500 C,

2 min. 15 seconds at 720 C, and a final 5 min extension at 720 C. ISSR primers were

obtained from the University ofBritish Columbia Biotechnology Laboratory. PCR

reactions were performed in an Ml-Research (Watertown, MA) PTC- 100 thermo cycler. 29

Table 3.1. Location ofpopulations and the number ofindividuals sampled.

Site Latitude Longitude Oregon Number Individuals Environment Name County ofplants Sampled 1992­ 1996

Paulina 43° 42' 121° 12' Deschutes 339 27 Subalpine VIew

Broken 44° 04' 121 0 41' Deschutes 493 49 Subalpine Top

Katati-2 43° 27' 121° 18' Lake 153 23 Montane

Table 3.2. Primers used in ISSR analyses ofBotrychium pumicola and size ofthe bands they produced. R = A, G; Y = C, T

Primer Name Primer Sequence ISSR band sizes in base pairs

UBC-813 (CT)gT 320,390,450,485,530,600,630 UBC-814 (CT)gA 350,450,500,520,540,610,730 UBC-824 (TC)gG 450,560,600,740,770,860 UBC-845 (CT)gRG 420,480 UBC-840 (GA)gYT 450,500,550,590,600,650 UBC-848 (CA)gRG 440 30

Products were analyzed on 1.2% agarose gels in 1X TBE buffer and stained with ethidium bromide. Band size was estimated from a 100 bp ladder (NEB, Beverly, MA). Loci were named based on the primer and observed band size.

Data were scored as presence and absence ofbands (Appendix A). Percent polymorphic loci, allele frequencies, Nei's genetic diversity, measures ofpopulation differentiation, and Shannon's index ofphenotypic diversity (King and Schaal, 1989) were computed with POPGENE 1.20 (Yeh et aI., 1997). NTSYSpc 2.02 (Rohlf 1997) was used to conduct a UPGMA analysis using the Dice coefficient and a Mantel test which examines the correlation between the matrix ofgenetic distance and spatial distance within a site. This test is a randomization procedure that compares the correlation between two matrices with the correlation between one ofthese and random permutations ofthe other.

By use ofallele frequencies, the probability that each genotype could arise independently was calculated following Parks and Werth (1993).

3.4. Results

Twenty two primers were evaluated for their ability to produce polymorphic bands

(putative loci) with a subset ofthe Botrychium pumicola samples. Six ofthe primers were

detennined to produce interpretable and variable banding patterns (Table 3.2). For the 99

samples, these six primers produced twenty nine scorable bands, fifteen ofwhich were

polymorphic. Four ofthese primers had a CT sequence repeat, producing eleven ofthe 31 fifteen bands. The polymorphic bands for each CT primer were ofa unique size, suggesting that different loci were amplified.

The spatial relationships ofthe shared and unique genotypes in each population are shown in Figs 3.1,3.2, and 3.3. Sixteen multilocus genotypes were shared by more than one plant within a population (Table 3.3). Two ofthese genotypes also had individuals from another population (not shown). Most shared genotypes were only represented by two plants (Table 3.3). The highest number ofindividuals sharing a genotype at a site was five. Two ofthese five were in a cluster, a group ofplants growing from the same point.

In nine ofthe thirteen plant clusters sampled, the individuals in the cluster shared a genotype. It cannot be determined whether these identical plants in a clusters originated from the reproduction ofgemmae or multiple intragametophyte selffertilization events.

Because ofthe difficulty ofdetermining reproduction modes in a cluster. spatially separated plants within a population may be more appropriate for the detection ofasexual reproduction in Botrychium pumicola. Such disjunct shared genotypes occurred in ten cases. The probability ofthese ten genotypes occurring a second time in each population by random mating (Parks and Werth, 1993) was relatively high ranging from 0.097945 to

0.427633 (Table 3.3). Two shared genotypes were represented by a pair ofindividuals each belonging to a different population. Because ofthe large geographic distance between these plants, they were not considered potential clones.

The Mantel test was used to test for correlations between the matrix ofgenetic diversity and spatial distance. All three populations have a very poor fit (Table 3.4).

Genetic diversity does not correlate with spatial distance. 32

Figure 3.1. Paulina view population ofsampled specimens ofBotrychium pumicola. Unique genotypes are those that occurred in only one individual. Genotypes represented by more than one individual are designated by symbols. Clustered plants that share the same genotype are in a gray circle.

: i I i i , I i I I i I I I ! I I I I I I i ! , I I ! I I , , I I i ! ! I L\ I I : i I I I ! ~-r [J ~ I I ' ~~:L : : I , ~~ , i i I , i ... I I I I , I : i I I lL J , : I I - I I ! I I I I Ll genotypes I I rl- I I I I I ~ I ! I ~ ~ ~ I ... "­ I I i I I ...... I I 0 I .. uruque ~ I I ! I !III' i i Q) : I I i ! I -I' ... i I I -/ Q) I I IC')J cluster i I I I I E ! i I i i , + PI I I I , I ! x P2 I i , I ~ ! I I i i I I I .L I ! I, I i ( ] ! : I . . I : "''''-' , I""'" I i ! I , ~ , i I ! I ,I I I I i I I : , I I i i I meters 33

Figure 3.2. Broken Top population of sampled specimens of Botrychium pumicola. Unique genotypes are those that occurred in only one individual. Genotypes that are represented by more than one individual are designated by symbols. Clustered plants that share the same genotype are in a gray circle. Plants with genotype 82, B3 , and B4 are growing in the same cluster. One ofeach ofthe B2 and B3 genotype plants were not mapped.

genotypes . , o unIque

I I' I cluster - I ! I I iill ...... LL + Bl I ! I l ' w ' T ~ en I ~ , I : L... c Q) I !! I B2 : i )(

Figure 3.3. Katati-2 population of sampled specimens ofBotrychium pumicola. Unique genotypes are those that occurred in only one individual. Genotypes that are represented by more than one individual are designated by symbols. Clustered plants that share the same genotype are in a gray circle.

I ; i ! i ,! I , I , , i I ... I i I -" I I I I I l' I I : I : i I I genotypes I i !, 1 : I I I , ~ I I i XI , 0 umque , ! I ! ~ ~.... . ' I "*" I ~ '\ ~ I I, . '.... cluster ~ ~ I ,Q en , '­ A Q) , ! Kl I ~ ... : Q) ! , i 1 X + K2 E 1 i , I I [ [ , I I : : ; i I I I • K3 I I i I I , I i I 1 x K4 ! I I I I I : I ! I ! I I * K5 I I I I I , , i , I : , ,..., '-' ~ meters ------

35

Table 3.3. The shared genotypes, the distance between individuals, and the probability of that genotype occurring a second time. Genotypes are listed in order ofprobability scores, going from lowest to highest. Cluster = Zero distance, NA = not available

Genotypes Number of Distances Between Probability of Second Individuals Plants in Meters Encounter

B7 2 cluster 0.007 Bl 2 cluster 0.010

K3 2 cluster 0.015 B9 2 cluster 0.028

B5 2 cluster 0.079

B2 2 NA (not cluster) 0.098 K5 2 1.8 0.109 PI 2 3.5 0.186

K2 4 2 clusters, 2.6 0.212 B3 3 1.7, NA 0.214

B6 2 20.2 0.226 P2 2 cluster 0.302 B8 4 cluster of3, 7.9 0.376 K4 5 cluster, 2.0-6.4 0.396 B4 2 22.1 0.406 KI 3 6.6 0.428 36

Table 3.4. The results ofa Mantel test ofthe comparison ofthe genetic and spatial distance matrices.

Site Name Product-moment Interpretation Probability correlation Paulina view r = 0.13715 very poor fit p = 0.257 Broken Top r = 0.07162 very poor fit p = 0.458 Katati-2 r = 0.19683 very poor fit p= 0.447

Table 3.5. Measures ofgenetic diversity in each population Botrychium pumico!a. Fis=0.95

Site Name Proportion of Polymorphic h=Gene I = Shannon's distinguishable loci diversity (Nei index genets 1973)

Paulina view 0.93 45% 0.1445 0.2196 Broken Top 0.90 48% 0.1622 0.2455 Katati-2 0.65 34% 0.1027 0.1588 37

Because previous isozyme studies ofother species ofBotrychium have demonstrated high degrees ofinbreeding (Soltis and Soltis, 1986; McCauley et aI., 1985), the genetic diversity statistics (Gst) were calculated twice. Once with the assumption of

Hardy-Weinberg equilibrium (Fis = 0) and once with the assumption that the populations are mostly selfing (Fis = 0.95). The among population differentiation estimates under both ofthese assumptions was low, Gst = O. 1147 (Hardy-Weinberg equilibrium) and Gst =

0.0950 (Fis = 0.95). These low values indicate that most ofthe genetic diversity ofB. pumicola is found within populations and there is little among population differentiation.

Similarly, a UPGMA analysis did not segregate individuals by the three populations (not shown).

Population-level genetic diversity statistics are summarized in Table 3.5. The least genetically diverse population is Katati with 34% polymorphic loci. Both Nei's gene diversity and Shannon diversity were calculated with Fis=0.95. These indexes are affected by the deviation from Hardy-Weinberg equilibrium. However, the same trends in diversity are seen ifHardy-Weinberg equilibrium is assumed. In general the genetic diversity within a site ofBotrychium pumicola is high when compared with isozyme results ofother species ofBotrychium (McCauley et aI., 1985; Soltis and Soltis, 1986, Watano and

Sahashi, 1992; Soltis et al. 1988). 38

3.5. Discussion

ISSR markers show a high degree ofgenetic diversity in Botrychium pumicola.

Among the 99 individuals are 71 ISSR phenotypes, in contrast to isozyme studies ofother species ofBotrychium in which few polymorphisms are observed. Four populations with

184 individuals ofB. virginianum (L.) Swartz (subg. Osmundopteris) had only 4 polymorphic loci among 18 loci analyzed (Soltis and Soltis 1986). McCauley et al. (1985) found only 5 polymorphic loci among 9 enzymes from 209 individuals in 3 populations of

B. dissectum Sprengel (subg. Sceptridium). Although these authors did not give the total number ofgenotypes in their examination ofthese species, there could not be more than

32 and 15 genotypes respectively. Watano and Sahashi (1992) examined 4 species of

Botrychium subgenus Sceptridium, B. multifidum (S.G. Gmelin) Ruprecht var. robustum

(Milde) Tagawa, B. nipponicum (Makino) Holub, B. triangularifolium Sahashi, and B. ternatum (Thunb.) Lyon. They found B. ternatum to be the most polymorphic ofthe 4 species. Three populations with 138 individuals included 30 genotypes, only 2 ofwhich were restricted to a single plant. Several species ofsubg. Botrychium, including B. pumicola, have also been examined and found to have low levels ofisozyme polymorphisms within a species (Farrar pers. comm.). It is expected that ISSR will have higher levels ofpolymorphism than isozymes (Wolfe et aI., 1998; Wolfe and Liston 1998).

The mechanism for maintaining this level ofdiversity in a supposedly self-fertilizing plant requires examination. Although dominant ISSR loci can potentially distinguish many individuals, they do not measure heterozygosity. For this reason, the level ofinbreeding ------

39 cannot be determined. Except for one population ofBotrychium multifidum var. robustum

(Watano and Sahashi, 1992), all species ofBotrychium studied, including a small sample ofB. pumicola (Farrar pers. comm.), have exhibited high levels ofhomozygosity

(McCauleyet aI., 1985; Soltis and Soltis, 1986). This appears to result from the most

extreme type ofinbreeding in plants, intragametophytic selfing (Klekowski, 1979). In this type ofself-fertilization the gametophyte, even ifthe parent sporophyte was heterozygous, will develop a homozygous sporophyte. Although it might be expected that this would

lead to a population oflow genetic diversity, it is not uncommon for selfing plants to have

high genetic diversity (Hedrick, 1998). There are mechanisms for maintaining

polymorphisms such as variable selection over space may maintain levels of

polymorphisms identical to random-mating (Hedrick, 1998). Another explanation for the

high levels ofISSR diversity in a selfing plant would be the non-Mendelian behavior of

ISSR markers (Tsumura et aI., 1996) (e. g. a chromosomal structural rearrangement)

(Wolfe and Liston, 1998). B. pumicola is a diploid with 90 chromosomes (Wagner and

Wagner, 1993). One could imagine that these ISSR bands may be in portions ofthe

chromosomes that do not segregate equally during meiosis, even in a completely

homozygous individual.

It is fairly common for up to seven plants ofBotrychium pumicola to arise in a

cluster from the same point ofsoil. Plant clusters were expected to have shared genotypes,

either because ofasexual reproduction ofgemmae or through multiple self-fertilization of

a single gametophyte. Except through the direct observation ofthe developing plants, it is

impossible to distinguish between the two modes ofreproduction, gemmae and multiple 40 self-fertilization. Most ofthe plant clusters sampled, 9 of 13, were made up ofplants with identical genotype. However, thirty percent ofsampled clusters contain plants from separate fertilization events. The fertilizations may be temporally independent or simultaneous. In the latter case, clusters may result from cross-fertilization ofadjacent gametophytes. It has been observed that the spores ofB. pumicola unlike other species of the genus, often remain in a tetrad (W.H. Wagner, pers comm.). Possibly this increases the probability ofmultiple gametophytes developing in a cluster.

The dispersal ofgemmae is important for determining their significance to the population. To determine dispersal, it is better to examine the shared genotypes that are not in a cluster but in the same site to better understand gemmae dispersal. Ten genotypes fit these criteria. None ofthese were very rare in their population and the probability ofa second, sexually developed plant was 9.8% or greater (Table 3.3). It is assumed that spatially disjunct plants with identical genotypes, and a 5% or greater chance ofbeing developed from a second sexual event, are not clones (Parks and Werth, 1993; Montalvo et aI., 1997). This method assumes that the species is mating at random, which does not fit the inbreeding ofspecies ofBotrychium. The probability oftwo different individuals producing the same genotype will decrease considering the high rate ofinbreeding.

However, the likelihood ofparents producing identical offspring should increase the probability ofidentical genotypes from independent sexual events. Therefore the probabilities ofa second sexually developed plant reported here are conservative values and the true values would be higher. 41

Most asexual plants that reproduce by stolons or rhizomes are expected to have a patchy distribution ofclones. However, plants that produce vegetative diaspores may have more intermingling ofclones (Gabrielsen and Brochmann 1998). The gemmae of

Botrychium pumicola probably only disperse through soil movement. Animals may be responsible for long distance dispersal events (e.g. more that a meter). We do not expect these dispersal events to occur often. Instead, we expect the gemmae to disperse only a short distance from the parent plant, producing a patchy distribution ofclones. The plant clusters are an extreme example ofthis type ofdistribution. However, none ofthe disjunct plants sampled less than a meter apart had identical genotypes. The Mantel test supports this by showing no correlation between genetic distance and spatial distribution. The data are consistent with the dispersal ofgemmae not being an important factor in the population structure ofthis species.

It is possible that some ofthese disjunct identical genotypes (Table 3.3) were formed by self-fertilization ofa gametophyte. Self-fertilization can mimic asexual reproduction. It should be easier for a spore to disperse long distances and for an identical genotype to develop through self-fertilization than for a gemma to be transported with soil. The self-fertilization ofgametophytes could produce the observed pattern of intermingled shared genotypes.

Like the isozyme studies ofspecies ofBotrychium, B. pumicola has a low Gst value (McCauley et aI., 1985; Soltis and Soltis, 1986; Watano and Sahashi, 1992). This low value shows little genetic differentiation among populations ofB. pumicola. The lack ofinterpopulational genetic differentiation in species ofBotrychium is assumed to be the 42 product ofhigh rates ofgene flow due to the long distance dispersal ofspores (Soltis et al., 1988).

The genetic diversity ofrare plant populations concerns natural resource managers.

We only sampled 3 populations ofthis species but the Katati-2 population had a lower genetic diversity than the other two. Several reasons may account for this, including sampling size. However, this population does stand out in two ways that need further investigation. The Katati-2 population is a montane habitat. The subalpine sites typically have more plants than the montane sites (Joslin 1997). This trend is observed in the three

populations ofthis study (Table 3.1). The Katati-2 population may be less diverse because

offewer individuals contributing to the gene pool. The other difference in the Katati-2

population is the recent disturbance ofsalvage wood cutting (Joslin, 1997). Species of

Botrychium are known to favor disturbed sites (Wagner and Wagner, 1993). The spores

need prolonged periods ofdarkness to germinate (Whitter, 1973), which may be facilitated

by soil disturbance. Why this might decrease genetic diversity is unclear, perhaps it results

from a more recent colonization ofthe site.

The main goal ofthis research was to evaluate the significance ofasexual

reproduction ofgemmae ofBotrychium pumicola. ISSR bands have provided useful

genetic markers for examining the population structure ofthis species. There were few

shared genotypes within the populations. Over half ofthe shared genotypes were spatially

disjunct. Because ofthe high probability ofa second sexual occurrence ofthese genotypes

and the lack ofa patchy pattern ofgenotypes expected from the distribution ofgemmae,

we assume that these shared genotypes are independent sexual events, and not 43 reproduction by gemmae. Gemmae are probably important in the temporal maintenance of a genet. The observation that 30% ofclusters ofplants that did not contain shared genotypes, suggests that two gametophytes may sometimes develop at the same point.

Some ofthese may be temporally separated, but it is suggestive ofpotential outcrossing between gametophytes ..

3.6. Acknowledgments

Thanks to Pat Joslin and Christine Hopkins for assistance with this project.

Funding was provided in part by Portland Garden Club and the Deschutes National Forest. 44

Chapter 4

The Mycorrhizae of Botrychium pumicola (Ophioglossaceae)

4.1. Abstract

The Genus Botrychium has long been considered mycorrhizal. Its achlorophyllous gametophytes are believed to receive their nutrition from nearby photosynthetic plants via shared mycorrhizal fungi. The sporophyte has large roots lacking root hairs, suggesting that the roots are poorly adapted to absorb nutrients. The roots are heavily colonized by endophytic fungi and have been previously reported as arbuscular mycorrhizae (AM).

Some species ofBotrychium, including B. pumicoia, produce achlorophyllous gemmae that depend on mycorrhzal fungi in much the same way as the gametophyte. This is the first report ofendophytic fungi from the roots ofB. pumicoia. The roots were highly colonized. Arbuscules oftypical AM were present in small amounts, but most fungal

structures in the root are intracellular septate hyphae, probably an ascomycete.

Amorphous masses ofstained fungal material representing disintegrating hyphae were

present in many root cells. These clumps offungal material were often associated with

septate hyphae and probably do not represent disintegrating arbuscules. 45

4.2. Introduction

Around 480 million years ago during the mid-Palaeozoic era, land plants came into being, forever changing global environments and the evolution ofterrestrial life.

During this time, unparalleled innovations occurred in plants. From the simple few-celled alga-like plant body evolved a complex array oforgans and tissues. By the end ofthe

Devonian (360 Mya) plants had evolved sexual organs, vascular tissue, wood, stomates, leaves and roots ofvarious kinds, sporangia, seeds, and the tree habit (Kenrick & Crane

1997). The presence ofvesicular-arbuscular mycorrhiza-like structures in Early Devonian megafossils (Kidston & Lang, 1921; Pirozynski & Dalpe, 1982; Sharma et aI., 1993;

Remy et aI., 1994; Taylor et aI., 1995) suggest that the mycorrhizal symbiosis also evolved early in the colonization ofland (Pirozynski & Malloch, 1975; Selosse & Tacon,

1998).

Extant plants today are highly diverse: Bryophytes, Pteridophytes,

Gymnosperms, and Angiosperms. The evolution ofplants probably progressed from a

Bryophyte-like ancestor to sporophyte-dominated plants similar to the Pteridophytes, and from that group evolved the seed plants, Gymnosperms and Angiosperms (Kenrick &

Crane, 1997). Each ofthese major plant groups has been reported to have mycorrhizae

(Pirozynski & Malloch, 1975; Selosse & Tacon, 1998). In fact mycorrhizae are so common in the plant kingdom (Trappe, 1987), it should be assumed that a plant is mycorrhizal unless it belongs to a family that is typically nonmycorrhizal or until proven otherwise. 46

Many extant taxa ofPteridophytes have intracellular fungi in their roots (Rayner,

1927; Burgeff, 1938; Boullard, 1957, 1979; Fontana, 1959; Hepden, 1960; Cooper,

1976; Mishra et al., 1980; Iqbal et ai., 1981; Laferriere and Koske, 1981; Berch and

Kendrick, 1982; Gemmae and Koske, 1990; Gemma et ai., 1992; Turnau et ai., 1993;

Schmid et al., 1995; Sharma, 1998). These have mostly been described as vesicular­ arbuscular mycorrhizai. Roots ofsome fern species contain ericoid-like or orchidoid-like, regularly septate ascomycete hyphae (Boullard, 1957; Cooper, 1976; Schmid et ai., 1995,

Sharma, 1998).

The genus Botrychium is a member ofthe Ophioglossaceae, an early lineage of vascular plants (Manhart, 1995; Wolf et al., 1998). The Flora ofNorth America (Wagner

& Wagner, 1993) characterizes the Ophioglossaceae as having nonphotosynthetic, subterranean, mycorrhizal gametophytes. Schmid and Oberwinkler (1994) found aseptate hyphae forming intracellular coils that degenerated within the gametophyte cells ofB. lunaria. Vesicles were present but no arbuscules were observed. The mycorrhiza morphology is similar to that ofthe gametophyte ofPsi/olum nudum (Peterson, et ai.

1981). The Psilotaceae is possibly a sister family to the Ophioglossaceae (Manhart 1995,

Wolfet at., 1998). It is hypothesized that these early plant lineages represent unique mycorrhizal relationships (Peterson et. at. 1981; Schmid and Oberwinkler, 1994). Most achiorophyllous mycotrophic plants have Ascomycete or Basidiomycete mycorrhizae

(Leake 1994), but the gametophyte ofOphiogossaceae has an aspetate fungus, probably a

Zygomycete. 47

The gametophytes ofBotrychium have received much attention regarding mycorrhizae, probably because ofthey are achlorophyllous, (Schmid and Oberwinkler,

1994). However, the sporophytes also depend highly on mycorrhizal fungi. The thick roots that lack root hairs and the high incidence offungal colonization in the roots

strongly suggest this dependence (Wagner & Wagner, 1993). These attributes are classic

characteristics ofobligate mycotrophic plants (Baylis, 1975). Previous reports of

Botrychium roots show them to be mostly colonized by fungi. Hepden (1960) found most

roots ofB. lunaria (subgenus Botrychium) to be colonized with endophytes that she

referred to as vesicular-arbuscular mycorrhizae but didn't describe. Berch and Kendrick

(1982) found almost 100% frequency ofarbuscules in root segments ofB. virginianum

(subgenus Osmundopteris) and B. oneidense (subgenus Sceptridium). Nair and

Mahabale (1975) observed intracellular septate hyphae and vesicles in roots ofB.

virginianum var. daucifolium. When the septate hyphae became old, they clumped

together and formed dark irregular bodies that Nair and Mahabale (1975) referred to as

arbuscules.

A few species produce subterranean sporophytic gemmae which are

achlorophyllous but can develop into an adult plant with chlorophyll (Farrar & 10hnson­

Groh, 1990). Farrar and 10hnson-Groh (1990) observed fungal hyphae growing through

the 6-to-8-cell-thick conduit from a parent stem to a gemma. After they detach from the

parent plant, the developing gemmae probably get carbohydrates from these mycorrhizal

fungi shared with neighboring plants. Demographic studies on a few species of

Botrychium show they can survive without photosynthesizing after herbivory and drought 48

(Montgomery, 1990; Muller, 1992). Possibly these species are receiving supplemental carbohydrates from their mycorrhizal fungi.

Botrychium pumicola is one species that forms subterranean achlorophyllous gemmae (Camacho, 1996). It grows mostly in central Oregon (Wagner and Wagner,

1993) with one report from Siskiyou Co. California (W.H.Wagner pers. comm.). Its habitat is sparsely vegetated pumice or pumice rich soils. This study reports the different

fungal morphologies found with the roots ofBotrychium pumicola.

4.3. Methods

Fourteen individuals were sampled from 5 populations (Table 4.1). The roots

were arbitrarily divided into sections about 4 cm long for use in DNA extraction, fungal

isolation and mycorrhizal morphology descriptions. Root samples designated for

microscopy were prepared as described by Koske and Gemma (1989). Roots were

washed with water then placed in 50% ethanol at 24° C until staining. The root segments

were placed in 10% KOH and steamed for approximately 30 minutes to clear them of

pigments and then neutralized in 1% HCI to acidifY the tissue. Finally, roots were

immersed in trypan blue (0.05% in lactoglycerol) in a steamer for 1 hour, then rinsed in

water, placed on a slide and longitudinally dissected in half. Stained roots were then

examined with a compound microscope for mycorrhiza morphology and endophytic

fungi. 49

Table 4.l. Location and general habitat type ofthe plants sampled.

Site Name Latitude Longitude Habitat Date sampled Katati-3 430 26' 121 0 18' Montane 30 June 1995 Paulina View 430 42' 121 0 12' Subalpine 30 June 1995 Broken Top 440 4' 121 0 41' Subalpine 16 August 1995

Icy 43 0 28' 121 0 25' Montane 30 June 1995 Loa Rock 420 58' 1220 8' Subalpine 23 August 1995 50

4.4. Results

The roots ofBotrychium pumicola are thick and fleshy. They often disintegrated during the staining process. The cells would separate, preventing a clear picture ofthe overall internal structures. When intact views were obtained, the hyphae often strongly colonized a layer 3-6 cells into the root cortex for most ofthe length ofthe root (Fig. 4.1 and 4.2).

Arbuscules (Fig. 4.3 and 4.4) were observed in one plant and were infrequent and patchy in that plant. The arbuscules were associated with intercellular aseptate hyphae

(Fig. 4.3). Vesicles were more common, less patchy and more scattered throughout the roots (Fig 4.5 and 4.6). Hyphae associated with vesicles were often septate and intracellular.

Intercellular septate hyphae were commonly observed in all roots (Fig. 4.7 and

4.8). The intracellular hyphae sometimes coiled around the inside ofcells (Fig. 4.9) but often were entangled (Fig. 4.10). Sometimes septate hyphae extended from long rectangular plant cells into shorter cells, forming arbuscular-like clumps (Fig. 4.11, 4.12, and 4.13). These structures were probably degenerating hyphae (Fig. 4.14). A continuum ofstructures was observed from coils to dense hyphae to the degenerated clumps of hyphae. Occasionally the smaller cells had large conglomerates ofhyphae. Very thin hyphae similar to Glomus tenue (Greenall) Hall (Hall, 1977) were occasionally present. 51

Figure 4.1-4.2. Longitudinal section ofBotrychium pumicola root stained with trypan blue. The darker portions are filled with fungal hyphae and in fig. 2 Most ofthe root is this dark color. The lower margin is clear, representing fungal free epidermal cells

Figure 4.3-4.4. Arbuscules and aseptate intercellular hyphae in a root ofBotrychium pumicola.

Figure 4.5-4.6. Vesicles in roots ofBotrychium pumicola. The vesicles are associated with septate intracellular hyphae. 52

Figure 4.7-4.8. Regularly septate intracellular hyphae in the roots of Botrychium pumicola.

Figure 4.9. Hyphal coils in cells of Botrychium pumicola roots.

Figure 4.10. Dense entangled hyphae in cells of Botrychium pumicola roots. 53

Figure 4.11-4.13 . Septate hyphae growing in elongated outer cells of Botrychium pumicola extending in to shorter cells and fonning amorphus masses of stained material.

Figure 4.14. Amorphous masses of fungal material in root cells of Botrychium pumicola. 54

4.5. Discussion

The roots ofBotrychium pumicola are heavily colonized by endophytic fungi.

Every root segment examined had hyphae along most ofthe root length. Similar high

levels offungi have been seen in other species ofBotrychium (Hepden, 1960; Nair and

Mahabale, 1975; Berch and Kendrick, 1982). With the thick roots, lack ofroot hairs and

high incidence offungi, the sporophyte ofB. pumicola appears to be an obligate

mycotroph.

The Paris-type ofAM is defined by the absence ofintercellular hyphae and the

presence ofcoils (Smith and Smith, 1997). Smith and Smith (1997) suggest that most

pteridophytes are reported to form this type ofAM. In fact they claim that the coils,

vesicles and remnants ofarbuscules described by Boullard (1958) for Botrychium are the

Paris-type. I do not believe that the intercellular hyphae and coils ofB. pumicola

represent the Paris-type. Although AM fungi can become septate (Smith and Smith,

1997), the regular septations in the hyphae suggest that this fungus is an ascomycete or

basidiomycete, not a zygomycete. Molecular studies collaborate this, suggesting a higher

proportion ofascomycetes than zygomycetes in B. pumicola roots (Chapter 5).

Arbuscules ofthe typical Arum-type were found in some plant roots of

Botrychium pumicola. The Arum-type is the typical AM, with arbuscules and extensive

intercellular hyphae (Smith and Smith, 1997). The low abundance ofAM structures

correlates with the low abundance ofAMF-like DNA sequences from B. pumicola roots

(Chapter 5). Most ofthe fungal structures were septate intracellular hyphae (often coiled) ------

55 and amorphous clumps ofstained material. The amorphous clumps could be degenerated arbuscules (Nair and Mahabale, 1975). However, the continuum ofcoils to clumps better fits the descriptions oforchid mycorrhizae (Burgeff, 1959).

Others report that species ofBotrychium are AM (Hepden, 1960; Berch and

Kendrick, 1982; Nair, 1989; Smith and Smith, 1997). B. pumicola has AM, but most of the fungal structures are not. The fungal community ofB. pumicola roots appears to consist ofseveral different taxa. It may be that B. pumicola differs from the other members ofthe genus. However, the descriptions ofthe above authors do not conflict with the dominant structures observed in this study. Except for Nair (1989), who describes the hyphae as being regularly septate, those authors are not clear whether septations are present or not. Nair (1989) isolated Fusarium oxysporum, an ascomycete, from the roots and claimed to reinoculate plants and get the vesicular-arbuscular morphology. Further studies are needed to elucidate the true nature ofthese endophytic fungi.

4.6. Acknowledgments

I'd like to thank Pat Joslin, Randy Molina, JeffStone, Jamie Platt, Joseph

Spatafora, and Jim Trappe for their assistance with this project. Funding was provided in part by NSF minority graduate student fellowship and the Deschutes National Forest.

Laboratory resources were provided by Randy Molina at the Corvallis Forestry Sciences 56

Laboratory ofthe Pacific Northwest Research Station, USDA Forest Service., Joseph

Spatafora, and Jeffiey Stone. 57

Chapter 5

DNA Examination of the Root Fungal Community of Pumice Grape Fern,

Botrych;um pum;cola (Ophioglossaceae).

5.1. Abstract

Species ofBotrychium (Ophioglossaceae) have an achlorophyllous gametophyte,

and they apparently depend on mycorrhizal fungi to complete their life cycle. Both the

achlorophyllous subterranean gametophyte and the roots ofthe sporophyte are

abundantly colonized by intracellular fungi. Some species, such as B. pumicola, can

reproduce asexually by achlorophyllous subterranean sporophytic gemmae. In both the

gametophyte and gemma stages, the plant is apparently nourished by mycorrhizal fungi.

Previous reports ofBotrychium mycorrhizae describe them as arbuscular mycorrhizae

(AM). This study focused on identification ofpotential mycorrhizal fungi ofB. pumicola.

All fungi, not just AM fungi, ofB. pumico!a roots were potentially examined. Direct peR

amplification, cloning and sequencing ofthe fungal ITS region ofthe nrDNA from the

roots were used to characterize the fungal community. The ITS region was used to

phylogenetically identify the fungi associated with B. pumicola. Because several fungi

may inhabit the same section ofroot, the PCR products from the root DNA were cloned

to segregate their various fungal ITS types. This technique ofPCR clone libraries provide

a method to examine the composition ofthe fungal community. 58

Seven plants from three different locations produced a total of26 different RFLP­ types. Sequence analysis ofthe RFLP-types showed several to be highly similar. It is believed that the RFLP's overestimated the diversity offungi. Sixteen phylogenetic groups (phylotypes) ofITS sequences were subsequently determined by their similarity.

Twelve are filamentous ascomycetes and the other four are related to Glomus sequences.

A root segment less than 2 cm long may contain as many as eight different phylotypes.

The two most abundant phylotypes, occurring in five and four ofthe root clone libraries

respectively, are commonly isolated, sterile filamentous ascomycetous fungi. The

glomalean phylotypes are never present in more than 20% ofthe total clones in a root

library. Near neighbor plant individuals, i.e. growing a few cm apart, had similar fungal

communities in their root systems. Plants from other sites, or spatially more distant within

a site, had different fungal communities.

5.2. Introduction

Several members ofthe fern genus Botrychium subg. Botrychium are rare

(Wagner and Wagner 1993). Nine species in the continental United States are candidates

for listing as threatened or endangered species under the Federal Endangered Species Act

of 1973 (USDI-Fish and Wildlife Service, 1993). One ofthe nine species is B. pumicola

(Fig. 1), found mostly in central Oregon, with a single known location in Northern

California. In subalpine areas it occurs in open to partially tree-covered sites, and at lower 59 montane sites it occurs in open frost pockets oflodgepole pine forests. These habitats are typically sparsely vegetated raw pumice and pumice-rich soils.

The genus Botrychium is a member ofthe Ophioglossaceae, an early lineage of vascular plants (Wolfet al. 1998). Wagner and Wagner (1993) characterized the

Ophioglossaceae as having non-green, subterranean, mycorrhizal gametophytes. Schmid and Oberwinkler (1994) found aseptate hyphae forming intracellular coils and degenerated masses within the gametophyte cells ofB. lunaria. Vesicles were present but no arbuscules were observed. This early lineage ofplants may represent a novel mycorrhizal relationship, because ofthe transfer ofcarbohydrates from fungus to host gametophyte (Schmid & Oberwinkler 1994).

The sporophyte also apparently depends on mycorrhizal fungi. The root morphology (Wagner & Wagner, 1993) and high incidence ofmycorrhizal fungal colonization (Berch & Kendrick, 1982; Nair, 1988) provide strong evidence for this dependence. A few species, including B. pumicola, produce subterranean sporophytic

gemmae which are achlorophyllous but can develop into an adult plant with chlorophyll

(Farrar & 10hnson-Groh, 1990; Camacho, 1996). The developing gemmae probably get

carbohydrates from nearby green plants via shared mycorrhizal fungi.

One fundamental question about this symbiosis is the host receptivity ofthese

ferns. Molina et al. (1992) describe host receptivity as " ..the numbers and diversity of

mycorrhizal fungi accepted by a particular host, also ranging from narrow (low number)

to broad (high number)". Direct PCR amplification ofthe fungal ITS region from the

mycorrhizae will provide insight to the breadth ofhost receptivity. This report compares 60 the ITS offungi within individual root segments between plants from the same soil excavation, more distant within a population, and in 3 different populations.

5.3. Methods

Specimens ofBotrychium pumicola were collected for DNA analysis from three populations (Table 5.1). Two additional sites, Icy and Loa Rock, were collected for

fungal isolation. Samples were extracted with surrounding soil, enclosed in zip-lock bags,

and kept cool in an ice chest and cold room until processing in the laboratory. Roots were

carefully dissected from the soil. Each root system was divided into samples for fungal

isolation, DNA isolation and microscopic examination. The latter is used in Chapter 4.

Portions ofthe divided root segments were used to isolate fungi into axenic

culture. One cm segments were surface-sterilized by submersion in hydrogen peroxide for

15-30 seconds and placed on modified Melin-Norkrans (MMN) medium (Marx & Zak

1965). Fungal colonies growing from root segments were transferred to new plates.

DNA was extracted from one- to two-cm root segments or fungal cultures. DNA

was extracted by the method ofDoyle and Doyle (1987). Samples were ground in CTAB

buffer (IOmM Tris-HCI (pH 8.0), l.4M NaCI, 20-mM EDTA, 2% CTAB) supplemented

0 with 2.0% Na03S and 2.0% polyvinylpyrrolidone (MW 40,000) at 65 C, extracted twice

in chloroform: isoamyl alcohol 24: 1, and precipitated in isopropanol containing 4.5 M

ammonium acetate for 60 min at -200 C. The samples were centrifuged at 14000 rpm for

10 min and the pellets washed in 70% EtOH and resuspended in 10 mM Tris buffer, pH 61

Table 5.1. Location, habitat type, and date of sample populations ofBotrychium pumicola.

Site Name Latitude Longitude Habitat Date sampled

Katati 43 0 26' 121 0 IS' Montane 30 June 1995

Paulina View 43 0 42' 121 0 12' Alpine 30 June 1995

Broken Top 440 04' 121 0 41' Alpine 16 August 1995 Icy 43 0 2S' 121 0 25' Montane 30 June 1995

LloaRock 42°5S' 1220 OS' Alpine 23 August 1995 62

8.0 containing ImM EDTA. Primer pairs used for amplification ofthe internal transcribed spacer region ofthe nrDNA were ITS4 and ITS1-f(Gardes and Bruns, 1993) and occasionally ITS 1 or ITS5 with ITS4 (White et aI., 1990). PCR reactions were carried out in 50 ilL volumes with 1.25 U Replitherm DNA polymerase (Epicentre

Technologies). PCR amplification conditions were 35 cycles of94° C denaturing, 50° C annealing, and 72° C extension, each at 60 s. The final extension was at 72° C for 5 minutes. The resulting product was cloned with the pCR 2.1 T A cloning kit (Invitrogen) following the manufactures instructions. DNA was extracted from each clone and the insert was reamplified for RFLP analysis and sequencing. RFLP's were generated by adding 2 units ofHaellI or MboI in 2X buffer 1:1 with 10 ul ofPCR reaction and incubating at 3'f for 6 hr to overnight. The digest was run on a 1.5% agarose gel with

100 bp DNA Ladder. All similar RFLP-types were run on the same gel to verifY similarity. Products were prepared for sequencing by precipitation in 0.5 volumes of4.5

M ammonium acetate and 1.5 volumes ofisopropanol. Cycle sequencing with dye terminator chemistry was performed with an ABI model 377A fluorescent sequencer.

Sequences were aligned with the program Clustal X (Thompson et al. 1997), with parameters gap openings 15 and gap extension 6 (Appendix B and C). Neighbor joining analyses were performed with the program neighbor in the Phylip package (Felsenstein,

1993). A BLAST search (Altschul et af. 1997) was preformed on all sequences to determine other similar sequences from sequence data bases. Taxa from GenBank and

EMBL used in the 5.8S analysis are in Table 5.2. ------

63

Table 5.2. DNA sequences from EMBL or GenBank used in the 5.8S rDNA analysis.

Species GenBank or Classification

EMBL

Accession

Scutellospora castanea AJOO2872 Zygomycota, Glomales Scutellospora heterogama AFOO4693 Zygomycota, Glomales Glomus coronatum X96846 Zygomycota, Glomales

Glomus Jasciculataum X96843 Zygomycota, Glomales Glomus mosseae U31996 Zygomycota, Glomales Glomus intraradices AFOO4681 Zygomycota, Glomales

Glomus etunicatum AFOO4683 Zygomycota, Glomales Glomus claroideum AFOO4688 Zygomycota, Glomales Glomus monosporum AFOO4689 Zygomycota, Glomales Gigaspora gigantea AFOO4685 Zygomycota, Glomales Gigaspora rosea AFOO4701 Zygomycota, Glomales Gigaspora albida AFOO4707 Zygomycota, Glomales Gigaspora margarita AJOO6850 Zygomycota, Glomales Entrophospora inJrequens U94714 Zygomycota, Glomales

Endogone pisiformis AFOO6511 Zygomycota, Endogonales Thermomyces lanuginosis MI0392 Ascomycota

Ashbya gossypii U09322 Ascomycota, Saccharomycetales

Kluyveromyces aestuarii U09324 Ascomycota, Saccharomycetales

Saccharomyces cerevisiae K04018 Ascomycota, Saccharomycetales

Candida albicans L07796 Ascomycota, Saccharomycetales ~~~~~~~~~~------

64

Table 5.2 (cont.)

Monoascus purpureus U18356 Ascomycota, Eurotiales

Trichoderma viride X93986 Ascomycota, Hypocreales

Neurospora crassa MI0692 Ascomycota, Sordariales

Glomerella cingulata X738 11 Ascomycota, Phyllachorales

Colletotrichum acutatum X738 10 Ascomycota, Phyllacorales

Nectria vilior U57673 Ascomycota, Hypocreales

Drechlera avenae X78123 Ascomycota, Pleosporales

Phyllactinia moricola D84384 Ascomycota, Erysiphales

Sphaerotheca cucurbitae D84377 Ascomycota, Erysiphales

Rutstroemia firma Z80893 Ascomycota, Leotiales

Morchella esculenta U51851 Ascomycota, Pezizales

Sclerotinia sclerotiorum M96382 Ascomycota, Leotiales

Phoma wasabiae L38711 Ascomycota, Pleosporales

Gaeumannomyces U17211 Ascomycota, Magnaporthales

cylindrosporus

Ophiostoma ulmi U23424 Ascomycota, Ophiostomatales Ceratocystis douglasii U75626 Ascomycota, Microascales

Claviceps purpurea U57669 Ascomycota, Hypocreales Cistella grevillei U57089 Ascomycota, Leotiales

Phialophora gregata U66727 Ascomycota

Dactylaria dimorphospora U51980 Ascomycota

Gelatinipulvinella astraoeca U72611 Ascomycota, Leotiales

Amanita muscaria Z54294 Basidiomycota, Agaricales

Cryptococcus neoformans L14067 Basidiomycota, Filobasidiales ,------­-- -

65

Table 5.2 (cont.)

Tremella foliacea AF042417 Basidiomycota, T remellales

Trichaptum abietinum U63475 Basidiomycota, Aphyllophorales

Ceratobasidium AJOOO194 Basidiomycota, Ceratobasidiales

oryzae-sativae

Thanatephorus cucumeris AJOO0202 Basidiomycota, Ceratobasidiales

Brassica napus DI0840 Magnoliophyta, Capparales

Taxus baccata X93991 Pinophyta, Coniferales

Pinus contorta U23956 Pinophyta, Coniferales

Isoetes histrix X91871 Lycopodiophyta, Isoetales

Selaginella denticulata X91873 Lycopodiophyta, Selaginellales

Marsilea quadrifolia X15139 Filicophyta, Marsileales

Osmunda regalis X63199 Filicophyta, Filicales

Gossypium hirsutum U12719 Magnoliophyta, Malvales Oryza sativa M16845 Magnoliophyta, Cyperales

Dolichosaccus symmetrus L01631 Eumetazoa, Platyhelminthes

Arion rufus XOO131 Bilateria, Mollusca

Homo sapiens J01866 Chordata, Primates Xenopus laevus K01369 Chordata, Amphibia 66

5.4. Results

Seven fungal ITS clone libraries were assembled from seven different individuals ofBotrychium pumicola representing 3 different locations, Paulina View, Broken Top, and Katati (Table 5.1). The number ofclones in each library ranges from 8 in the Katati 3,

Broken Top 17, and Broken Top 18 plants to 39 in the Paulina View 12 plant (Table

5.3). Twenty-six unique RFLP-types were observed (Table 5.4). At least one

representative ofeach RFLP-type was sequenced (Table 5.5). Isolated fungi were also

sequenced through the ITS region (Table 5.5).

Neighbor-joining analysis ofthese sequence data sets showed that different RFLP­

types have highly similar sequence composition, noted by the short branch lengths

separating the operational taxonomic units (Fig. 5.1). This high degree ofsimilarity is

used to group sequences into phylotypes. Each phylotype contains sequences that are

more that 95.84% similar (Table 5.6), which falls within the range ofintraspecific

similarity for ascomycetous fungi (Seifert et ai., 1995). The 26 RFLP-types represent 16

phylotypes (Table 5.4). The phylotypes are used for interpreting the fungal community in

the root instead ofthe RFLP-types, because they may better represent the potential

species. The distribution ofthese phylotypes in the root libraries are shown in Table 5.3

and the relative abundance is shown in Fig. 5.2.

Three ofthe ITS clone sequences proved to be chimeric peR products oftwo

different phylotypes (Table 5.5). In two clones from different roots, Katati 2 and Broken

Top 18, the chimeric amplicon has the ITSI ofphylotype Al and the ITS2 ofphylotype 67

Table 5.3. The number ofclones in a phylotype for each root library ofBotrychium pumicola. The 0.5 value represents a chimeric ITS ofthe two phylotypes. Phylotypes are the alpha-numeric code in the left column.

Location Katati Katati Paulina Paulina Paulina Broken Broken

View View View top top

plant 2 3 12 13 14 17 18

Al .05 28 18 16 1

A2 4 4 1 3

A3 0.5 6

A4 1 1

A5 3 1

A6 3 1

A7 3

A8 7

A9 1

AlO 4

All 3

A12 2 Zl 1 1

Z2 3

Z3 4

Z4 2 1 1 ------

68

Table 5.4. The different RFLP-types observed in the fungal internal transcribed spacer (ITS) clones ofBotrychium pumicola roots and their respective phylotype. ITS size and restriction band sizes were determined directly from the sequence.

RFLP- ITS5 - ITS4 Mbol band fragments Haelll band fragments Phylotyp

type amplicon in in base pairs in base pairs e

base pairs

A 550 297,149,53,51 455,95 AI,

AlIA3 B 623 296,256,36,24,11 294,155,110,64 A2/A7

C 628 299,290,49 295,159,110,64 A2

D 626 346,290 295,157,110,64 A2

E 630 301,256,38,24,11 456,110,64 A2

F 629 341,236,52 458,107,64 A2

G 631 302,273,56 463,104,64 A2

H 551 348,152,51 366,95,90 A3

I 559 350,149,60 279, 176, 104 A3

J 602 356,190,65 602 A4

K 555 298,151,55,51 279, 176, 100 AS

L 527 342,138,47 420, 107 A6 M 523 290,138,52,43 420, 103 A6 N 596 255,251,52,38 275,157,59,47 A7 0 960 (intron) 294,225,212,91 333,252,172,65 A8

P 960 (intron) 298,282,91 242,176,162,59 A8

Q 556 355,201 285,157,95,19 A9

R 605 332,224,49 436, 169 AIO

S 605 204,201,127,49,24 437, 168 AlO ------.

69

Table 5.4 (cont.)

T 558 297,179,50,31 464,94 All U 610 323,214,49,24 277,172,52,48 A12

V 538 389,69,63,17 538 Zl W 543 398,65,63,17 543 Z2 X 549 400,65,43,24,17 549 Z4 Y 538 151,98,92,72,52,32, 538 Z3 24 Z 538 223,98,92,52,32,24, 538 Z3 17 70

Table 5.5. DNA sequences ofthe ITS region from Botrychium pumicola fungi, isolated fungi and clones from the PCR root fungallihrary. Their corresponding phylotype and RFLP-type are shown. The chimeric sequences are shown. Isolates were not involved in RFLP analyses.

Sequence Phylotype RFLP-type Site Name Plant Chimeric

sequence clone 12-5 Al A Paulina View 12

clone13-1 Al A Paulina View 13 clonel4-21 Al A Paulina View 14 isolateB 1-3 Al Loa Rock Bl clone2-8 A2 C Katati 2 clone2-20 A2 D Katati 2 isolate 11-2 A2 Paulina View 11 clonel2-9 A2 F Paulina View 12

clonel3-17 A2 E Paulina View 13 iso late 14-1 A2 Paulina View 14

clonel7-1 A2 G Broken Top 17 clonel7-6 A2 H Broken Top 17 isloateB 1-4 A2 Loa Rock BI clone2-12 A2/A7 B Katati 2 Chimera clone2-13 A3/Al A Katati 2 Chimera clonel8-5 A3/AI A Broken Top 18 Chimera clonel8-1 A3 H Broken Top 18

clone I 8-4 A3 I Broken Top 18

clonel7-3 A4 J Broken Top 17

clone2-24 A5 K Katati 2 71

Table 5.5 (cont.) clone 17-5 A5 K Broken Top 17 clone3-12 A6 L Katati 3 clone3-6 A6 L Katati 3 clone3-1 A6 M Katati 3 clone2-4 A7 N Katati 2 clone2-23 A8 P Katati 2 clone2-2 A8 0 Katati 2 clone2-10 A9 Q Katati 2 clone3-4 AI0 S Katati 3 clone3-10 AlO S Katati 3 clone3-15 AlO R Katati 3 clonel7-4 All T Broken Top 17 clonel4-1 A12 U Paulina View 14 clone 14-6 A12 U Paulina View 14 isolate2-7 Katati 2 clone3-14 ZI V Katati 3 clone 12-6 Z2 W Paulina View 12 clone2-31 Z3 Y Katati 2 clone2-35 Z3 Z Katati 2 clone12-41 Z4 X Paulina View 12 clone 13-3 Z4 X Paulina View 13

isolatel-l Katati 1

isolate2-1 Katati 2

isolate5-2 Icy 5

isolate7-1 Icy 7 72

Figure 5.1 Neighbor-joining analysis ofthe ITS region of fungi from the roots of Botrychium pumicola. Sequences from the root clone libraries are designated a clone and sequences from isolated fungi are designated as isolate. Sequences from the clone library that have short branch lengths between neighbors are grouped in to phylotypes. The sequences in phylotypes are highly similar, greater the 5%. Phylotypes with A are consider ascomycetes and those with Z are considered zygomycetes. Bootstrap values above 70% are shown above the branches.

clone 12-5 clone 14-2 1 99 l'h~·III-I' P" \ I

I'ln·lu-l~ 1)(' \3

78 c1one2-24 ;5---- clone17-4 I'h~·III-1\ pI' \ II '----- isolate2-7 '------clone2-10 I'lnlll-I'I)(' \') clone12-9 clone 17-1 clone 17-6 isolate 11-2 isolatel4-1 clone13-1 7 100 isolateB 1-4 clone2-8 c1one2-20 100 • c1one3-4 99 c1one3-10 I'h~IIt-I~ pl' \ III clone3-15 '------clone17-3 Pin 111-1\ pI' \-1 ,..=1-=.00-"--______---l c1onel4-1 1'1·1 . \ I ., ,..=1...::.00-=--____--1 clone 14-6 I~ fH~ P" ­ '------clone2-23 l'Il\lu-l~ pI' \S '------c1one2-4 J>hyllt-I\IH' \ i clone3-1 100 '------f clo0e3-12 I'h~·III-lyJl" .\6

1'11\·lu-IYPl' I'.-t clone I 2-4 I '----- clone3-14 l'lnlll-l, P" 1'.1 100 L..-__ clonel2-6 I'hylfl-hp" I'.'!. clone2-31 L..::..:'-"-____---j clone2-35 I'lnlll-tYJll' Z3 isolate2-1 100

0.1 73

Table 5.6. Phylotypes with multiple RFLP-types. The range ofsequence similarity observed within these phylotypes.

Phylotype RFLP-types Similarity ofsequences A2 C,D,E,F,G 96.24%-99.45%

A3 H, I 95.84%

A6 L,M 99.55%-lOO%

AlO R,S 97.98% - 99.42%

Z3 Y,Z 99.88%

Table 5.7. BLAST results ofBotrychium pumicola fungal phylotypes. Three ofthe 16 phylotypes had known fungal ITS sequences ofhigh similarity, BLAST score greater than 200. These represent sequences closely related species.

Phylotype GenBank or Species Blast score Probability

EMBL

Accession A2 U66727 Phialophora gregata 264 6 x lO-69 A3 U51980 Dactylaria 202 1 x lO-50 dimorphospora

AlO X93986 Trichoderma viride 355 2 x lO-96 74

Figure 5.2. Stacked bar graph showing the percentage of phylotype clones in a root segment. Ascomycete phylotypes are represented by colors and Zygomycete phylotypes are represented by patterens. The Paulina View plants are the only plants that have similar species composition.

100 80 ...... ::: OJ 60 ....Q tU 40 0.- 20 0 Br()ken l()p 17 Katati 2 Paulina View 12 Paulina View 14 Broken t()P 18

Phylotypes II Al [J A7 ~ ZI D A2 II A8 D Z2 A3 D A9 ~ Z3 D A4 0 AIO ~ Z4 II AS II All D A6 D A12 75

A3. The other chimeric amplicon (from the root ofKatati 2) has the ITS1 ofA2 and the

ITS2 ofA7. It is assumed that all ofthe RFLP types that matched these chimeric amplicons are ofthe same chimeric type. The total percentage ofchimeric clones is 4%.

In the latter chimeric amplicon both phylotypes that contributed to the chimera were present in the library. However the other chimeric amplicons represented the only observance ofat least one ofthe contributing phylotypes in its root library. The chimeras were scored as being halfpresent in their corresponding phylotypes (Table 5.3). The presence ofchimeric gene products from PCR has commonly been reported from rDNA

PCR libraries ofmixed microbial communities (Komatsoulis & Waterman 1997).

Several fungi were isolated from surfaced sterilized roots. A few isolates representing some ofthe common colony morphologies were sequenced for the ITS region (Table 5.5). These fungal sequences were included with the clone sequences in the neighbor joining analysis in Figs. 5.1. The isolated fungi formed 4 groups. Two ofthe more common phylotypes ofisolated fungi belong to the two most common ITS clone

phylotypes, Al and A2. The other two sequence groups did not match any ofthe clone

phylotypes. In fact, one is quite distantly related.

A neighbor joining analysis ofthe 5.8S region ofthe different phylotypes shows

that they represent taxonomically diverse fungi (Fig. 5.3). Most ofthe phylotypes are

placed within the filamentous ascomycetes. Four ofthe phylotypes are nested within the

arbuscular mycorrhizal genus Glomus. The ITS 1 ofall phylotypes were analyzed by use

ofBLAST. Three phylotypes had a high score (Table 5.7). Phylotype AlO was almost

identical to Trichoderma viride (X93986), possible the same species. Phylotype A2 was 76

Figure 5.3 Neighbor-joining analysis ofthe 5.8S rDNA. Representatives ofthe Botrychium pumicola fungal phylotypes are denoted with their alpha numeric code. The relationship ofthe phylotypes to ascomycetes and zygomycetes is shown. Single representatives ofthe two fungal isolates that did not belong to a phylotype are included.

AI2 Phialophora american AS Drechslera A6 A4 Phoma Dactvlaria A3 . SP'llaer.olheca PhV aclima ~steiia Al ~Jr;rotinia A9 Rutstroemia A7 Gelatinipulvinella Claviceps Ascomycetes Ne«rospora CoLletofrichum Glomerella Nectria AIO TriChOde~ viride eralocyslis 'Phiostoma fhia10pGa~anomycesra gregata Isolate2- A2 Thermomyces Monas~sMorc ella romvces s :Y9' ,andliia Saccharomyces~ Z4 Z2 ZI Z3 omus inlraradices mus elutu~i~atum F mus cJarou;leum omus mosseae omus monosp,orum rpmus Jasciculataum I011lUS coronalum Zygomycetes I r-- ndogone isolate~-l~ Gigaspora rosea Gigaspora gJganlea GI1!{lspora marJ!.{lMla Gilgaspora a151(ia Scutel ospora castanea Sculellospora helerogama Entrppho~pora Ceraloba#dium Thanatephorus Trif~'3:,~t~: Basidiomycetes TremelJa .L-__ ryptococcus

L-Animals______~.------r.:-;- HomoXenopus Arum '------Dolichosaccus Plants Brassica Gossypium Orna I r--~ Taxus '----- Pinus Marsiltza usmunda L_--r---;:;-;-/soeJes SeiagmelLa 0.1 77 similar to Phialophora gregata (U66727), a stem parasite. Phylotype A3 was similar to

Dactylaria dimorphospora (U51980), isolated from soil.

5.5. Discussion

It is difficult to distinguish the number offungal taxa in a root by microscopy. In

addition, culturing methods can bias which fungi are isolated from a root and restrict the

number of fungal taxa from a small root segment to those able to grow on the media

used. An alternative method for examining the fungi present in a root is to PCR amplify

fungal DNA from the root. Total genomic DNA ofa root will include the DNA ofany of

its symbiotic organisms. Because several fungi maybe in any root segment, the PCR

amplicon can be cloned and the resulting clone library should represent the fungal

community ofthe root, with the DNA ofmore dominant fungi being in the highest

percentage ofclones.

RFLP patterns ofthe ITS region are widely adopted in the use ofdistinguishing

different species ofmycorrhizal fungi (Erland et aI., 1994; Gardes and Bruns, 1996;

KAren et al., 1997; Pritsch et aI., 1997). Most ofthese studies show low intraspecific

variation ofthe RFLP patterns. However, KAren et a1. (1997) did find that 16% ofthe

species they analyzed had multiple RFLP patterns and Pritsch et a1. (1997) found multiple

RFLP patterns in Paxillus rudicundulus . Different RFLP's can result from DNA

sequences that are highly similar due to a few substitution. Overall similarity ofthe ITS

sequence can vary with in a species (Seifert et aI., 1995). A species could have sequences 78 as divergent as 15.8% (Viljoen et al., 1993) and 10.5% (Neuveglise et al., 1994). It is therefore realistic to believe that each phylotype in Table 5.6, even with multiple RFLP­ types, represents a single species. Even ifthey are not the same species, their close relationship would suggest that they have some similar functions.

Four ofthe fungal phylotypes, Zl, Z2, Z3 and Z4, appear to be related to the

Glomales. They form a distinct group nested within Glomus and probably are members of that genus. These related fungi may represent a phylogenetic specificity in Botrychium pumicola fungi, where the mycorrhizal fungi ofa plant species are restricted to a genus or family offungi. This result would be similar to what is found in some nonphotosynthetic orchids (Taylor and Bruns, 1997). The phylogenetic specificity hypothesis needs to be tested further. These plants formed associations with 4 AMF-like fungi. One root segment ofB. pumicola contained 3 phylotypes ofAMF-like fungi. It was expected that there would be multiple AMF, as AM plants often associate with several AMF (Molina et ai, 1992).

The abundance ofAMF-like sequences in the clone hbraries was never greater

that 20%. This implies that this group offungi represents a small portion ofthe total

fungal community in the roots. The AMF-like phylotypes occurred only in 5 ofthe 7

libraries. The low abundance ofAMF-like sequences agrees with microscopic

examination ofthe roots: arbuscules are not the dominant fungal structures. They were

infrequent and patchy. Aseptate hyphae typical ofAMF also were patchy and occasionally

abundant. Therefor. it is not surprising that the AMF are not dominant in the root clone 79 libraries. However, There may not need to be a high abundance ofAMF for a plant to receive a mycorrhizal effect.

The dominant components ofthe root clone libraries are ascomycetes representing broadly diverse taxa (Table 5.3). Two ascomycete phylotypes occurred in over half ofthe clone libraries. Phylotype Al is the most frequent fungus, occurring in 5 ofthe 7 clone libraries. It comprised 72% to 90% ofthe fungi in root segments ofPaulina View plants collected in a cluster. It was of low abundance in root from the Broken Top (12.5%) and Katati (2.2%). No close relatives were found in the BLAST search, but the 5.8S rDNA analysis shows it associated with species classified in the Helotiales. Phylotype A2 was the next most frequent. It is in four ofthe roots segments and ranged from 5% to

37.5%. BLAST results show that this fungus is closely related to Phialophora gregata.

Phialophora gregata, also know as brown stem rot, is a latent pathogen ofsoybean. Both phylotypes Al and A2 were commonly isolated from B. pumicola roots. These isolates were sterile.

The other 10 ascomycete phylotypes are less frequent in the roots. Four are present in two ofthe roots. Phylotype A3 has a high similarity to the hyphomycete

Dactylaria dimorphospora. The other 3 ofthese phylotypes show no close matches. Six ofthe phylotypes occur in only one root segment. Ofthese, only phylotype AIO has a high similarity to an existing fungal sequence. It is almost identical to Trichoderma viride and represents this or a closely related species. Species of Trichoderma are mycoparasites. This fungus maybe living offother fungi in or on the root. 80

Because ofthe high abundance ofphylotype AI, Paulina View 12, 13, and 14, have similar occurrence and abundance ofphylotypes. These three plants were growing in a cluster and were extracted in the same soil clump. The other four root communities were sampled from plants ofdifferent soil extractions at two different sites. Root communities from the same site but different excavations do not share similar fungal species composition. This suggests that fungal communities within roots ofdifferent plants may depend more on micro habitat than broader geographic location and reflect the patchy distribution ofmany soil fungi.

Many fungi are associated with the roots ofBotrychium pumicola. As many as 8 different taxa can inhabit a root segment ofless than 2cm. Several fungi appear to be uncommon, half ofthe 16 phylotypes were only present in one root. Two fungi were isolated from surface-sterilized roots but not observed in the clone libraries. One was isolated from several plants. Perhaps this fungus was present in the roots but not in enough abundance to be observed in the libraries or difficult to clone. More fungi could well be found with increased sampling.

5.6. Acknowledgments

I'd like to thank Randy Molina, Jeff Stone, Jamie Platt, Joseph Spatafora, Jim

Trappe, and Aaron Liston for their assistance with this project. Sequencing was preformed by the Central Services Laboratory within the Center for Gene Research and

Biotechnology at Oregon State University. Funding was provided in part by NSF minority graduate student fellowship, the Hardman Foundation and the Deschutes 81

National Forest. Laboratory resources were provided by Pacific Northwest Research

Station ofthe National Forest Service, Joseph Spatafora and Aaron Liston. Special thanks goes to Pat Joslin ofthe Deschutes National Forest and Christine Hopkins for efforts in getting this project started, collecting the plant material, and general knowledge ofBotrychium pumicola. 82

Chapter 6

Conclusion

6.1 Abstract

This research has increased our knowledge ofthe biology ofBotrychium pumicola. It is now understood the this species can reproduce asexually. Although this species produces asexual propagules (gemmae), these gemmae do not apparently disperse far from the parent plant. The gemmae are important in the maintenance ofgenets, allowing for their survival after the death ofindividuals. Mycorrhizae are an important aspect ofthe biology ofB. pumicola. Vesicular arbuscular mycorrhizae (V AM) are present in the roots, but not in abundance. The most abundant fungi are ascomycetes, in

particular, two that are commonly isolated from sterilized roots. The role ofascomycetous

fungi is unclear, but the roots do not appear to be harmed by them. Maybe only a smaIl

amount ofV AM fungi are necessary to provide B. pumicola with nutrients. However, the

high incidence ofAscomycetes should be investigated further for possible functional

relationships. 83

6.2 Introduction

Botrychium pumicola Coville is a member ofthe Ophioglossaceae. Along with other species ofBotrychium it has a complex life cycle which mostly takes place below ground. Many ofthese species are rare (USDI-Fish and Wildlife Service, 1993). It is important to understand the biology ofBotrychium to make better management decisions for the conservation ofthese species. The little knowledge that we have ofB. pumicola

(Hopkins and O'Neil, 1993) and other species ofBotrychium is primarily from above ground observations (Lesica and Ahlenslager, 1996). Lesica and Ahlenslager (1996) describe it well: "Once more is known about their subterranean life history, we will be better able to understand the causes ofrarity in these interesting ferns."

Observations and knowledge ofBotrychium is hindered at ground level.

Reproduction is underground. Both the subterranean gametophytes and gemmae develop underground (Wagner and Wagner, 1993). Isozyme studies suggest that the gametophytes usually self-fertilize (McCauley et aI., 1985; Soltis and Soltis, 1986; Watano and Sahashi,

1992). Self-fertilization and the low allelic diversity in these studies is suggestive of reduced genetic variability. Gemmae are vegetative propagules that abscise at maturity from the parent plant (Farrar and Johnson-Grob, 1990). These subterranean structures can develop into a mature plant. They are commonly observed at the base ofthe parent plant

(Farrar and Johnson-Grob, 1990; Camacho, 1996), but their ability to disperse across the landscape is unclear. 84

Another below ground feature ofthe biology ofBotrychium is the mycorrhizal symbiosis. Both the gametophyte (Boullard, 1979; Schmid and Oberwinkler, 1993) and the sporophyte (Hepden, 1960; Berch and Kendrick, 1982; Nair, 1989) are observed to be densely colonized by endophytic fungi. The gametophyte mycorrhizae have coiled, aseptate hyphae with occasional vesicles, similar to vesicular arbuscular mycorrhizae

(VAM). The sporophyte has also been described as V AM. One such report (Nair, 1989) describes the hyphae as septate, which is not typical ofV AM. This author then suggests that the ascomycete, Fusarium oxysporum can produce these arbuscules. Evaluations of the mycorrhizae ofother species ofBotrychium need to be interpreted with care to avoid ambiguity.

The goals ofthese studies are to better understand the reproductive biology and the mycorrhizal symbiosis ofBotrychium pumicola. This is achieved with observations of below ground structures and DNA techniques. Inter-simple sequence repeats (ISSR) are used to study the population genetics ofthis species. The internal transcribed spacer (ITS) ofthe rDNA offungi from root is used to characterize the fungal community.

6.3 Discussion

It was discovered that Botrychium pumicola produced asexual subterranean gemmae (Camacho, 1996). However, the significance ofthese gemmae were not

understood. The use ofISSR suggest that the gemmae do not disperse from their parent

plant (Chapter 3). Identical disjunct genotypes are probably maintained by the dispersal 85 and selfing ofspores. Spatially the gemmae may not be important, but they are probably important on a temporal scale. Other species in the subgenus Botrychium, in which B. pumicola is classified, have a short life span with a half-life up to 3 years. The gemmae of

B. pumicola will allow a genet to exceed the life span ofanyone individual.

The root fungal community is diverse (Chapter 4 and 5). Staining ofthe roots revealed several different fungal structures, including the typical VAM. The VAM were

infrequent and the dominant hyphae are septate, probably not zygomycetous. The fungal

ITS from the roots collaborated this observation. These analysis suggest that the dominant

fungi are ascomycetes. The most frequent fungal ITS sequences from the roots matched

commonly isolated B. pumicola root fungi. These fungi are sterile ascomycetes. Although

ascomycetes can form mycorrhizae (Harley and Smith, 1983), it is not clear ifthese fungi

are functioning in a mycorrhzial manor.

Botrychium pumicola may be intermediate between autotrophic plants and

nonphotosynthetic plants that receive all oftheir nourishment via fungi. The gemma stage

ofthe life-cycle is nonphotosynthetic and plant is assumed to receive carbohydrates

through fungi shared with neighboring photosynthesizing plants. It is reasonable to believe

that flow ofcarbohydrates from these plants to B. pumicola through shared fungi does not

completely stop when the plant produces a photosynthesizing leaf. This would help explain

the ability ofBotrychium individuals to survive without producing a leaf for one or more

years.

My research has provided new insight to the biology ofBotrychium and increases

our knowledge ofB. pumicola. It has generated new questions to focus on in the future. 86

For example, it would be interesting to detennine the mechanisms that produce the high levels oflSSR variation within this selfing species and the role ofthe ascomycetes in the roots need to be addressed. 87

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APPENDICES 99

Appendix A ISSR data set

Table includes individual monitored plants and their ISSR bands. The bands are labeled p

(for primer) the primer number - and the size ofthe band in base pairs. Ifa 1 is present in the band column they the plant in that row has an ISSR band ofthe corresponding size.

Ifa 0 is present then there was no band present.

Plant p814-350 p814-450 p814-500 p814-520 p814-540 p814-61 0 p814-730 3003#527 1 1 1 0 1 1 1 3003#525 1 1 1 1 0 1 1 3003#555 1 1 1 1 0 1 1 3003#565 1 1 1 1 0 1 1 3003#577 1 1 1 1 0 1 1 3003#518 1 1 1 0 0 1 1 3003#578 1 1 1 1 0 1 1 3003#517 1 1 1 1 0 0 1 3003#521 1 1 0 1 0 0 1 3003#519-1 1 1 1 0 1 1 1 3003#519-2 1 1 1 0 1 1 1 3003#520 1 1 1 1 0 1 1 3003#566 1 1 1 0 1 1 1 3003#558 1 1 1 0 0 1 1 3003#529-1 1 1 1 1 0 1 1 3003#529-2 1 1 1 1 0 1 1 3003#574 1 1 1 1 0 1 1 3003#572-1 1 1 1 0 0 1 1 3003#572-2 1 1 1 0 0 1 1 3003#532 1 1 1 0 0 1 1 3003#546 1 1 0 0 1 1 1 3003#562-1 1 1 1 0 0 1 1 3003#562-2 1 1 1 0 0 1 1 3004#500-1 1 1 1 0 1 1 1 3004#500-2 1 1 1 0 1 1 1 3004#548-1 1 1 1 1 0 1 1 3004#548-2 1 1 1 1 0 1 1 BT228 1 1 1 1 0 1 1 BT285 1 1 1 0 0 1 1 BT224 1 1 1 0 0 0 1 100

BT289 1 1 1 0 0 1 1 BT257 1 1 1 0 0 1 1 3004#475 1 1 1 0 1 0 1 3004#458 1 1 1 0 1 1 1 3004#435 1 1 1 0 1 1 1 3004#448 1 1 1 0 1 1 1 3004#459 1 1 1 0 1 1 1 3004#538 1 1 0 0 1 1 1 3004#534-1 1 1 1 0 1 1 1 3004#534-2 1 1 1 0 1 1 1 3004#527 1 1 1 0 1 1 1 3004#437 1 1 1 0 0 1 1 3004#415 1 1 1 1 0 0 1 3004#450 1 1 1 0 0 1 1 3004#449 1 1 1 1 0 1 1 3004#441 1 1 1 1 0 1 1 3004#440-1 1 1 1 1 0 1 1 3004#440-2 1 1 1 1 0 1 1 3004#439 1 1 1 0 1 1 1 3004#553 1 1 1 1 0 1 1 3004#453 1 1 1 0 1 1 1 3004#454 1 1 1 1 0 1 1 3004#493-1 1 1 1 1 0 1 1 3004#493-2 1 1 1 1 0 1 1 3004#492 1 1 1 0 1 1 1 BT195 1 1 1 0 1 1 1 BT191 1 1 1 0 1 1 1 BT230 1 1 1 0 1 1 1 BT220 1 1 1 0 1 0 1 BT217 1 1 1 0 1 1 1 BT264 1 1 1 0 0 1 1 BT247 1 1 1 1 0 1 1 BT225 1 1 1 1 0 1 1 BT22 1 1 1 1 0 1 1 BT227 1 1 1 1 0 1 1 BT237 1 1 1 1 0 1 1 BT221 1 1 1 0 0 1 1 BT265 1 1 1 0 0 1 1 BT267 1 1 1 0 0 1 1 BT256 1 1 1 0 0 1 1 BT288 1 1 1 0 0 1 1 BT293 1 1 1 0 0 1 1 BT275-1 1 1 1 1 0 1 1 BT275-2 1 1 1 1 0 1 1 BT276 1 1 1 1 0 1 1 BT016 1 1 1 0 1 0 1 BT019 1 1 1 0 0 1 1 BT018-1 1 1 1 0 0 1 1 BT018-2 1 1 1 0 0 1 1 101

BT023-1 1 1 1 1 0 1 1 BT023-2 1 1 1 1 0 1 1 BT023-3 1 1 1 1 0 1 1 BT116 1 1 1 0 1 1 1 BT312 1 1 1 1 0 1 1 BT082 1 1 1 0 0 0 1 BT113 1 1 1 0 1 1 1 BT115 1 1 1 0 1 1 1 BT117 1 1 1 0 0 1 1 BT119 1 1 0 0 0 1 1 BT203 1 1 1 0 1 1 1 BT182 1 1 1 0 0 0 1 BT324 1 1 1 1 0 1 1 BT336-1 1 1 1 1 0 1 1 BT336-2 1 1 1 1 0 1 1 BT164 1 1 1 0 1 1 1 BT152 1 1 1 0 0 1 1 BT327 1 1 1 1 0 1 1 BT149-1 1 1 1 0 0 0 1 BT149-2 1 1 1 0 0 0 1

Plant p824450 p824-560 p824-600 p824-740 p824-770 p824-860 3003#527 1 1 1 1 1 0 3003#525 1 1 1 1 1 0 3003#555 1 1 1 1 1 1 3003#565 1 1 1 1 1 1 3oo3#5n 1 1 1 1 1 1 3003#518 1 1 1 1 1 0 3003#578 1 1 1 1 1 1 3003#517 1 1 1 1 1 1 3003#521 1 1 1 1 1 0 3003#519-1 1 1 1 1 1 1 3003#519-2 1 1 1 1 1 1 3003#520 1 1 1 1 1 1 3003#566 1 1 1 1 1 0 3003#558 1 1 1 1 1 0 3003#529-1 1 1 1 1 1 1 3003#529-2 1 1 1 1 1 1 3003#574 1 1 1 1 1 1 3003#572-1 1 1 1 1 1 0 3003#572-2 1 1 1 1 1 0 3003#532 1 1 1 1 1 1 3003#546 1 1 1 1 1 0 3003#562-1 1 1 1 1 1 0 3003#562-2 1 1 1 1 1 0 3004#500-1 1 1 1 1 1 1 3004#500-2 1 0 1 1 1 1 3004#548-1 1 1 1 1 1 0 3004#548-2 1 1 1 1 1 0 102

BT228 1 1 1 1 1 0 BT285 1 0 1 1 1 0 BT224 1 0 1 1 1 0 BT289 1 0 1 1 1 0 BT257 1 0 1 1 1 0 3004#475 1 0 1 1 1 0 3004#458 1 1 1 1 1 0 3004#435 1 1 1 1 1 1 3004#448 1 0 1 1 1 0 3004#459 1 1 1 1 1 1 3004#538 1 1 1 1 1 1 3004#534-1 1 1 1 1 1 1 3004#534-2 1 1 1 1 1 1 3004#527 1 1 1 1 1 0 3004#437 1 1 1 1 1 0 3004#415 1 1 1 1 1 0 3004#450 1 1 1 1 1 0 3004#449 1 1 1 1 1 0 3004#441 1 1 1 1 1 0 3004#440-1 1 0 1 1 1 0 3004#440-2 1 1 1 1 1 1 3004#439 1 1 1 1 1 0 3004#553 1 1 1 1 1 0 3004#453 1 1 1 1 1 1 3004#454 1 1 1 1 1 1 3004#493-1 1 1 1 1 1 0 3004#493-2 1 1 1 1 1 1 3004#492 1 1 1 1 1 0 BT195 1 0 1 1 1 0 BT191 1 1 1 1 1 0 BT230 1 1 1 1 1 0 BT220 1 0 1 1 1 1 BT217 1 0 1 1 1 0 BT264 1 0 1 1 1 0 BT247 1 0 1 1 1 1 BT225 1 0 1 1 1 0 BT22 1 1 1 1 1 0 BT227 1 0 1 1 1 1 BT237 1 1 1 1 1 0 BT221 1 1 1 1 1 0 BT265 1 0 1 1 1 0 BT267 1 0 1 1 1 0 BT256 1 1 1 1 1 0 BT288 1 0 1 1 1 0 BT293 1 1 1 1 1 0 BT275-1 1 1 1 1 1 0 BT275-2 1 1 1 1 1 0 BT276 1 1 1 1 1 0 BT016 1 1 1 1 1 0 103

BT019 1 1 1 1 1 1 BT018-1 1 1 1 1 1 0 BT018-2 1 1 1 1 1 0 BT023-1 1 1 1 1 1 0 BT023-2 1 1 1 1 1 0 BT023-3 1 1 1 1 1 0 BT116 1 1 1 1 1 1 BT312 1 1 1 1 1 0 BT082 1 1 1 1 1 0 BT113 1 1 1 1 1 0 BT115 1 1 1 1 1 1 BT117 1 1 1 1 1 0 BT119 1 1 1 1 1 0 BT203 1 1 1 1 1 0 BT182 1 0 1 1 1 0 BT324 1 1 1 1 1 0 BT336-1 1 0 1 1 1 0 BT336-2 1 0 1 1 1 0 BT164 1 1 1 1 1 0 BT152 1 1 1 1 1 0 BT327 1 1 1 1 1 0 BT149-1 1 1 1 1 1 1 BT149-2 1 1 1 1 1 1

Plant p813-320 p813-390 p813-450 p813-485 p813-530 p813-600 p813-630 3003#527 1 1 0 1 1 1 1 3003#525 1 1 1 1 1 1 1 3003#555 1 1 1 1 1 0 1 3003#565 1 1 1 1 1 0 1 3003#577 1 1 1 1 1 0 1 3003#518 1 1 1 1 1 0 1 3003#578 1 1 1 1 1 1 1 3003#517 1 1 1 1 1 1 1 3003#521 1 1 1 1 1 1 1 3003#519-1 1 0 1 1 1 0 1 3003#519-2 1 0 1 1 1 0 1 3003#520 1 1 1 1 1 1 1 3003#566 1 1 1 1 1 1 1 3003#558 1 1 1 1 1 0 1 3003#529-1 1 1 1 1 1 0 1 3003#529-2 1 1 1 1 1 0 1 3003#574 1 1 1 1 1 1 1 3003#572-1 1 1 1 1 1 1 0 3003#572-2 1 1 1 1 1 1 0 3003#532 1 1 1 1 1 1 0 3003#546 1 1 1 1 1 1 1 3003#562-1 1 1 1 1 1 1 0 3003#562-2 1 1 1 1 1 1 0 104

3004#500-1 1 1 1 1 1 1 1 3004#500-2 1 1 1 1 1 1 1 3004#548-1 1 1 1 1 1 1 1 3004#548-2 1 1 1 1 1 1 1 BT228 1 0 0 1 1 1 0 BT285 1 1 1 1 1 1 0 BT224 1 1 1 1 1 1 1 BT289 1 1 1 1 1 1 0 BT257 1 1 1 1 1 1 0 3004#475 1 1 0 1 1 1 1 3004#458 1 1 0 1 1 1 1 3004#435 1 1 1 1 1 1 1 3004#448 1 1 1 1 1 1 1 3004#459 1 1 1 1 1 0 1 3004#538 1 0 1 1 1 0 1 3004#534-1 1 1 1 1 1 1 1 3004#534-2 1 0 1 1 1 0 1 3004#527 1 1 1 1 1 0 1 3004#437 1 1 1 1 1 1 0 3004#415 1 1 1 1 1 1 1 3004#450 1 1 1 1 1 0 1 3004#449 1 1 1 1 1 1 1 3004#441 1 1 1 1 1 1 1 3004#440-1 1 1 1 1 1 0 1 3004#440-2 1 1 1 1 1 0 0 3004#439 1 0 0 1 1 1 1 3004#553 1 1 1 1 1 0 1 3004#453 1 1 1 1 1 1 1 3004#454 1 1 1 1 1 0 1 3004#493-1 1 1 1 1 1 0 1 3004#493-2 1 0 0 1 1 1 1 3004#492 1 1 1 1 1 1 1 BT195 1 1 0 1 1 0 1 BT191 1 1 0 1 1 0 1 BT230 1 1 1 1 1 1 0 BT220 1 1 1 1 1 1 1 BT217 1 1 1 1 1 1 1 BT264 1 1 1 1 1 1 0 BT247 1 1 1 1 1 1 0 BT225 1 1 1 1 1 1 0 BT22 1 1 1 1 1 1 0 BT227 1 1 1 1 1 1 0 BT237 1 1 1 1 1 1 0 BT221 1 1 1 1 1 1 0 BT265 1 1 1 1 1 1 0 BT267 1 1 0 1 1 1 0 BT256 1 1 0 1 1 1 0 BT288 1 1 0 1 1 1 0 BT293 1 1 0 1 1 1 1 105

BT275-1 1 1 0 1 1 0 1 BT275-2 1 1 0 1 1 0 1 BT276 1 1 0 1 1 0 1 BT016 1 0 1 1 1 1 0 BT019 1 0 1 1 1 0 1 BT018-1 1 0 0 1 1 1 1 BT018-2 1 0 0 1 1 1 1 BT023-1 1 1 1 1 1 1 0 BT023-2 1 1 1 1 1 1 0 BT023-3 1 1 1 1 1 1 0 BT116 1 1 1 1 1 1 0 BT312 1 1 1 1 1 0 1 BT082 1 1 1 1 1 1 0 BT113 1 1 1 1 1 1 0 BT115 1 0 0 1 1 1 0 BT117 1 1 1 1 1 0 1 BT119 1 0 0 1 1 1 0 BT203 1 0 0 1 1 0 1 BT182 1 1 0 1 1 0 0 BT324 1 1 1 1 1 0 1 BT336-1 1 1 0 1 1 0 1 BT336-2 1 1 0 1 1 0 1 BT164 1 0 1 1 1 1 0 BT152 1 0 1 1 1 1 0 BT327 1 1 1 1 1 0 1 BT149-1 1 0 1 1 1 1 0 BT149-2 1 0 1 1 1 1 0

Plant p840-450 p840-500 p840-550 p840-590 p840~OO p840~50 3003#527 1 1 1 0 1 1 3003#525 1 1 0 0 1 1 3003#555 1 1 1 0 1 1 3003#565 1 1 1 0 1 1 3003#577 1 1 1 0 1 1 3003#518 1 1 1 0 1 1 3003#578 1 1 0 0 1 1 3003#517 1 1 1 0 1 1 3003#521 1 1 1 0 1 1 3003#519-1 1 1 1 0 1 1 3003#519-2 1 1 1 0 1 1 3003#520 1 1 0 0 1 1 3003#566 1 1 1 0 1 1 3003#558 1 1 1 0 1 1 3003#529-1 1 1 1 0 1 1 3003#529-2 1 1 1 0 1 1 3003#574 1 1 1 0 1 1 3003#572-1 1 1 1 0 1 1 106

3003#572-2 1 1 1 0 1 1 3003#532 1 1 1 0 1 1 3003#546 1 1 1 0 1 1 3003#562-1 1 1 1 0 1 1 3003#562-2 1 1 1 0 1 1 3004#500-1 1 1 1 0 1 1 3004#500-2 1 1 1 0 1 1 3004#548-1 1 1 1 0 1 1 3004#548-2 1 1 1 0 1 1 BT228 1 0 1 0 1 1 BT285 1 1 1 0 1 1 BT224 1 0 1 0 1 1 BT289 1 1 1 0 1 1 BT257 1 1 1 0 1 1 3004#475 1 1 1 1 1 1 3004#458 1 1 1 0 1 1 3004#435 1 1 1 1 1 1 3004#448 1 1 1 1 1 1 3004#459 1 1 1 0 1 1 3004#538 1 1 1 1 1 1 3004#534-1 1 1 1 1 1 1 3004#534-2 1 1 1 1 1 1 3004#527 1 1 1 0 1 1 3004#437 1 1 1 0 1 1 3004#415 1 1 1 0 1 1 3004#450 1 1 1 0 1 1 3004#449 1 1 1 0 1 1 3004#441 1 1 1 1 1 1 3004#440-1 1 1 1 0 1 1 3004#440-2 1 1 1 0 1 1 3004#439 1 1 1 0 1 1 3004#553 1 1 1 0 1 1 3004#453 1 1 1 0 1 1 3004#454 1 1 1 1 1 1 3004#493-1 1 1 1 1 1 1 3004#493-2 1 1 1 1 1 1 3004#492 1 1 1 0 1 1 BT195 1 1 1 0 1 1 BT191 1 1 1 0 1 1 BT230 1 1 1 0 1 1 BT220 1 1 1 0 1 1 BT217 1 1 1 0 1 1 BT264 1 1 1 0 1 1 BT247 1 1 1 0 1 1 BT225 1 1 1 0 1 1 BT22 1 1 1 0 1 1 BT227 1 1 1 0 1 1 BT237 1 1 1 0 1 1 BT221 1 1 1 0 1 1 107

BT265 1 1 1 0 1 1 BT267 1 1 1 0 1 1 BT256 1 1 1 0 1 1 BT288 1 1 1 0 1 1 BT293 1 1 1 0 1 1 BT275-1 1 0 1 0 1 1 BT275-2 1 0 1 0 1 1 BT276 1 1 0 0 1 1 BT016 1 1 1 0 1 1 BT019 1 1 0 0 1 1 BT018-1 1 1 1 0 1 1 BT018-2 1 1 1 0 1 1 BT023-1 1 1 1 0 1 1 BT023-2 1 1 1 0 1 1 BT023-3 1 1 1 0 1 1 BT116 1 0 1 0 1 1 BT312 1 1 0 0 1 1 BT082 1 1 1 0 1 1 BT113 1 1 1 0 1 1 BT115 1 1 0 0 1 1 BT117 1 0 1 0 1 1 BT119 1 1 0 0 1 1 BT203 1 1 1 0 1 1 BT182 1 1 1 0 1 1 BT324 1 1 0 0 1 1 BT336-1 1 1 1 0 1 1 BT336-2 1 1 1 0 1 1 BT164 1 1 1 0 1 1 BT152 1 1 1 0 1 1 BT327 1 1 1 0 1 1 BT149-1 1 1 1 0 1 1 BT149-2 1 1 1 0 1 1

Plant p848-440 p845-420 p845-480 3003#527 1 1 1 3003#525 1 1 1 3003#555 1 1 1 3003#565 1 1 1 3003#577 1 1 1 3003#518 1 1 1 3003#578 1 1 1 3003#517 1 1 1 3003#521 1 1 1 3003#519-1 1 1 1 3003#519-2 1 1 1 3003#520 1 1 1 108

3003#566 1 1 1 3003#558 1 1 1 3003#529-1 1 1 1 3003#529-2 1 1 1 3003#574 1 1 1 3003#572-1 1 1 1 3003#572-2 1 1 1 3003#532 1 1 1 3003#546 1 1 1 3003#562-1 1 1 1 3003#562-2 1 1 1 3004#500-1 1 1 0 3004#500-2 1 1 0 3004#548-1 1 1 1 3004#548-2 1 1 1 BT228 1 1 1 BT285 1 1 1 BT224 1 1 0 BT289 1 1 0 BT257 1 1 0 3004#475 1 1 1 3004#458 1 1 1 3004#435 1 1 1 3004#448 1 1 0 3004#459 1 1 1 3004#538 1 1 1 3004#534-1 1 1 1 3004#534-2 1 1 1 3004#527 1 1 1 3004#437 0 1 1 3004#415 1 1 0 3004#450 1 1 1 3004#449 0 1 0 3004#441 1 1 1 3004#440-1 1 1 1 3004#440-2 0 1 1 3004#439 1 1 1 3004#553 1 1 1 3004#453 1 1 1 3004#454 1 1 1 3004#493-1 1 1 0 3004#493-2 0 1 0 3004#492 1 1 1 BT195 1 1 1 BT191 0 1 1 BT230 1 1 1 BT220 1 1 1 BT217 0 1 1 BT264 1 1 1 109

BT247 1 1 1 BT225 1 1 0 BT22 0 1 0 BT227 0 1 0 BT237 1 1 1 BT221 1 1 1 BT265 1 1 0 BT267 1 1 0 BT256 1 1 0 BT288 1 1 0 BT293 1 1 0 BT275-1 1 1 1 BT275-2 1 1 1 BT276 1 1 1 BT016 1 1 1 BT019 1 1 1 BT018-1 1 1 1 BT018-2 1 1 1 BT023-1 1 1 1 BT023-2 1 1 1 BT023-3 1 1 1 BT116 1 1 1 BT312 1 1 0 BT082 1 1 1 BT113 1 1 1 BT115 1 1 1 BT117 0 1 0 BT119 1 1 0 BT203 1 1 1 BT182 1 1 1 BT324 0 1 1 BT336-1 1 1 1 BT336-2 1 1 1 BT164 1 1 1 BT152 0 1 0 BT327 0 1 1 BT149-1 1 1 1 BT149-2 1 1 1 110

Appendix B DNA alignment ofthe complete ITS nrDNA region.

clone17C-l clone17C-3 clone17C-4 isolate23-5 clone2C-4 ...... TT CCGTAGGTGA clone2C-23 clone2C-2 clone2C-13 ...... TT CCGTAGGTGA clone18C-l ...... T CCGTAGGTGA clone18C-5 ...... T AACAAGGTTT CCGTAGGTGA clone18C-4 ...... T AACAA.GTTT CNGTAGGTGA clone12C-5 ...... T CCGTAGGTGA clone14C-21 ...... T CCGTAGGNGA clone13C-l ...... GTAGGNGA clone2C-lO isolatell-3b TAAAAGTCGT AACAAGGTTT CCGTAGGTGA isolateBl-3 CCNTTGTCAT TTAGAGGAAG TAAAAGTCGT AACAAGGTTT CCGTAGGTGA clone17C-5 ...... TTT CCGTAGGTGA clone2C-24 ...... TTT CCGTAGGTGA isolate2-7 clone2C-8 ...... TTT CCGTAGGTGA clone12C-9 ...... TT CCGTAGGTGA clone17C-6 ...... TT CNGTAGGTGA clone2C-20 ....TTT CCGTAGGTGA clone13C-17 ...... TTT CCGTAGGTGA isolate15-1 ...... CAAGG .. T CCGTAGGTGA clone2C-12 ...... TTT CCGTAGGTGA isolateBl-4 CCTTTTGCAT TTAGAGGAAG TAAAAGTCGT AACAAGGTTT CCGTAGGTGA isolatell-2 .AAAGTCGT AACNAGGTTT CCGTAGGTGA isolate14-1 ...AGGTGA isolate9-1 isolate3-8 clone3C-4 ••••••• T CCGTTGGTGA clone3C-15 ...... TT CNGTTGGNGA clone3C-lO •••••••• T CCGTTGGTGA clone14C-l ... .TTT CCGTAGGTGA clone14C-6 ...... TTT CCGTAGGTGA clone3C-l ••••••••• T CCGTAGGTGA clone3C-12 ...... TTT CCGTAGGTGA clone3C-6 ...... TTT CCGTAGGTGA isolate5-2 .. GGCGGTGA isolate2-1 ...... GGA AGGGAAGTCT CTGGNGGTGA isolatel-l ...CCCCGGA AGGGNAGTC. CNGGCGGTGA isolate7-1 ...... GA AGGGCNGTCC CCGGTGGTGA clone2C-31 ...... TTT CCGTAGGTGA clone2C-35 ...... TTT CCGTAGGTGA clone12C-6 ••••• TT CNGTAGGTGA clone3C-14 ...... TT CCGTAGGTGA clone13C-3 •••••••• TT CNGTAGGTGA clone12C-41 ...... TTT CCGTAGGTGA ------

111

clone17C-l ...... · ...CATTAC TAGAGCAAAG GATANACAGC ACCCGCGGA. clone17C-3 · ...... CATTAC · ...... TG A...... · ...G..... clone17C-4 ...... · ...CATTAC ...... AG AGAT ...... CATG ... isolate23-5 ·...... CCATTGA ...... GG NCTNG ... TG NNAGNGGGAG clone2C-4 AC.TGCGGAA GGN.CATTAG T .... CTATG NGNGG ...... CCCGTGAGG clone2C-23 ... TGCGGAA GGATCATTAA ...... CG A...... G TGAGGGG ... clone2C-2 ...... CATTAA ...... CG A...... A TGANGG .... clone2C-13 AC.TGCGGAA GGA.CATTAC · ...... CG AGTT ...... CATG ... clone18C-l AC.TGCGGAA GGA.CATTAC ...... CG AGTT ...... CA.G ... clone18C-5 AC.TGCGGAA GGA.CATTAC · ...... CG AGTT ...... CATG ... clone18C-4 AC.TGCGGAA NGA.CATTAC · ...... CG AGTT ...... CATG ... clone12C-5 AC.TGCGGAA GGA.CATTAC ...... AG AGTT ...... CATG ... clone14C-21 AC.TGCGGAA GGN.CATTAC ...... AG ANNT ...... CA.G ... clone13C-l AC.TGCGGAA GGN.CATTAC ...... AG NGNT ...... CA.G ... clone2C-lO · ...... CATTAC · ...... AG AGTT ...... CGTG ... isolatell-3b ACCTGCGGAA GGATCATTAC · ...... AG AGTT ...... · .. CATG ... isolateBl-3 ACCTGCGGAA GGATCATTAC ...... AG AGTT ...... CATG ... clone17C-5 ACCTGCGGAA GGATCATTAC ...... AG AGTT ...... CATG ... clone2C-24 ACCTGCGGAA GGATCATTAC ...... AG AGTT ...... CATG ... isolate2-7 · ...... · .ATCATTAA ...... AG AGAT ...... CATG ... clone2C-8 ACCTGCGGAA GGATCATTAC TAGAGCAAAG GATAGACAGC GCCCGCGGA. clone12C-9 AC.TGCGGAA GGA.CATTAC TAGAGCAAAG GATAA.CAGC ACCCGCGGAA clone17C-6 AC.TGCGGAA GGA.CATTAC TAGAGCAAAG GATAA.CAGC ACCCGCGGA. clone2C-20 ACCTGCGGAA GGATCATTAC TAGAGCAAAG GATAGACAGC GCCCGCGGA. clone13C-17 ACCTGCGGAA GGATCATTAC TAGAGCAAAG GATAGACAGC ACCCGCGGA. isolate15-1 ACCTGCGGAA GGATCATTAC TAGAGCAAAG GATAGACAGN GNCCGTGGA. clone2C-12 ACCTGCGGAA GGATCATTAC TAGAGCAAAG GATAGACAGC GCCCGCGGA. isolateBl-4 ACCTGCGGAA GGATCATTAC TAGAGCAAAG GATAGACAGC GCCCGCGGA. isolatell-2 ACCTGCGGAA GGATCATTAC TAGAGCNAAG GATAGACNGC ACCCGCGGA. isolate14-1 ACCTGCGGAA GGATCATTAC TAGAGCAAAG GATAGACAGC ACCCGCGGA. isolate9-1 ...... · . ACCATTAC TAGAGCAAAG GATAGACAGC ACCCGCGGA. isolate3-8 · ...... ATCTATTAC TANAGCAAAG GATAGACAGC GCCCGCGGA. clone3C-4 ACCAGCGGAG GGATCATTAC · ...... CG A...... G..... clone3C-15 AC.AGCGGAA GGN.CATTAC ...... CG A...... G..... clone3C-lO ACCAGCGGAG GGATCATTAC ...... CG A...... · ...G..... clone14C-l ACCTGCGGAA GGATCATTAC ...... CG A...... G TTAGGG .... clone14C-6 ACCTGCGGAA GGATCATTAC ...... CG A...... G TTAGGG .... clone3C-l AC.TGCGGAA GGA.CATTAT T ...... AG TTTA ...... GG .... clone3C-12 ACCTGCGGAA GGATCATTAT T ...... AG TTTA ...... GGG ... clone3C-6 ACCTGCGGAA GGATCATTAT T ...... AG TTTA ...... GGG ... isolate5-2 ACCTGCGGAA GGANCNTTAC CA ...... AG AGAA ...... · ...... isolate2-1 ACCTGCGGAA GGACCNTTAC CA ...... AG AGAA ...... isolatel-l ACCTGCGGAA GGACCCTTAC CA ...... AG AGAA ...... isolate7-1 ACCTGCGGAA GGATCATTAC CA ...... AG AGAA ...... · ...... clone2C-31 ACCTGCGGAA GGATCATTAT T ...... G A...... clone2C-35 ACCTGCGGAA GGATCATTAT T ...... G A ...... ·...... clone12C-6 NC.TGCGGAA GGA.CATTAC T ...... G ATTT ...... clone3C-14 NC.TGCGGAA GGA.CATTAC C...... A ATAT ...... clone13C-3 AC.TGCGGAA GGA.CATTAC T ...... G ATTT ...... clone12C-41 ACCTGCGGAA GGATCATTAC T ...... G ATTT ......

clone17C-l GCTCGCTCCC GGGGCTACCC TACTCCGGTA GGGTTTANAG TCGTCGGGCC clone17C-3 ...... TCG ...... T AAAAA ••••• clone17C-4 .....CCCTT CGGGGTA ...... isolate23-5 ACTTGCCCTG ATCGAAAGAG AGCCGTGAGG TGAACTCTCG NGGCNAGGAC clone2C-4 GCTTACACGN CGGGN .... C TCGTCTT ...... TTT ... G TCGCCGAAAG 112 clone2C-23 ...... TCG ...... TCC AGGCC ..... clone2C-2 · ...... TCG ...... · ...... · ...... TCC ANGCC ..... clone2C-13 · ....CCCTC . CGGGTA ... · ...... · ...... · ...... clone18C-l · ....CCCTT CGGGGTA ... · ...... · ...... · ...... clone18C-5 · .... CCCTC ACGGGTA ...... clone18C-4 .... . CCCTC ACGGGTA ...... clone12C-5 .... . CCCTT . CGGGTA ...... clone14C-21 · .... CCCTT ACGGGNA ... · ...... · ...... clone13C-l .....CCCTT ACGGGNA ... · ...... · ...... · ...... clone2C-lO .....CCCTT CGGGGTA ... · ...... · ...... · ...... isolatell-3b .....CCCTT ACGGGTA ... ·...... · ...... isolateBl-3 ..... CCCTT ACGGGTA ... · ...... clone17C-5 ..... CCCTT ACGGGTA ...... clone2C-24 · ....CCCTC ACGGGTA ... ·...... ·...... ·...... isolate2-7 · .... CCCTC ACGGGTA ... ·...... ·...... · ...... clone2C-8 GCTCGCTCCC GGGGCTACCC TACTCCCGTA GGGTTTAGAG TCGTCGGGCC clone12C-9 GCTCGCTCCC GGGGCTACCC TACTCCGGTA GGGTTTANAN TCGTCGGGCC clone17C-6 GCTCGCTCCC GGGGCTACCC TACTCCGGTA GGGTTTANAG TCGTCGGGCC clone2C-20 GCTCGCTCCC GGGGCTACCC TACTCCCGTA GGGTTTAGAG TCGTCGGGCC clone13C-17 GCTCGCTCCC GGGGCTACCC TACTCCGGTA GGGTTTAGAG TCGTCGGGCC isolate15-1 GGTTGCTCCC GGGGGTACCC TACAACCN.A GGGTTTAGAG TCGGCGGNNC clone2C-12 GCTCGCTCCC GGGGCTACCC TACTCCCGTA GGGTTTAGAG TCGTCGGGCC isolateBl-4 GCTCGCTCCC GGGGCTACCC TACTCCCGTA GGGTTTAGAG TCGTCGGGCC isolatell-2 GCTCGCTCCC GGGGCTACCC TACTCCGGTA GGGTTTAGAG TCGTCGGGCC isolate14-1 GCTCGCTCCC GGGGCTACCC TACTCCGGTA GGGTTTAGAG TCGTCGGGCC isolate9-1 GCTCGCTCCC GGGGCTACCC TACTCCGGTA GGGTTTAGAG TCGTCGCCCC isolate3-8 GCTCGCTCCC NGGGCTACCC TACTCCGGTA GGGTTTAGAG TCGNCGCCCC clone3C-4 · ...... TT ...... · ...... · ...... T ACAA ...... clone3C-15 ...... NT ...... T ACA ...... clone3C-lO ...... TT ...... T ACAA ...... clone14C-l ...... TCC ...... CCT GGGcc ..... clone14C-6 · ...... TCC ...... · ...... · ...... CCT GGGcc ..... clone3C-l · ...... T GTTGAT .... · ... c ...... CA GCGCCCA ... clone3C-12 · ...... T GTTGAT .... · ...c ...... CA GCGCCCA ... clone3C-6 · ...... T GTTGAT ...... c ...... CA GCGCCCA ... isolate5-2 · ...... ATCNT ...... isolate2-1 ...... ATCCT ...... isolatel-l ...... ATCCT ...... isolate7-1 ...... ATCTT ...... clone2C-31 ...... clone2C-35 ...... clone12C-6 ...... GGGG ...... clone3C-14 ...... AGCG ...... clone13C-3 ...... AGCG ...... clone12C-41 ...... AGCG ...... clone17C-l TCTCGCAGAA . GCTCGGTC. .CTGAACTCC AC .. CCT ... · ...... T. clone17C-3 · ...... A ...... CTCC ... CA AC .. CCA ...... T clone17C-4 ...... GAC. · .TC .... cc AC .. CCT ...... AT isolate23-5 TATTGNACGA GTGGNGNTC. .ATCNAGGCA GCGACCNAAA ANAAAAAATT clone2C-4 GC.CGCAAAT TGGTGGGGNG TCTGAA ...A AC .. CCT ... · ...... T. clone2C-23 ...... CGA ...... c . · CTCC .•. CA AC .. CCT ... · ...... TT clone2C-2 ...... CGA ...... c . .CTCC ... CA AC .. CCT ...... TT clone2C-13 ...... GAT. .CTC .... cc AC .. CCT ...... TT clone18C-l ...... GAT. .CTC .... cc AC .. CCT ...... TT clone18C-5 ...... GAT. .CTC ....cc AC .. CCT ... · ...... TT 113 clone18C-4 ...... GAT. · CTC .... CC AC .. CCT ... · ...... TT clone12C-5 ...... GAT . · CTC ....CC AC ..CCT ... · ...... TT clone14C-21 ...... GAT . · CTC ....CC AC .. CCT ...... TT clone13C-l ...... GAT . · CTC ....CC AC ..CCT ... · ...... TT clone2C-lO ...... GAC. .CTC ....CC AC .. CCT ...... TT isolatell-3b ...... GAT . · CTC .... CC AC .. CCT...... TT isolateBl-3 ...... GAT. · CTC .... CC AC .. CCT ...... TT clone17C-5 ·...... · .....GAT. .CTC ....CC AC ..CCT ... · ...... T . clone2C-24 ...... GAT . · CTC .... CC AC .. CCT ... · ...... T . isolate2-7 ...... · .....GAC. · CTC .... CC AC .. CCT ... · ...... TT clone2C-8 TCTCGGAGAA . GCTCGGTC. .CTGAACTCC AC ..CCT ... · ...... T . clone12C-9 TCTCGCAGAA . GCTCGGTC. . CTGAACTCC AC .. CCT. .. · ...... T . clone17C-6 TCTCGCAGAA . GCTCGGTC. . CTGAACTCC AC .. CCT ...... T . clone2C-20 TCTCGGAGAA . GCTCGGTC. . CTGAACTCC AC ..CCT ... · ...... T. clone13C-17 TCTCGCAGAA . GCTCGGTC. .CTGAACTCC AC .. CCT ... · ...... T . isolate15-1 TCTCGGAGAA . GTTCGGNC. . CNGAACTCC AN ..CCT ... · ...... T . clone2C-12 TCTCGGAGAA . GCTCGGTC. . CTGAACTCC AC .. CCT ...... T . isolateBl-4 TCTCGGAGAA . GCTCGGTC. . CTGAACTCC AC .. CCT ...... T . isolatell-2 TCTCGCAGAA . GCTCGGTC. . CTGAACTCC AC .. CCT ...... T . isolate14-1 TCTCGCAGAA . GCTCGGTC. . CTGAACTCC AC .. CCT ...... T . isolate9-1 TCTCGCAGAA . GCTCGGTC. . CTGAACTCC AC ..CCT ... · ...... T . isolate3-8 TCTCGCAGAA . GCTCGGTN. . CTGAACTCC AG ..CCT ...... T. clone3C-4 ...... CTCC ... CA AC .. CCAA ...... T clone3C-15 ·...... CTCC ... CA AC .. CCAA ...... T clone3C-lO ...... CTCC ... CA AC .. CCAA ...... T clone14C-l · ...... CGA · ...... C. .CTCC ....A AC .. CCT. .. · ...... TT clone14C-6 ...... CGA · ...... C. .CTCC ....A AC ..CCT. .. · ...... TT clone3C-l ...... AA .... .CTT ....CA AC ..CCT ... · ...... TT clone3C-12 ...... AA .... .CTT .... CA AC .. CCT ...... TT clone3C-6 · ...... AA .... .CTT .... CA AC .. CCT ...... TT isolate5-2 ...... · .TC .....A ACN ..CT ...... isolate2-1 ·...... · .TC .....A ACN .. CT ...... isolatel-l · ...... · .TC .....A ACC .. CT ...... isolate7-1 ...... · . TC .....A ACA .. CT ...... clone2C-31 ...... A CCT ...... clone2C-35 ...... A CCT ...... clone12C-6 ...... A CC ...... clone3C-14 ...... · ...... A AC ...... clone13C-3 ...... · ...... A AC ...... clone12C-41 ...... A ACC ......

clone17C-l GAATAAA ..C TACCTT .. T. GTTGCTTTGG CGGGCC .... · ...... G clone17C-3 GTGAAT .... TACCTT .. T. GTTGCCTCGG CGG ...... T ..AC ... C clone17C-4 GTTTACA .. T TACCTT .. T. GTTGCTTTGG CGGGCC ...... GTTCAG isolate23-5 GTNTCCC ..G GACCTC ..T. GTTCCCNCGN CGGACNGNCA TN ..AC ..GN clone2C-4 GAATATA .. A . AACTT ..A. GTTGCTTTGG CGGGCC ...... G clone2C-23 GTTTACC .. G AACCTC .. T. GTTGCTTCGG CGGACCCGCC TC ..AC .. GG clone2C-2 GTTTACC .. G AACCTC .. T. NTTGCTTCGG CGGGACCGCC TC ..AC .. NG clone2C-13 GTATACT .. A TACCTT .. T. GTTGCTTTGG CGGGCC ...... G clone18C-l GTATACT ..A TACCTT .. T. GTTGCTTTGG CGGGCC ...... G clone18C-5 GTATACT .. A TACCTT .. T. GTTGCTTTGG CGGGCC .... · ...... G clone18C-4 GTATACT ..A TACCTT .. T. GTTGCTTTGG CGGGCC ...... G clone12C-5 GAGTACT ..A TACTTT ..T. GTTGCTTTGG CAGGCC .... · ...... G clone14C-21 GAGNACT .. A TACTTT .. T. GNTGCTTTGG CAGGCC ...... G clone13C-l GAGCACT .. A TACTTT .. T. GTTGCTTTGG CAGGCC ...... G clone2C-lO GTATAC ... C TACCTT .. T. GTTGCTTTGG CGGGCC ...... G 114 isolatell-3b GAGTACT .. A TACTTT .. T. GTTGCTTTGG CAGGCC ...... G isolateBl-3 GAGTACT .. A TACTTT .. T. GTTGCTTTGG CAGGCC .... · ...... G clone17C-5 GAATATT .. A TACCTT .. T. GTTGCTTTGG CGGGCC .... · ...... G clone2C-24 GAATATTTTA TACCTT .. T. GTTGCTTTGG CGGGcc .... · ...... G isolate2-7 GTTTACA .. A TACCTT .. T. GTTGCTTTGG CGGGCcc ...... GTTTGG clone2C-8 GAATAAA .. c TACCTT .. T. GTTGCTTTGG CGGGCC ...... G clone12C-9 GAATAAA .. c TACCTT .. T. GTTGCTTTGG CGGGCC ...... G clone17C-6 GAATAAA .. c TACCTT .. T. GTTGCTTTGG CGGGCC .... · ...... G clone2C-20 GAATAAA .. c TACCTT .. T. GTTGCTTTGG CGGGCC .... · ...... G clone13C-17 GAATAAA .. C TACCTT .. T. GTTGCTTTGG CGGGCC .... · ...... G isolate15-1 GAATAAA .. c TACCTT .. T. GTTGGTTTGG GGGGCC .... · ...... G clone2C-12 GAATAAA .. c TACCTT .. T. GTTGCTTTGG CGGGCC ...... G isolateBl-4 GAATAAA .. c TACCTT .. T. GTTGCTTTGG CGGGcc ...... G isolatell-2 GAATAAA .. C TACCTT .. T. GTTGCTTTGG CGGGCC .... · ...... G isolate14-1 GAATAAA .. C TACCTT .. T. GTTGCTTTGG CGGGCC .... · ...... G isolate9-1 GAATAAA .. C TACCTG .. N. GTTGCTTNGG CGGGCC .... · ...... G isolate3-8 GAATAAA .. C TACCTN .. T. GGTGCGTTGG CGGGCN ...... G clone3C-4 GTGAACC .. A TACCAAACT. GTTGCCTCGG CGGGG ..... TC .. AC ... G clone3C-15 GNGAACC .. A TACCAAACT. GNTGCCTCGG CGGGG ..... NC .. AC ... G clone3C-1O GTGAACC .. A TACCAAACT. GTTGCCTCGG CGGGG ..... TC ..AC ...G clone14C-l GTCTACC .. T TACCTC .. TT GTTGCTTCGG CGGGCCCGTC TTTAACCAGA clone14C-6 GTCTACC .. T TACCTC .. TT GTTGCTTCGG CGGGCCCGTC TTTAACCAGA clone3C-l GACTTAA ... TCAATT .. T. GTTTCTTTGC CGG ...... ·...... clone3C-12 GACTTAA ... TCAATT .. T. GTTTCTTTGC CGG ......

clone3C-6 GACTTAA ... TCAATT .. T. GTTTCTTTGC CGG ...... oooooooo .... oo ...... isolate5-2 . G ..AAA ... GATCTTTTCC TTTGTGCTGG CTTTG ..... · ...... A isolate2-1 .G .. AAA ... GATCNTTTCC TTTGTGCTGG CNTTG ...... A isolatel-l .G .. AAA ... GATCNTTTCC TTTGTGCTGG CNTTG ..... · ...... A isolate7-1 . G .. AAA ... GATCTTTTCC TTTGTGCTGG CTTTG ...... A clone2C-31 ...... AAGCA ..AGC CTGGCT.TG. CG ...... · ...... C clone2C-35 ...... AAGCA .. AGC CTGGCT.TG. CG ...... c clone12C-6 ...... GAGCGTCAGC GAGGTTCCG. CGATC ...... A clone3C-14 ...... CAGCGTTAGC GAGGTTTCG. CGATC ...... A clone13C-3 ...... GAGCATTAGC GAGGTTCTG. CGATC ...... A clone12C-41 ...... GAGCATTAGC GAGGTTCTG. CGATC ...... A

clone17C-l CCTCGTG ...... CCAGC. GGCTTCGGCT GTTGAGTGCC CGCCAGAGG. clone17C-3 TACTCGGTGC TTTA.AGCT. ...A.CCT .. TGCA ... GTT CCCCGGTGG. clone17C-4 CCCCGTG.CT GA.ACTACC. GGCTTATGCT GGTAAGCGCC TGCCAGAGG. isolate23-5 CCGCCGGAGG ATTGCCGACA GTCGTCCTCT GNCCCGCGTC CNCCGATGG. clone2C-4 CCCTTGG ...... GCGNC. GGCCCCGGCT GAC.AGTGCC CGCCAAAAG. clone2C-23 CCGCCGGAGG ATTGCCGACA GGCGTCCTCT GGCCCGCGTC CGCCGACGG. clone2C-2 CCGCCGGAAG ATTGCCGACA NGCGTCCTCT GGCCC.CGTC C.CCNACGG. clone2C-13 CCT ...... A.NCTACT. GGCTTCGGCT GGTAAGTGCC CGCCAGANG. clone18C-l CCT ...... A.GCTACT. GGCTTCGGCT GGTAAGTGCC CGCCAGANA. clone18C-5 CCT ...... A.GCTACT. GGCTTCGGCT GGTAAGTGCC CGCCAGAGG. clone18C-4 CCT ...... A.GCTACT. GGCTTCGGCT GGTAAGTGCC CGCCAGAGG. clone12C-5 CTTCG ...... GCTACC. GGCTTCGGCT GGTGAGTGCC TGCCAGAG .. clone14C-21 CTTCG ...... GCTACC. GGCTTCGGCT GGTGAGNGCC TGCCAGAN .. clone13C-l CTTCG ...... GCTACC. GGCTTCGGCT GGTGAGTGCC TGCCANAN .. clone2C-1O TCGCAAGA ...... CCGGC. GGCTTCGGCT GTCGTGTGCC CGCCAGAGG. isolatell-3b CTTCG ...... GCTACC. GGCTTCGGCT GGTGAGTGCC TGCCAGAG .. isolateBl-3 CTTCG ...... GCTACC. GGCTTCGGCT GGTGAGTGCC TGCCAGAGG . clone17C-5 CTTCG ...... GCTACC. GGCTACGGCT GGTGAGTGCC CGCCAGAGG. clone2C-24 CTTCG ...... GCTACC. GGCTTCGGCT GGTGAGTGCC CGCCAGAGG. isolate2-7 CCCCGCGACT GA.ACAACC. GGCCCCGGCT GGTCAGTGCC CGCCAGAGA. 115 clone2C-B CCTCGTG ...... CCAGC. GGCTTCGGCT GTTGAGTGCC CGCCAGAGG . clone12C-9 CCTCGTG ...... CCANC. GGCTTCGGCT GTTGANTGCC CGCCAGANG. clone17C-6 CCTCGTG ...... CCAGC. GGCTTCGGCT GTTGAGTGCC CGCCAGAGG. clone2C-20 CCTCGTG ...... CCAGC. GGCTTCGGCT GTTGAGTGCC CGCCAGAGG. clone13C-17 CCTCGTG ...... CCAGC. GGCTTCGGCT GTTGAGTGCC CGCCAGAGG. isolate15-1 CCNCGTG ...... CCAGC. GGCTTCGGCT TTTGNGTGCC CNNCAGAGG . clone2C-12 CCTCGTG ...... CCAGC. GGCTTCGGCT GTTGAGTGCC CGCCAGAGG . isolateBl-4 CCTCGTG ...... CCAGC. GGCTTCGGCT GTTGAGTGCC CGCCAGAGG . isolatell-2 CCTCGTG ...... CCAGC. GGNTTCGGCT GTTGAGTGCC CGCCAGAGG. isolate14-1 CCTCGTG ...... CCAGC. GGCTTCGGCT GTTGAGTGCC CGCCAGAGG. isolate9-1 CCTCGCG ...... GCAGC. GGCTTCGCCT GTCGAGTGCC CGNCAGAGG. isolate3-B CCTCGTG ...... GNAGC. GGCTTCGGCT GTTGAGGGNC TGCCAGCGG . clone3C-4 CCCCGGGTGC GTCGCAGCCC CGGAACCA .. GGC ....GCC CGCCGGAGGG clone3C-15 CCCCGGGNGC GNCGCAGCCC CGGAACCA .. GGC ....GCC CGCCGGAGGG clone3C-1O CCCCGGGTGC GTCGCAGCCC CGGAACCA .. GGc .... GCC CGCCGGAGGG clone14C-l CCGTCGGAGG GT ....GTCA CACACCCTCT GGCCCGCGCC CGCCGGTGG. clone14C-6 CCGTCGGAGG GT ....GTCA CACACCCTCT GGCCCGCGCC CGCCGGTGG. clone3C-l ...... · .TTTCGG ...... c CGGCAGAAGT clone3C-12 ...... · . TTTCGG ...... c CGGCAGAAGT clone3C-6 ...... · .TTTCGG ...... c CGGCAGAAGT isolate5-2 CCGTA ...... CGTAA TTTTGGGACT TTAAAATGGT TCGCAAGGGC isolate2-1 CCGTA ...... CGTAA TTTTGGGACT TTAAAATGGT TCGCAAGGGC isolatel-l CCGTA ...... CGTAA TTTTGGGACT TTAAAATGGT TCGCAAGGGC isolate7-1 CCGTA ...... NGTAA TTTTGGGACT TTAAAATGGT TCGCAAGGGC clone2C-31 ...... GTA. · ...... T TTAAAA .... .CCCCA .... clone2C-35 ...... GTA. · ...... T TTAAAA .... .CCCCA .... clone12C-6 c .. T ...... · ....TGTA. · ...... T TTAAAA .... .CCCA ..... clone3C-14 AATT ...... TGTA. · ...... T TTAAAA .... .CCCAA .... clone13C-3 c .. T ...... TATA ...... T TTAAAA .... .CCCA ..... clone12C-41 c .. T ...... TATA ...... T TTAAAA .... .CCCA ..... clone17C-l ACCAC ..AA. CTCTT ...... G.... TT T.TTAGTGAT clone17C-3 ACTAATCAA. CTCTT ...... GTTAT TTT.ACGGAA clone17C-4 ACCCC.AAC. CTCTG ...... A....AT .GTTATTGTC isolate23-5 .CCAACCA .. · .CT .....A AA .. CCCT .. .GAATGCAAC CGTGTCGTGT clone2C-4 CCCGA .. AA. CTCCA ...... TT G.TCTCTGAC clone2C-23 .CCAACCA .. · .CTT ....A AA .. CCCT .. .GAAT.CAAC CGTGTCGTGT clone2C-2 . GCAACCA .. · .CTT .... T AA .. CCCT .. .GAAT.CAAC CCTGTCNTGT clone2C-13 ACCCA.AAA. CCCTG ...... A....AT .ATTAGTGTC clone18C-l ACCCA.AAA. CCCTG ...... A....AT TATTAGTGTC clonelBC-5 ACCCA.AAA. CCCTG ...... A....AT .ATTAGTGTC clonelBC-4 ACCCA.AAA. CCCTG ...... A....AT .AT.AGTGTC clone12C-5 ACCCC.AAA. TTCTG ...... A....AT TAT.AGTGTC clone14C-21 ACCCC.AAA. TTCTG ...... A....AT TAT.AGTGTC clone13C-l ACCCC.AAA. TTCTG ...... A....AT TAT.AGTGTC clone2C-1O ACCCC .. AA. ACTCT ...... G....AA T.ACAGTGTC isolatell-3b ACCCC.AAA. TTCTG ...... A....AT TAT.AGTGTC isolateBl-3 ACCCC.AAA. TTCTG ...... A.... AT TAT.AGTGTC clone17C-5 ACCCCCAAA. CTCTG ...... A....AT TAT.AGTGTC clone2C-24 ACCCC.AAA. CTCTG ...... A....AT TAT.AGTGTC isolate2-7 ACCGA.AAA. CTCTG ...... A....AT . .TAAATGTC clone2C-B ACCCC .. AA. CTCTT ...... G.... TT T.TTAGTGAT clone12C-9 ACCAC .. AA. CTCTC ...... G.... TT T.TTAGTGAT clone17C-6 ACCAC .. AA. CTCTT ...... G.... TT T.TTAATGAT clone2C-20 ACCCC .. AA. CTCTT ...... G.... TT T.TTAGTGAT clone13C-17 ACCAC .. AA. CTCTT ...... G.... TT T.TTAGTGAT ------

116

isolate15-1 ACCAN .. AA. NTCTN ...... G.... TT N.TTAGTGAT clone2C-12 ACCCC .. AA. CTCTT ..... ·...... · .. G.... TT T.TTAGTGAT isolateBl-4 ACCCC ..AA. CTCTT ..... · ...... · .. G....TT T.TTAGTGAT isolatell-2 ACCAC .. AA. CTCTT ..... · ...... · .. G.... TT T.TTAGTGAT isolate14-1 ACCAC .. AA. CTCTT ...... G.... TT T.TTAGTGAT isolate9-1 ACCAC .. AA. NTCNT ..... · ...... G.... GT T.TTAGTGAT isolate3-8 ACCAC .. AA. NTCTG ...... G.... TT T.TTAGTNAN clone3C-4 ACCAACCAAA CTCTTTCTGT AGTCCTCTCG CGGACGTTAT TTTTACAGC. clone3C-15 ACCAACCAAA CTCTTTCTGT AGTCCCCTCG CGGACGCTAT TTTTACAGC. clone3C-lO ACCAACCAAA CTCTTTCTGT AGTCCCCTCG CGGACGTTAT TTTTACAGC. clone14C-l .CCCTACAA. .ACTA ....A AA .. CTCT .. .TGTTAAAAT CGTGTCGTCT clone14C-6 .CCCTACAA . . ACTA ....A AA .. CTCT .. .TGTTAAAAT CGTGTCGTCT clone3C-l TTTCT.CAAA CTCAT ...... T AGAAATTTGT clone3C-12 TTTCT.CAAA CTCAT ..... · ...... · ...... T AGAAATTTGT clone3C-6 TTTCT.CAAA CTCAT ..... ·...... · ...... T AGAAATTTGT isolateS-2 CGGTCCCAAA AACAA .. TAT ATCATCCTT. ...ATGAAAT TTTTTCTGAA isolate2-1 CGGTCCCAAA AACAA .. TAT ATCATCCTT. ... ATGAAAT TTTTTCTGAA isolatel-l CGGTCCCAAA AACAA .. TAT ATCATCCTT. ...ATGAAAT TTTTTCTGAA isolate7-1 CGGTCCCAAA AACAA .. TAT ATCATCCTT. ...ATGAAAT TTTTTCTGAA clone2C-31 CACTTTTT ...... TGA AA ...... ATAACAT GTATTTT ... clone2C-3S CACTTTTT ...... TGA AA ...... ATAACAT GTATTTT ... clone12C-6 CACTTTGAAT GTAAA .. TGT AT ...... ATAATAT TTTGTAT ... clone3C-14 CTCTTTAAAT ...... GT AT ...... ATTATAT TAT. TGT ... clone13C-3 CTCTTTAAAT ...... TGT AT ...... ATAA.AT ATT ...... clone12C-41 CTCTTTAAAT ...... TGT AT ...... ATAA.AT ATT ......

clone17C-l GT.CTGAGTA C.T ..AT ... . ATAAT .... .AGTTAAAA . TTTCAACAAC clone17C-3 AT.CTGAGCG T.CTTATT .. · .AAATA ... .AGTCAAAAC TTTCANCAAC clone17C-4 GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC isolate23-S GTACTCAGTC C.ATGANT .. .AAATTA ... .AAGCAAAAC TTTCANNTAC clone2C-4 GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC clone2C-23 GT.CTCAGTC C.ATGATT .. .AAATTA ... .AATTAAAAC TTTCAACAAC clone2C-2 TT.CTCAGTC C.NTGAAT .. .AAATTA ... .AATTAAAAC TTTCAACAAC clone2C-13 GT.CTGAGTA A.A .. AT ... TTTAAT .... .ATTTAAAAC TTTCAACAAC clone18C-l GT.CTGAGTA A.A .. ATA .. TTTAAT .... .ATTTAAAAC TTTCAACAAC clone18C-S GT.CTGAGTA A.A .. AT ... TTTAAT .... .ATTTAAAAC TTTCAACAAC clone18C-4 GT.CTGAGTA A.A .. AT ... TTTAAT .... .ATTTAAAAC TTTCAACAAC clone12C-5 GT.CTGAGAA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC clone14C-21 GT.CTGANAA C.T ..AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC clone13C-l GT.CTGANAA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC clone2C-lO GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC isolatell-3b GT.CTGAGAA C.T ..AT ... .ATAAT .... .AGTTAAAAC TTTCAANAAC isolateBl-3 GT.CTGAGAA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC clone17C-5 GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC clone2C-24 GT.CTGAGTA C.T ..AT ... .AAAAT .... .AGTTAAAAC TTTCAACAAC isolate2-7 GT.CCGAGTA C.T .. AT ... .GTAAT .... .AGTTAAAAC TTTCAACAAC clone2C-8 GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC clone12C-9 GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC clone17C-6 GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC clone2C-20 GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC clone13C-17 GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC isolate15-1 GT.CTGAGTA C.T .. AT ... .NNNNN .... .NNNNNNNN . ... . NNNNNN clone2C-12 GT.CTGAGTA C.T ..AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC isolateBl-4 GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC isolatell-2 GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTCCAACAAC isolate14-1 GT.CTGAGTA C.T .. AT ... .ATAAT .... .AGTTAAAAC TTTCAACAAC ~------

117

isolate9-1 GT.CTGANTA C.N .. NN ... . NNNNN .... .NNNNNNNN . .... NNNNNN isolate3-8 GT.CTGAGTA C.T ..AT ... . ATAAT .... .TGTTAAAA . . ... CNTTNN clone3C-4 . T.CTGAGCA A.AA.ATT .. CAAAATG ... .AATCAAAAC TTTCAACAAC clone3C-15 .T.CTGAGCA A.AA.ATT .. CAAAATG ... .AATCAAAAC TTTCAACAAC clone3C-lO .T.CTGAGCA A.AA.ATT .. CAAAATG ... .AATCAAAAC TTTCAACAAC clone14C-l GA ....AGTA A.TCAAAC .. .AAATAA ... .AATAAAAAC TTTCAACAAC clone14C-6 GA ....AGTA A.TCAAAC .. .AAATAA ... .AATAAAAAC TTTCAACAAC clone3C-l CTTCTGAACT C.CAAAAA .. .ATAAT .... .AATTAAAAC TTTCAACAAC clone3C-12 CTTCTGAACT C.CAAAAA .. .ATAAT .... .AATTAAAAC TTTCAACAAC clone3C-6 CTTCTGAACT C.CAAAAA .. .ATAAT .... .AATTAAAAC TTTCAACAAC isolate5-2 CAATTAAACA AAATGATTTT AATAATCTGT TTAAAACAAC TTTCAACAAC isolate2-1 CAATTAAACA AAATGATTTT AATAATCTGT TTAAAACAAC TTNCAACAAC isolatel-l CAATTAAACA AAATGATTTT AATAATCTGT TTAAAACAAC TTTCAACAAC isolate7-1 CAATTAAACA AAATGATTTT AATAATCTGT TTAAAACAAC TTTCAACAAC clone2C-31 . ACTTAAA .. · .ATAAGT .. AATTAA .... · .AAGATCAC TTTCAACAAC clone2C-35 . ACTTAAA .. · .ATAAGT .. AATTAA .... · .AAGATCAC TTTCAACAAC clone12C-6 . TTTTTTA .. · .ATAATA .. AAAA ...... · .AAGATCAC TTTCAACAAC clone3C-14 . ATTTTAA .. · .ATAATA .. AAAA ...... GATCAC TTTCAACAAC clone13C-3 ... TTTAA .. · .ATGATA .. AAAATT .... · .ANGATNAC TTTCAACAAN clone12C-41 ... TTTAA .. · .ATGATA .. AAAATT .... · .AAGATCAC TTTCAACAAC

clone17C-l GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone17C-3 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone17C-4 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT isolate23-5 GGAT ...... ·...... ·...... ·...... clone2C-4 GGATCTCTTG GTTCTGGCAT CGATNAAAAA CGCAGCGAAA TGCGATAAGT clone2C-23 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone2C-2 NGATCTCTTG GTTCT ...... ·...... clone2C-13 GGATCTCTTG GCTCTGGCAT CGATGAGGAA CGCAGCGAAA TGCGATAAGT clone18C-l GGATCTCTTG GCTCTGGCAT CGATGAANAA CGCAGCGAAA TGCGATAAGT clone18C-5 GGATCTCTTG GCTCTGGCAT CGATGAGGAA CGCAGCGAAA TGCGATAAGT clone18C-4 GGATCTCTTG GCTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone12C-5 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone14C-21 GGATCTCTTG GTTCTGGCAT CGATNAAAAA CGCAGCGAAA TGCGATAAGN clone13C-l GGATCTCTTG GTTCTGGCAT CGATGAANAA CGCAGCGAAA TGCGATAAGT clone2C-lO GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT isolatell-3b GGATCTCTTG · ...... · ...... ·...... isolateBl-3 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone17C-5 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone2C-24 GGATCCCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT isolate2-7 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone2C-8 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone12C-9 GGATCTCTTG GTTCTGGCAT CGATGAAAAA CGCAGCGAAA TGCGATAAGT clone17C-6 GGATCTCTTG GTTCTGGCAT CGATGAANAA CNCNCCGAAA TGCGATAAGT clone2C-20 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone13C-17 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT isolate15-1 NNNN ...... ·...... clone2C-12 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT isolateBl-4 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT isolatell-2 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGNGAAA TGCGATAAGT isolate14-1 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT isolate9-1 NNN ...... ·...... ·...... isolate3-8 NNN ...... ·...... · ...... ·...... clone3C-4 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone3C-15 GGATCTCTTG GTTCTGGCAT CGATNAAAAA CGCAGCGAAA TGCGATAAGT clone3C-lO GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT ------

118

clone14C-l GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone14C-6 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone3C-l GGATCTCTTG GTTCTGGCAT CGATGAANAA CGCAGCGAAA TGCGATAAGT clone3C-12 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT clone3C-6 GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT isolate5-2 GGATCTCTTG GTTCTCGCAT CGATGAAGAA CGCAGCGAAA TGCGATACGT isolate2-1 GGATCTCTTG GTTCTCGCAT CGATGAAGAA CGCAGCGAAA TGCGATACGT isolatel-l GGATCTCTTG GTTCTCGCAT CGATGAAGAA CGCAGCGAAA TGCGATACGT isolate7-1 GGATCTCTTG GTTCTCGCAT CGATGAAGAA CGCAGCGAAA TGCGATACGT clone2C-31 GGATCTCTTG GTTCTCGCAT CGATGAAGAA CGTAGCGAAG TGCGATAAGT clone2C-35 GGATCTCTTG GTTCTCGCAT CGATGAAGAA CGTAGCGAAG TGCGATAAGT clone12C-6 GGATCTCTTG GCTCTCGCAT CGATGAANAA CGTAGCGAAG TGCGATAAGT clone3C-14 GGATCTCTTG GCTCTCGCAT CGATGAAGAA CGTAGCGAAG TGCGATAAGT clone13C-3 GGATCTCTTG GCTCTCGCAT CGATGAAGAA CNTANCGAAG TGCGATAAGT clone12C-41 GGATCTCTTG GCTCTCGCAT CGATGAAGAA CGTAGTGAAG TGCGATAAGT

clone17C-l AATGTGAATT GCANAATTCA NTGAATCATC GAATCTTTGA ACGCACATTG clone17C-3 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone17C-4 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG isolate23-5 · ...... · ...... · ...... ·...... ·...... clone2C-4 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone2C-23 AATGCGAATT GCAGAATTCA GTGAGTCATC GAATCTTTGA ACGCACATTG clone2C-2 ...... ·...... ·...... ·...... clone2C-13 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone18C-l AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone18C-5 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone18C-4 AATGTGAATC GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone12C-5 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone14C-21 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone13C-l AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone2C-1O AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG isolatell-3b · ...... · ...... · ...... ·...... ·...... isolateBl-3 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone17C-5 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone2C-24 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG isolate2-7 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone2C-8 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone12C-9 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone17C-6 AATGTGAATT GCANAATTCA GCGAATCATC NAATCTTTGA ACGCNCNTTG clone2C-20 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone13C-17 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG isolate15-1 ...... clone2C-12 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG isolateBl-4 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG isolatell-2 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG isolate14-1 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG isolate9-1 · ...... · ...... ·...... isolate3-8 ...... · ...... clone3C-4 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone3C-15 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone3C-1O AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone14C-l AATGCGAATT GCAGAA.TCC GTGAGTCATC GAATCTTTGA ACGCACATTG clone14C-6 AATGCGAATT GCAGAA.TCC GTGAGTCATC GAATCTTTGA ACGCACATTG clone3C-l AATGTGAATT GCAGAATTCA GTGAATCATC GAATTTTTGA ACGCATATTG clone3C-12 AATGTGAATT GCAGAATTCA GTGAATCATC GAATTTTTGA ACGCATATTG clone3C-6 AATGTGAATT GCAGAATTCA GTGAATCATC GAATTTTTGA ACGCATATTG 119 isolate5-2 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG isolate2-1 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG isolatel-l AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG isolate7-1 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG clone2C-31 AGTGTGAATT GCAGAATTCC GTGGATCATC GAATTTTTGA ACGCAAATTG clone2C-35 AGTGTGAATT GCAGAATTCC GTGGACCATC GAATTTTTGA ACGCAAATTG clone12C-6 AATGTGAATT GCAGAATTCC GTGAATCATC GAATCTTTGA ACGCAAATTG clone3C-14 AATGTGAATT GCAGAATTCC GTGAATCATC GAATTTTTGA ACGCAAATTG clone13C-3 AATGTGAATT GCAGTATTCC GTGAATCATC NAATCTTTGA ACGCAAATTG clone12C-41 AATGTGAATT GCAGTATTCC GTGAATCATC GAATCTTTGA ACGCAAATTG

clone17C-l CGCCCTCTGG TATTCGCGGG GGCATGCCTG TTCGANCGTC .TTATTACCA clone17C-3 CGCCCATTAG TATTCTATTG GGCATGCCTG TTCGAGCGTC ATT.TCAACC clone17C-4 CGCCCCTTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTACAACCC isolate23-5 ...... clone2C-4 CGCCCCCTGG TATTCCGGGG GGCATGCCTG TTCNAACGTC ATTATNACCA clone2C-23 CGCCCTTTGG TATTCCGAAG GGCATGCCTG TTCGAGCGTC ATTATCACCC clone2C-2 ...... clone2C-13 CGCCCCTTGG TATTCCGGGG GGCATGCCTG TTCGANCGTC ATTATAACCC clone18C-l CGCCCCTTGG TATTCCGAGG GGCATGCCTA TTCGAGCGTC ATTATCACCC clone18C-5 CGCCCCTTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTATAACCC clone18C-4 CGCCCCTTGG TATTCCGAGG GGCATGCCTA TTCGAGCGTC ATTATCACCC clone12C-5 CGCCCCTTGG TAT.CCGGGG GGCATGCCTG TTCAANCGTC ATTATAACCC clone14C-21 CGCCCCTTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTATAACCC clone13C-l CGCCCCTTGG TATTCCGGGG GGCATGCCTG TTCGANCGTC ATTATAACCC clone2C-1O CGCCCCTTGG TATTCCGGGG GGCATGCCTC TTCCAGCGTC ATT.TCACCC isolatell-3b ...... isolateBl-3 CGCCCCTTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTATAACCC clone17C-5 CGCCCCTTGG TATTCCGAGG GGCATGCCTG TTCGAGCGTC ATTATAACCC clone2C-24 CGCCCCTTGG TATTCCGAGG GGCATGCCTG TTCGAGCGTC ATTATAACCC isolate2-7 NGCCCTCTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTACAACCC clone2C-8 CGCCCTCTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTATAACCA clone12C-9 CGCCCTCTGG TATTCCGGGG GGCATGCCTG TTCGANCGTC ATTATAACCA clone17C-6 CGCCCTCTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTATAACCA clone2C-20 CGCCCTCTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTATAACCA clone13C-17 CGCCCTCTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTATAACCA isolate15-1 ...... clone2C-12 CGCCCCCTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTATGACCA isolateBl-4 CGCCCTCTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTATAACCA isolatell-2 CGCCCTCTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTATAACCA isolate14-1 CGNCCTCTGG TATTCCGGGG GGCATGCCTG TTCGAGCGTC ATTATAACCA isolate9-1 ...... isolate3-8 ...... clone3C-4 CGCCCGCCAG TATTCTGGCG GGCATGCCTG TCCGAGCGTC ATT.TCAACC clone3C-15 CGCCCGCCAG TATTCTGGCG GGCATGCCTG TCCGANCGTC ATT.TCAACC clone3C-1O CGCCCGCCAG TATTCTGGCG GGCATGCCTG TCCGAGCGTC ATT.TCAACC clone14C-l CGCCCTTTGG TATTCCGAAG GGCATGCCTG TTCGAGCGTC ATTATCACCC clone14C-6 CGCCCTTTGG TATTCCGAAG GGCATGCCTG TTCGAGCGTC ATTATCACCC clone3C-l CGCCCTTTGG CATTCCGAAG GGCATACCTG TTCGAGCGTC ATTA.CACCC clone3C-12 CGCCCTTTGG CATTCCGAAG GGCATACCTG TTCGAGCGTC ATTA.CACCC clone3C-6 CGCCCTTTGG CATTCCGAAG GGCATACCTG TTCGAGCGTC ATTA.CACCC isolate5-2 NACTCCTTGG TATTCCGAGG AGTATGCCTG TTTCAGTATC ATGAGCACTC isolate2-1 NACTCCTTGG TATTCCGAGG AGTATGCCTG TTTCAGTATC ATGAGCACTC isolatel-l NACTCCTTGG TATTCCGAGG AGTATGCCTG TTTCAGTATC ATGAGCACTC isolate7-1 CACTCCTTGG TATTCCGAGG AGNATGCCTG TTTCAGTATC ATGAGCACTC clone2C-31 CACTCTCTGG TATTCCGGAG AGTATGCCTG TTTGAGGGTC A.T.CAAATA 120 clone2C-3S CACTCTCTGG TATTCCGGAG AGTATGCCTG TTTGAGGGTC A.T.CAAATA clone12C-6 CACTCTCTGG TATTCCGGAA AGTATGCCTG TTTGAGGGTC AGT.TAAATA clone3C-14 CACTCTCTGG TACTCCGGAN AGTATGCCTG TTTGAGGGTC AGT.AAAATA clone13C-3 CACTTTCTGG TATTCCGGAA AGTATGCCNG TTTGAGGGTC ANT.CNAATA clone12C-41 CACTTTCTGG TATTCCGGAA AGTATGCCTG TTTGAGGGTC AGT.CAAATA clone17C-l CTCAAGCTCT CG ...... C TTGGTATTGG GGTT ...... clone17C-3 CTTAAGCTCA G...... c TTAGTGTTGG GAATCTACCG TGAGCGGTGC clone17C-4 . TCAAGCTCT G...... c TTGGTATTGG GTATC ..... · ...... isolate23-S ...... · ...... oOoO •••••••• ·...... oOoOoOoOoOoO •• oO • clone2C-4 ATCCCGCAAG G...... · .GGTCTTGG GGTCT ..... oOoO .. oOoO.oOoOoO • clone2C-23 CTCGGGCCCC ...... GTGC TCGGTGTT .. GGACG ..... oOoO •• oOoOoO.oO • clone2C-2 ...... ·...... ·...... ·...... · ...... clone2C-13 . TCAAGCCTA A...... C TTGGTGTTGG AG ...... ·...... clone18C-l CTCAAGCCTA G...... C TTGGTGTTGA NA ...... · ...... clone18C-S . TCAAGCCTA G...... C TTGGTGTTGG AG ...... oOoOoO •• oOoO ••• clone18C-4 CTCAAGCTTC GG ...... C TTGGTGTTGA NG ...... clone12C-S . TCAANCCTA C...... C TTGGTGTTGG AA ...... oO ...... oOoO • clone14C-21 . TCAAGCCTA N...... C TTGGTGTTGG AG ...... clone13C-l . TCAAGCCTA G...... C TTGGTGTTGG AG ...... · ...... clone2C-lO CTCAAGCTCT G...... C TTGGTTTTGG GC ...... isolatell-3b oO .. oO ...... oO • ...... ·...... oO ..... isolateBl-3 . TCAAGCCTA G...... C TTGGTGTTGG AG ...... oOoO ...... oOoO .. clone17C-S CTCAAGCTTA G...... C TTGGTATTGG GG ...... ·...... clone2C-24 CTCAAGCTCG G...... C TTGGTGTTGG GG ...... oOoO .. isolate2-7 . TCAAGCTCT G...... C TTGGTATTGG GCTAC ...... oOoO ...... clone2C-8 CTCAAGCTCT CG ...... C TTGGTATTGG GGTT ...... clone12C-9 CTCAAGCTCT CG ...... C TTGGTATTGG GGTT ...... oOoO ... clone17C-6 CTCAANCTCT CG ...... C TTGGTATTGG GGTT ...... oO ...... clone2C-20 CTCAAGCTCT CG ...... C TTGGTATTGG GGTT ...... oOoOoO ...... clone13C-17 CTCAAGCTCT TG ...... C TTGGTATTGG GGTT ...... ·...... isolatelS-l ...... oO ...... clone2C-12 ATCCCGCAAG G...... · .GGTCTTGG GGTCT ..... · ...... isolateBl-4 CTCAAGCTCT CG ...... C TTGGTATTGG GGTT ...... isolatell-2 CTCAAGCTCT CG ...... C TTGGTATTGG GGTT ...... oO ...... isolate14-1 CTCAAGCTCT CG ...... C TTGGTATTGG GGTT ...... oOoO ...... oO .... isolate9-1 ...... · ...... ·...... isolate3-8 ...... clone3C-4 CTCGAACCCC TCCGGGGGGT .CGGCGTTGG GGACT ..... · ...... clone3C-1S CTCGAACCCC TCCGGGGGGN TCTGCGTTNG GGACT ...... clone3C-lO CTCGAACCCC TCCGGGGGGT .CGGCGTTGG GGACT ...... clone14C-l CTCAAGCTCC ...... G.GC TTGGTGTT .. GGGCT ...... clone14C-6 CTCAAGCTCC ...... G.GC TTGGTGTT .. GGGCT ...... clone3C-l CTCAAGCTTA GG ...... T TTGATGTTGG GCA ...... clone3C-12 CTCAAGCTTA GG ...... T TTGATGTTGG GCA ...... clone3C-6 CTCAAGCTTA GG ...... T TTGATGTTGG GCA ...... isolateS-2 . TCACACCTA AC ...... C TTNGGNTTTA TGG ...... isolate2-1 . TCACANCTA AC ...... C TTNGGGTTTA TGG ...... isolatel-l . TCACACCTA AC ...... C TTNGGGTTTA TGG ...... · ...... isolate7-1 . TCACACCTA AC ...... C TTNGGGTTTA TGG ...... ·...... clone2C-31 AAAAATCGAT ...... TTGTTACT .. · . T ...... clone2C-3S AAAAATCGAT ...... TTGTTACT .. · .T ...... clone12C-6 AAAA .. CGGT G...... TTGTTGCTGC CTT ...... clone3C-14 AAAA.TCGAT G...... TTGTTGTCAC C...... clone13C-3 AAAA.TCGGT G...... TTGTNANTAC CCTTAN ...... clone12C-41 AAAAATCGGT G...... TTGTTATTAC CCTTAG ...... 121 clone17C-l · .CGCGG ...... TT. TCN CGGc ...... TC.CTA clone17C-3 TACCTGGTAC CTACCTGTAA CGGGCGTAAG CTACCTGTAG CTACCCTGTA clone17C-4 .ACCTT ...... TGG TGGT.G ...... TGCCCCA isolate23-5 ...... clone2C-4 · .CGCGGT .. · ...... TCG CTGC ...... G...... GCCCTTA clone2C-23 · . GCCGGTTC G.. GGTGAC. CGAAC ...... CCCTCCT .. A clone2C-2 ...... clone2C-13 · .CCTG ...... CC.TCT GGGc ...... · .AGCTCTTA clone18C-l · .CCTG ...... CTGTCA AGGC ...... · .AGTCTCTA clone18C-5 · .CCTG .... · ...cc. TCT GGGC ...... · .AGCTCTTA clone18C-4 · .CCTG ...... CTGTAA AGGC ...... · . ACCCTCTA clone12C-5 · .CCTG ...... CC.TCT GGGc ...... · .ANCTCTTA clone14C-21 · . CCTG ...... CC.TCT GGGc ...... · .AGCTCTTA clone13C-l · .CCTG ...... cc. TCT GGGc ...... · .AGCTCTTA clone2C-1O · .CGCG ...... CCAGCAA CGGCGG ...... GCCTCGA isolatell-3b ...... isolateBl-3 · .CCTG ...... CC.TCT GGGc ...... · .AGCTCTTA clone17C-5 · . CCTG ...... CCATCC TGGC ...... · .AGCCCTTA clone2C-24 · .CCTG ...... CCGCAC GGGc ...... · .AGCCCTTA isolate2-7 .ACCCG ...... ACT GGGT.G ...... GGACTTA clone2C-8 · .CGCGG ...... TT.TCG CGGc ...... CC.CTA clone12C-9 · .CGCGG ... · ...TT. TCG CGGc ...... TC.CTA clone17C-6 · .CNCGG ...... TT.TCG CGGc ...... TC.CTA clone2C-20 · .CGCGG ...... TT.TCG CGGc ...... CC.CTA clone13C-17 .. CGCGG ...... TT.TCG CGGc ...... TN.CTA isolate15-1 ...... clone2C-12 · .CGCGGT ...... TCG CTGC ...... G...... GCC. TTA isolateBl-4 · .CGCGG ...... TT.TCG CGGC ...... TC.CTA isolatell-2 · .CGCGG ...... TT.TCG CGGC ...... TC.CTA isolate14-1 · .CGCGG ...... KT.TCG CGGc ...... TC.CTA isolate9-1 ...... isolate3-8 ...... clone3C-4 .. TCGGGAAC c .. CCTAAGA CGGGAT ...... c ... CCGGCCCCGA clone3C-15 ..TCCGGAAC c ..CCTAAGA CGGAAT .... · .....c ... CCGGCCCCGA clone3C-1O .. TCGGGAAC c .. CCTAAGA CGGGAT ...... c ... CCGGCCCCGA clone14C-l .. CCAGGTGG T .. TTTACC . CAAACT ...... G... CCGGCCTC.A clone14C-6 .. CCAGGTGG T .. TTTACC. CAAACT ...... G... CCGGCCTC.A clone3C-l .. . CTG ...... CTGTAA GGGCAT ...... GCC .. TA clone3C-12 ...CTG ...... CTGTAA GGGCAT ...... GCC .. TA clone3C-6 ...CTG ...... CTGTAA GGGCAT .... · ...... GCC .. TA isolate5-2 · .NGTGGGAA · ...... NT GGGAAT .... ·...... GNGCCGA isolate2-1 · .NGTGGGA. · ...... TT GGGAAT .... · ...... GCGCCGA isolatel-l · .NGTNGGA...... T TGGNAT .... · ...... GNGNCGA isolate7-1 · .CGTGGGA...... NT GGGAAT ...... GCGCCGA clone2C-31 ....TGGTAA ... . C.ATTT CGGAATTGGG TCATCTTAA. ...ACCTTT . clone2C-35 .... TGGTAA ....C.ATTT CGGAATTGGG TCATCTTAA. ...ACCTTT . clone12C-6 . GCGTGGTGA ....C.GCAT CGGATTTGGG TCGTCTT ...... ACCTTT . clone3C-14 ...GTGATAA .... C.GCGT CGGAATTGAG TCGTCTT ...... ACCTTC. clone13C-3 .TGGTGGTGA .... C.GCNT CNGAGTTGAT TCGTCTTT .. ...ACCTNTC clone12C-41 .TGGTGGTGA ....C.GCAT CGGAGTTGAG TCGTCTTT .. ...ACCTTTC

clone17C-l ...... AA ATC ..AGTGG .NGTG .. CCT ....ATC.GG c .... T .CTA clone17C-3 GTTCCTCAAA TTC .. AACGG CGGGG .. TTA TA ..GTCAT. c .... T. CTG clone17C-4 ...... AA ATC .. AG.GG CGGTG ..CC. ....ATCTGG c .... T .CTA isolate23-5 ...... ------

122

clone2C-4 •••••••• AA ACC •• AGTGG CG.CG •• CCA •••• GCGGTG C •••• T .CTC clone2C-23 • ••••••• AA GAC •• AATGA CGGCGGCCT. GT •• GGTTCC CCCGGTACAC clone2C-2 ·...... ·...... · ...... clone2C-13 •••••••• AA ATC •• AGTGG CGGTG •• CC • ••• • GTCTGG c.... T.CTA clone18C-l •••••••• AA ATC •• AGTGG CAGTG •• CT • ••• • GTCAGG c .... T.CTA clone18C-5 •••••••• AA ATC •• AGTGG CGGTG •• CC • ••• • GTCTGG C •••• T .CTA clone18C-4 •••••••• AA ATC •• AGTGG CAGTG •• CT • ••• • GTCAGG C •••• T.CTA clone12C-5 • ••••••• AA ATC •• ANTGG CGGTG •• CC. •••• GTCTGG c ....T .CTA clone14C-21 •••••••• AA ATC •• AGTGG CGGTG •• CC • ••• • GTCTGG c.... T.CTA clone13C-l • •••••••AA ATe •• AGTGG CGGTG •• CC • ••• • GTCTGG C •••• T.CTA clone2C-1O •••••••• AA AT ••• AGTGG CGACG •• CCA ••••• TCGTG C •••• T .CTC isolatell-3b ...... · ...... isolateBl-3 •••••••• AA ATC •• AGTGG CGGTG •• CC. • ••• GTCTGG C •••• T .CTA clone17C-5 • •••••••AA ATC •• AGTGG CGGTG •• CC. •••• ATCCGG C •••• T.CTA clone2C-24 •••••••• AA ATC •• AGTGG CGGTG •• CC. • ••• ATCTGG C •••• T .CTA isolate2-7 •••••••• AA ATC •• AGTGG CGGTG •• CC. •••• ATCTGG C •••• T.CTA clone2C-8 •••••••• AA ATC •• AGTGG CGGTG •• CCT •••• GTC.GG ••••• T.CTA clone12C-9 •••••••• AA ATC •• AGTGG CGGTG •• CCT •••• ATC.GG C •••• T.CTA clone17C-6 •••••••• AA ATC •• ANTGG CGGTG •• CCT •••• ATC.GG C •••• T.CTA clone2C-20 •••••••• AA ATC •• AGTGG CGGTG •• CCT •••• GTC.GG C •••• T.CTA clone13C-17 • ••••••• AA ATC •• AGTGG CGGTG •• CCT • ••• ATC.GG C •••• T.CTA isolate15-1 ...... · ...... · ...... ·...... clone2C-12 •••••••• AA ACC •• AGTGG CGGCG •• CCA •• • • GCGGTG C •••• T .CTC isolateBl-4 •••••••• AA ATC •• AGTGG CGGTG •• CCT •••• GTC.GG C •••• T.CTA isolatell-2 •••••••• AA ATC •• AGTGG CGGTG •• CCT •••• ATC.GG C •••• T.CTA isolate14-1 •••••••• AA ATC •• AGTGG CGGTG •• CCT •••• ATC.GG N •••• T.CTA isolate9-1 · ...... · ...... ·...... ·...... ·...... isolate3-8 ·...... · ...... · ...... · ...... clone3C-4 •••••••• AA TAC •• AGTGG CGG ••• TCTC GC •• CGCAGC C •••• TCTTA clone3C-15 •••••••• AA TAC •• AGTGG CGGG •• TCTC GC •• CGCAAC C •••• TCTCA clone3C-1O •••••••• AA TAC •• AGTGG CGG ••• TCTC GC •• CGCAGC C •••• TCTCA clone14C-l • •••••••AA GAT •• AGTGA CGGCGTCCCA GA •• GGGAAC C •• GGT.CNA clone14C-6 •••••••• AA GAT •• AGTGA CGGCGTCCCA GA •• GGGAAC C •• GGT .CNA clone3C-l ••••••• AAA ATT •• AGCGA TGGTA.ACC • ••• • GATAAC C ••••••ACA clone3C-12 ••••••• AAA ATT •• AGCGA TGGTA.ACC. •••• GATAAC C •••••• ACA clone3C-6 ••••••• AAA ATT •• AGCGA TGGTA.ACC. •••• GATAAC C •••••• ACA isolate5-2 C ••••••• TT GTC •• ATGGG TGGN ••• CC. •••• TTC ••• ••••• T •• TA isolate2-1 C ••••••• TT GTC •• ATGGG TGGG ••• CC. •••• TTC ••• • •••• T •• TA isolatel-l · ••••••• NT NTC •• ATGGG TGG •••• CC. •••• TTC .•• • •••• T •• NA isolate7-1 C ••••••• TT GTC •• ATGGG TGGG ••• CC • ••• • NNC ••• ••••• T •• AA clone2C-31 •••••••• AG GTTCAAGAGG CTTAAAATTG ATCGTTTTTG GTATGACTTA clone2C-35 •••••••• AG GTTCAAGAGG CTTAAAATTG ATCGTTTTTG GTATGACTTA clone12C-6 ••••••••• G GTT.AAGTGA CCTAAAA.T. •••• TTTGCA TGATTTTTCG clone3C-14 ••••••••• G GTT.AAGTGA CTTAAAATT • •••• TTTACG CGAATTCG.A clone13C-3 T ••••••• CG GTT.AAGTGA CTTAAAAAT • •••• TTNGTN CGATNTTC.G clone12C-41 T ••••••• TG GTT.AAGTGA CTTAAAAAT. •••• TTTGTA CGATTTTT.G

clone17C-l CGCGTAGTAA •••••• TACT • .CC.CCCGA T. TGA •• GTC CNGTT ••••• clone17C-3 AGCGCANTAA T ••••• TTTA •• TCACGCTT T. TGA •• ANG .TGC •••••• clone17C-4 AGCGTAGTAA •••••• CT.T •• TCTCGCTA T.GGA •• GTC CTCA •••••• isolate23-5 ·...... ·...... ·...... · ...... clone2C-4 ANCGTANTAA ••••• • TACT • .CCTCGCTC G.GGA •• AC. ACG ••••••• clone2C-23 TGAGCCTTTG ••••••• GCA •• CGT.GCCG G.ACG •• AGG GCGCC ••••• clone2C-2 ...... clone2C-13 ANCGTAATAA ••••• • TTTT •• TCTCGCTA C.ANA •• ATC CTGG •••••• clone18C-l AGCGTAGTAA A ••••• TTCA • .TC •• GCTA T .AGA•• CAC CTGG •••••. 123 clone18C-5 AGCGTAGTAA ...... TTTT · . TCTCGCTA C.AGA .. NTC CTGG ...... clone18C-4 AGCGTANTAA T ..... TTCA · .TC .. GCTA T .AGG .. GTC CTGG ...... clone12C-5 ACCNTATTAN ...... TTTT · . NCTCNCTA C.A.A .. ATC CCGG ...... clone14C-21 ANCGTAATAA ...... TTTT .. TCTCGCTA C.ANA ..ATC CCGG ...... clone13C-l ANCGTANTAA ...... TTTT · . TCTCGCTA C.AGA ..ATC CCGG ...... clone2C-1O AGCGTAGTAA ...... TTCT · . TCTCGCTG T.TGG .. GTC CCGG ...... isolatell-3b ...... isolateBl-3 AGCGTAGTAA ...... TTTT ..TCTCGCTA C.AGA ..GTC CTGG ...... clone17C-5 AGCGTAGTAA ...... CTCT .. TCTCGCTA T.GGA .. GTC CTGG ...... clone2C-24 AGCGTAGTAA ...... TTCT .. TCTCGCTA T .AGA .. GTC CCGG ...... isolate2-7 AGTGTAGTAA ...... TTCT ..TCTCGCTC T .GGA .. GAT CTAGG ..... clone2C-8 CGCGTAGTAA ...... TACT · . NCTCGCGA A.TGA .. ATN CCGTA ..... clone12C-9 CGCGTATTAA ...... TACT · .CCTCCCGA T.TGA .. ATC CGGTN ..... clone17C-6 CGCGTNGTAA ...... TACT · .CCTCGCGA T. TGA ..ATC CGGTA ..... clone2C-20 CGCGTAGTAA ...... TACT ..CCTCGCGA A.TGA ..GTC CNGTA ..... clone13C-17 CGCGTAGTAA ...... TACT .. TCTNGCGA A.TGA .. ATC CCGTA ..... isolate15-1 ·...... ·...... ·...... ·...... clone2C-12 A.CGTANTAA ...... TACT · . NCTCGCTT N.GGA ..ACC ACGG ...... isolateBl-4 CGCGTAGTAA ...... TACT · .CCTCGCGA T.TGA .. GTC CGGTA ..... isolatell-2 CGCGTAGTAA ...... TACT .. CCTCGCGA T. TGA .. GTC CGGTA ..... isolate14-1 CGCGTAGTAA ...... TACT · .CCTCGCGA T. TGA .. GTC CGGTA ..... isolate9-1 · ...... · ...... ·...... · ...... · ...... isolate3-8 ·...... · ...... ·...... ·...... · ...... clone3C-4 TGCGCAGTAG T .. TTGCACA · . ACTCGCAC C.GGG .. AGC GCN.G ..... clone3C-15 TGCGCANTAN T .. TTGCACA · . ACTCGCAC C.GGG .. ANC GCN.G ..... clone3C-1O TGCGCAGTAG T .. TTGCACA · . ACTNGCAC C .GGG ..AGC GCN.G ..... clone14C-l GGAGCTTTTA A.. CCGAACA .. TGTCGCNG G.ACG .. ACT NTGCG ..... clone14C-6 GGAGCTTTTA A.. CCGAGCA ..TGTCGCNG G.ACG .. ACT NTGCG ..... clone3C-l GGCGCACAGA ·...... · .TGTCGC .. · ...A..ATG ATGGA ..... clone3C-12 GGCGCACAGA ...... · .TGTCGC .. · ...A..ATG ATGGA ..... clone3C-6 GGCGCACAGA ...... · .TGTCGC ...... A.. ATG ATGGA ..... isolate5-2 AATGTGGN.C C...... TGG · .GT.GGCAA C..TA ..ATA CAGGA ..... isolate2-1 AATGTNGN.C C...... TTG · .GTTGGCAA C.. TA .. ATA CAG.A ..... isolatel-l AATGNGGN.C ...... TTG · .GTTGGCAC C.. TA ..ATA CAGGA ..... isolate7-1 ATTGTNGGTC C...... TGG · .GNTGGCAA C.CTA ..ATA CAGGA ..... clone2C-31 AGCGTATTTA AGATCTCTTA TCACGCGCTT GATGAGTATC GGATGTTTAT clone2C-35 AGCGTATTTA AGATCTCTTA TCACGCGCTT GATGAGTATC GGATGTTTAT clone12C-6 AACGTATTTA AA.TGATTTA .CGTACGTTT G.TGA.TAT. CGA.A .. TAT clone3C-14 AACGTATTTA A..TGTTT.A .TGTACGTTT G.CGA.TATT CCA.ATATAT clone13C-3 AACGTATTTA A..ACTTTTA .CGTACGTTC N.TAA.TATC ATATATCTAT clone12C-41 AACGTATTTA A..ACTTTTA .CGTACGTTC T.TAA.TATC ATATATCTAT clone17C-l .CG.TCNACT T .. GCCCAC . . CCACCC .. T AA.ATTTTTT TN.ANGT.TG clone17C-3 .TA.TAGCCC C.. G.CCGC. TAAACCCC.C AA.ATTTTTA AT .. GGT. TG clone17C-4 .TG.GTCGCT T .. G.CCAA . . CAACCCC.C AA.TTCTTTC A...GGT. TG isolate23-5 · ...... · ...... · ...... · ...... clone2C-4 . TG.GAT.CT C.. G.CCAG . .CAACCT .. T TA.CTTTCTT A... NGT .TG clone2C-23 .CA.GGACCC G.. GTCCTC. TCCTCTCAGC AG.GAAACTT CTTAGGT.TG clone2C-2 · ...... · ...... · ...... ·...... · ...... clone2C-13 .CG.GTTGCT T .. G.CCAA. .CAACCCC.A AA.TTTTCT. AT .. GGT. TG clone18C-l . TG.GCCACT C.. G.CCAG . .AACCCCC.C CA.TTTTTTA AT .. GAT.TG clone18C-5 . CG.GTTGCT T .. G.CCAA . .CAACCCC.A AA.TTTTCT. AT .. GGT. TG clone18C-4 .TG.GATACT C.. G.TCAA. .AACCCCC.C CA.TTTTTTA AT .. GAT.TG clone12C-5 . CG.GTTGCT T .. G.CCAA . .CAACCCC.A AN.TTTCCT. NT .. GGT .TN clone14C-21 .CG.GTTGCT T .. G.CCAA. .CAACCCC.A AA.TTTTCT. AT .. GGT.TG clone13C-l . CG.GTTGCT T .. G.CCAA. .CAACCCC.A AA.TTTTCT . AT .. GGT .TG 124 clone2C-lO . TGGTTGGTC c .. G.CCAG . .CAACCCT.C AA.CTTTCTT AA .. GTT.TG isolatell-3b ...... isolateBl-3 . CG.GTTGCT T .. G.CCAA. . CAACCCC. A AA.TTTTCT . AT ..GGT. TG clone17C-5 . TG.GACGCT T .. G.CCAT. . TAATCCT.T TA.ACTTCT . AT .• GGT. TG clone2C-24 .TG.GATGCT T .. G.CCAT. .TAA.CCC.C CA.ATTTCA . AT .. GGT. TG isolate2-7 .TG.TTTGCT T .. G.CCAG. . CAACCCC.C A.. TTTTATC AA.AGGT.TG clone2C-8 .GG.TCTACT T .. G.GCAA. .CAACCC .. T AA.TTTTTTN .A.AGGG.TG clone12C-9 .GG.TCTACT T ....CCAA. .CAANCCC.T AA.TTTTTTT .A.AGGT.TG clone17C-6 .NG.TCTACT T .. G.CCAA. .CAAANACCT AA.ATTTTTT TA.AGGT.TG clone2C-20 .GG.TCTACT T .• G.CCA .. .CAACCCC.T AA.TTTTTTA .A.GGG .. TG clone13C-17 .GG.TCTACT T .. G.GCAG. .CAACCCCTT AA.TTTTTTN .A.AGG .. TG isolate15-1 ...... clone2C-12 . TG.GATGCT C .. G.CCAG. .CAACCT .. T TA.CTTTCTT A... NGT.TG isolateBl-4 .GG.TCTACT T .. R.CCAG . . CAACCCC.T AA.TTTTTTT TA.AGGT.TG isolatell-2 .GG.TCTACT T .. G.CCAG . . CAACCCC.T AA.TTTTTTT .A.AGGT.TG isolate14-1 .GG.TCTACT T .. G.CCAG . . CAACCCC.T AA.TTTTTTT .A.AGGT.TG isolate9-1 ...... isolate3-8 ...... clone3C-4 . CG.CGTNCA C .. GTNCGT . . AAACACCCA AC.T .. TCTG AA.TGGT .. G clone3C-15 . C ..CGTCCA C..NTCCGT . TAAANACCCN AC. T .. TCTG AAATGTT .. N clone3C-lO . CG.CGTNCA C .. GTCCGT . .AAACACC.A AC.T .. TTTG AAATGGT.TG clone14C-l . GG.CGAC .. G.• GTCTT .. TATACATAG. AC.TTATTTA CAAGTGT.TG clone14C-6 . GG.CGAC .. G.. GTCTT .. TATACATAG. AC.TTATTTA CAAGTGT.TG clone3C-l . AGTTGGGC. ....ACCAG . TCTTAAC .. T AA.ATTTCTA AA .. TGC.TG clone3C-12 . AGTTGGGC. ....ACCAG . TCTTAAC .. T AA.ATTTCTA AA .. TGT.TG clone3C-6 . AGTTGGGC . ....ACCAG . TCTTAAC .. T AA.ATTTCTA AA .. TGT.TG isolate5-2 . GGNNGGGCT A.. NTGGNTT GGNATCCANN GGCAANTCTT .... GGG. TT isolate2-1 .GGNNGGGCT A..NTNGNTT GGGATTCATG GC ..AATCTT ....GGG. TT isolatel-l . GGNGGG.CT A.. NTGGNT . GGGAT.CATG GC ..AANNTT .... GGG.T. isolate7-1 .GGTNGGNCT A.. ATNGNTN GGNATCCATG GC.AAACCTT .... GGG.TT clone2C-31 TAGGCGTGAC CATGATCATG GTTTCGCGCC CATAATCTTT CATGAAA.TG clone2C-35 TAGGCGTGAC CATGATCATG GTTTCGCGCC CATAATCTTT CATGAAA.TG clone12C-6 TAGGTGTGGT C.TTCTCATG ATT.CGCGCT CATAATTATT CATCAAAATG clone3C-14 TAGGTGCGGT CATTCCTATG ATT.CGCGTC TATAATTTTT CATTTCT.TG clone13C-3 TACATGTGGT CATTCCNATG ATT.CGCGTC TATAATTTNN CATTATT.TG clone12C-41 TAGATGTGGT CATTCCTATG ATT.CGCGTC TATAATTTTT CATTATT.TG clone17C-l AACT ...... clone17C-3 AACT ...... clone17C-4 ACCT ...... isolate23-5 ...... clone2C-4 ACT.CGGAT. CAGGTAGGGA TACCCGCTGA A.CTTAAGCA TATCAATAA. clone2C-23 ACCTCGGAT. CAGGTAGGAA TACGCGCTGA A.CTTA ...... clone2C-2 ...... clone2C-13 AACTCGGAT. CAGTTAGGGA TACCCGCTGA A.CTTAAGCA TATCAATAAG clone18C-l ACCTCGGAT. TAAGTAGGGA TACCCGCTGA A.CTTAA.CA TATCAATAAG clone18C-5 ACCTCGGAT. CANGTAGGGA TACCCGCTGA A.CTTAAGCA TATCAATAAG clone18C-4 ACCTCGGAT. TAAGTAGGGA TACCCGCTGA A.CTTAAGCA TATCAATAAG clone12C-5 ACCCCGGAT. CAGGTAGGGA TACCCNCTNA A.CTTAANCA TTTCANTAAC clone14C-21 ACCTCGGAT. CANGTNAGGA TACCCGCTGA A.CTTAAGCA TATCAATTA. clone13C-l ACCTCGGAT. CAGGTAGGGA TACCCGCTGA A.CTTAAGCA TATCAATAA. clone2C-lO ACCTGGGAT. GAGGTAGGGA TACCCGCTGA A.. TTA ...... isolatell-3b ...... isolateBl-3 ACCTCGGAT. CAGGTAGGGA TACCCGCTGA A.CTTAAGCA TATCAATAAG clone17C-5 ACCTCGGAT. CAGGTAGGGA TACCCGCTGA A.CTTAA.CA TATCAATANG clone2C-24 ACCTCGGAT. CANGTAGGGA TACCCGCTGA A.CTTAAGCA TATCAATAGG 125 isolate2-7 ACCTCGGAT. CAGGTAGGGA TACCCGCTGA A.CTTAAGCA TATCAATANG clone2C-8 ACCTCGGAT. CAGGTAGGGA TACCCGTTGA · .CTTAAGCA TNT.AATAGG clone12C-9 AACTCGGAT. CAGGTAGGGA TACCCGCTGA A.CTTAAGCA TATCAATAAC clone17C-6 ACCTCGGAT. CAGGTAGGGA TACCCGCTGA A.CTTAAGCA TATCAATNA. clone2C-20 ACCTCGGAT. CANGTAGGGA TACCCGCTGA A.CTTAA.CA TAT.NATACG clone13C-17 ANCTTGGAT. CANGTNGGGA TCCCCGTTGA · .CTTAAGCA TTT.NATAGN isolate15-1 ...... clone2C-12 ACCTCGGAT. CANGTAGGGA TCCCCGTTGA A.CTTA.GC. TTTTAATACG isolateBl-4 ACCTCGGAT. CAGGTAGGGA TACCCNCTGA A.CTTAAGCA TATCAATAAG isolatell-2 ANCTCGGAT. CAGNTAGGGA TACCCGNTGA A.CTTAAGCA TATCAATAAG isolate14-1 ACCTCGGAT. CAGNTAGGGA TACCCGNTGA A.CTTAAGCA TATCAATAAG isolate9-1 ...... isolate3-8 ...... clone3C-4 ACCTTNGAT. CAG.TAGGAA TACCCGTTGA A.CTTAA.CA T.TCATAGGG clone3C-15 AACTCCGAT. CAGGTTNGAA TACC.GCTGA A.CTTAAGCA TATCATACA. clone3C-1O ACCTCGGAT. CAGGTAGGAA TACCCGTTGA · .CTTAAGCA TATCAATAAC clone14C-l ACCTTGGAT. CAGGTAGGAA TCCCCGTTGA · .CTTAAGCA T.TTAATAGG clone14C-6 ACCTCGGAT. CAAGTAGGAA TCCC.GCTGA · .CTTAAGCA T.TCAATAGG clone3C-l ACT.CGGAT. CAGGTAGGGA TACCCGCTGA A.CTTAA.CA TATCAATAAG clone3C-12 ACCTCGGAT. CAGGTAGGGA T.CCCGCTGA A.CTTAAGCA TATCAATANG clone3C-6 ACCTCGGAC. CAGGTAGGGA TACCCGCTGA A.CTTAAGCA TATCAATANG isolate5-2 ACATNGG.TC CAGGG .. GCA G.CTTGGTAT AAAGNAATCA TC ..AATN.G isolate2-1 ACAATGG.TT CAGGG .. GCA G.CTTGGTAN NCAGNAATCA TC ..AATN.G isolatel-l ACATNGG.T. CAGGG .. GCA N.CTTG.TAT N.AGNAATCA TC .. NNTN.G isolate7-1 ACANTNGGTC CAGGN .. GCA G.CTTGGTAT NCAGAAACCA TC .. AATTTG clone2C-31 ACCTCAAAT. CAGGTAGGAA TACCCGCTGA A.CTTAAGCA TATCAATAGG clone2C-35 ACCTCAAAT. CAGGTAGGAA TACCCGCTGA A.CTTAAGCA TATCAATANG clone12C-6 ACCTCAAAT. CAGTTA.GAN TACCCGCTGA A.CTTAAGCA TATCAATAA. clone3C-14 ACCTCAAAT. CAGGTAAGAG CACCCGCTGA A.CTTAAGCA TATCAATAAG clone13C-3 ACCTCNAAT. CAGGTAAGAN CANCCGCTGA A.CTTAAGCA TATCAATAAG clone12C-41 ACCTCAAAT. CAGGTAAGAG TACCCGCTGA A.CTTAAGCA TATCAATANG clone17C-l ...... clone17C-3 ...... clone17C-4 ...... isolate23-5 ...... clone2C-4 .GNAGAAAA ...... clone2C-23 ...... clone2C-2 ...... clone2C-13 CGANNAAAA...... clone18C-l CGGAGNAAA...... · ...... clone18C-5 CGGGGAAAG...... oooooo .. oooo ......

clone18C-4 CGGAAAAG ...... oooooooo ...... oo ......

clone12C-5 CNANAAAAA ...... oooo .. oo ...... oo ...... clone14C-21 CAAAAAAAA...... clone13C-l CGAGAAAAA...... clone2C-1O ...... isolatell-3b ...... isolateBl-3 CGG.AGGAAA AGAAACCAAC AGGGATTGNC TCAGTAACGG NGAGTGAAGC

clone17C-5 GGGGAGGAA ...... oo ...... clone2C-24 GGGGGGGAA...... isolate2-7 CGG.AGGAAA AGAAACCAAC AGGGATTGCC TCAGTATCGG CGAGTGAAGC clone2C-8 GGGGGGGAA......

clone12C-9 .GAGNAAAA ...... oo .... oo • oo ......

clone17C-6 . GCGGAGNA...... oooooooooooo ...... clone2C-20 GGGG .. GAA ...... 126 clone13C-17 GGGGGGGAA. isolate15-1 clone2C-12 GGGGGGA ...... isolateBl-4 CGGAGGAAAA GAAACCAACA GGGATTACCT CAGNAACGGN GAGTGAA ... isolatell-2 CGGAGGAAAA GAAACCANCA GGGATTACCT CAGTAACGGN GAGTGAAGCG isolate14-1 CGGAGGAAAA GAAACCAACA GGGATTACCT CAGTAACGGN GAGTGAAGCG isolate9-1 ...... isolate3-8 ...... clone3C-4 GGGGAGAA...... clone3C-15 .AAAAAAA ...... clone3C-10 GGGGGGAA ...... clone14C-l GGGGGNGA ...... clone14C-6 GGGGGGAA...... clone3C-l CGGAGNAA...... clone3C-12 GGGGGGGAA ...... clone3C-6 GGGGGGGAA ...... isolate5-2 GGCTGNACCA NGGGGNTCCG isolate2-1 GGCTGANC.A GGGGGGTCCG isolatel-l GGCTGNNC.A GNNGGTNCCN isolate7-1 GGCTGAAC.A GNNGGGTCC. clone2C-31 GGGGGGGA...... clone2C-35 GGGNGGAA ...... clone12C-6 CGAGNAAA ...... clone3C-14 CGAGNAAA ...... clone13C-3 CGGAGAAN...... clone12C-41 GGGGGGGAA ...... 127

Appendix C DNA alignment of the 5.88 nrDNA

1 50 Isoetes . ACTCGGCAA CGGATATCTT GGCTCTTGCC ACGATGAAGA ACGCAGCG.A Selaginella . ACTCGGCAA CGGATATCTT GGCTCTTGCA ACGATGAAGA ACGCAGCG.A Marsilea CTCTCAGCAA CGGATATCTT GGCTCTTGCA ACGATGAAGA ACGCAGCG.A Osmunda CTCTCAGCAA CGGATATCTT GGCTCTTGCA ACGATGAAGA ACGCAGCG.A Taxus CTCTCGGCAA CGGATATCTC GGCTCTCGC. ACGATGAAGA ACGTAGCG.A Pinus CTCTCGTCAA CGGATATCTC GGCTCTTGTT ACGATGAAGA ACGTANCG.A Brassica CTCTCGGCAA CGGATATCTC GGCTCTCACA TCGATGAAGA ACGTAGCG.A Gossypium CTCTCGGCAA CGGATATCTC GGCTCTCGCA TCGATGAAGA ACGTAGCG.A Oryza CTCTCGGCAA CGGATATCTC GGCTCTCGCA TCGATGAAGA ACGTAGCG.A Cistella CTTCCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGGG.A Al CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Scler CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A All CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Rutstroe CTTTCAACAA CGGATCTCTT GGTTCTGG.A TCGATGAAGA ACGCAGCG.A A9 CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Phial greg CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A A2 CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Bopu2-7 CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A A10 CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Trichod.vi CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Cerato CTTTCAACAA CGGATCTCTT GGCTCTAGCA TCGATGAAGA ACGCAGCG.A Ophios CTTTCAACAA CGGATCTCTT GGCTCTGGCA TCGATGAAGA ACGCAGCA.A Claviceps CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Neurospora CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Nect CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Colleto CTTTTAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Glomerel CTTTTAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Gaeuman CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Thermo CTTTCAACAA TGGATCTCTT GGTTCCGGCA TCGATGAAGA ACGCAGCG.A Monascus CTTTCAACAA CGGATCTCTT GGTTCCGGCA TCGATGAAGA ACGCAGCG.A Morchella CTTTCAACAA CGGATCTCTT GGTTCCCACA TCGATGAAGA ACGCAGCG.A A7 CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATNAAAA ACGCAGCG.A Gelatinipulvinella CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Dactylaria CTTTCAACAA CGGATCTCTT GGCTCTGGCA TCGATGAAGA ACGCAGCG.A A3 CTTTCAACAA CGGATCTCTT GGCTCTGGCA TCGATGAANA ACGCAGCG.A Sphaero CTTTCAACAA CGGATCTCTT GGCTCTGGCA TCGATGAAGA ACGCAGCG.A Phyllac CTTTCAACAA CGGATCTCTT GGCTCTGGCA TCGATGAAGA ACGCAGCG.A A5 CTTTCAACAA CGGATCCCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A A4 CTTTCANCAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Phoma CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A A12 CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Phial amer CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A A8 CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A Drechsler CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAAGA ACGCAGCG.A A6 CTTTCAACAA CGGATCTCTT GGTTCTGGCA TCGATGAANA ACGCAGCG.A Kluyv CTTTCAACAA CGGATCTCTT GGTTCTCGCA TCGATGAAGA ACGCAGCG.A Ashbya CTTTCAACAA CGGATCTCTT GGTTCTCGCA TCGATGAAGA ACGCAGCG.A Candida CTTTCAACAA CGGATCTCTT GGTTCTCGCA TCGATGAAGA ACGCAGCG.A Saccharomyces CTTTCAACAA CGGATCTCTT GGTTCTCGCA TCGATGAAGA ACGCAGCG.A Endogone CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Bopul-l CTTTCAACAA CGGATCTCTT GGTTCTCGCA TCGATGAAGA ACGCAGCG.A Glomus.mosseae CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Glomus.fasciculataum CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Glomus.momosporum CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Glomus.coronatum CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Glomus.claroideum CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Glomus.intratadices CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Glomus.etunicatum CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Z4 CTTTCAACAA NGGATCTCTT GGCTCTCGCA TCGATGAAGA ACNTANCG.A 128

Z2 CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAANA ACGTAGCG.A ZI CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGTAGCG.A Z3 CTTTCAACAA CGGATCTCTT GGTTCTCGCA TCGATGAAGA ACGTAGCG.A Giga.rosea CTTTCAACAA TGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Giga.gigantea CTTTCAACAA TGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Giga.margarita CTTTCAACAA TGGATCTCTT GGCTCTCACA TCGATGAAGA ACGCAGCG.A Giga.albida CTTTCAACAA TGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCA.A Scut.castanea CTTTCAACAA TGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Scut.heterogama CTTTCAACAA TGGATCTCTT GGTTCTCGCA TCGATGAAGA ACGCAGCG.A Entrophospora CTTTCAACAA CGGATCTCTT GGTTCCAGCA TCGATGAAGA ACGCAGCG.A Ceratobasidium GTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Thanatephorus GTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Trichaptum.laricinum CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Amanita CTTTCAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Tremella CTTTTAACAA CGGATCTCTT GGCTCTCGCA TCGATGAAGA ACGCAGCG.A Cryptococ CTTTCAACAA CGGATCTCTT GGCTTCCACA TCGATGAAGA ACGCAGCG.A Xenopus CTCTTAGCGG TGGATCACTC GGCTCGTGCG TCGATGAAGA ACGCAGC .. T Homo CTCTTAGCGG TGGATCACTC GGCTCGTGCG TCGATGAAGA ACGCAGCGCT Arion CTTTGTGCGG TGGATCACTC GGCTCGTGCG TCGATGAAGA GCGCAGC .. C Dolichosaeeus CTCTGAGCGG TGGATCACTC GGCTCGTGTG TCGATGAAGA GCGCAGC .. C

51 100 Isoetes AAT.GCGATA CGTGATGTGA ATTGCAGAAC T.CCGTGAAT CATTGAATCT Selaginella AAT.GTGATA CGTGATGTGA ATTGCAGAAT T.CCGTGAAT CATCAAATGT Marsilea AAT.GCGATA CGTAATGTGA ATTGCAGAAT T.CCGCGAAT CATCGAATCT Osmunda AAT.GCGATA CGTAGTGTGA ACTGCAGAAT T.CCGCGAAT AATCGAGTCT Taxus AAT.GCGATA CTTAGTGTGA ATTGCAGAAT C.CCGTGAAT CATCGAGTCT Pinus AAT.GCGATA CTTAGTGTGA ATTGCAGAAT C.CCGTGAAT CATCGAGTTT Brassica AAT.GCGATA CTTGGTGTGA ATTGCAGAAT C.CCGTGAAC CATCGAGTCT Gossypium AAT.GCGATA CTTGGTGTGA ATTGCAGAAT C.CCGTGAAC CATCGAGTCT Oryza AAT.GCGATA CCTGGTGTGA ATTGCAGAAT C.CCGTGAAC CATCGAGTCT Cistella AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Al AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Seler AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT All AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Rutstroe AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT A9 AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Phial greg AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAAT CT A2 AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Bopu2-7 AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT AlO AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Triehod.vi AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Cerato AAT.GCGATA CGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Ophios AAT.GCGATA CGTAATGCGA ATTGCAGAAT T.CAG.GAGT CATCGAATCT Clavieeps AAT.GCGATA CGTAATGTGA ATTGCAGACT T.CAGTGAAT CATCGAATCT Neurospora AAT.GCGATA GGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Neet AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CCGTGAAT CATCGAATCT Colleto AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Glomerel AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Gaeuman AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Thermo AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CCGTGAAT CATCGAATCT Monaseus AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAAT CT Morehella AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT A7 AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Gelatinipulvinella AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Daetylaria AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT A3 AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Sphaero AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.TAGTGAAT CATCGAATCT Phyllae AAT.GCGATA AGTAATGTGA ATTGCAGAAT C.TAGTGAAT CATCGAATCT AS AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT A4 AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Phoma AAT.GCGATA AGTAGTGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT A12 AAT.GCGATA AGTAATGCGA ATTGCAGAAT .. CCGTGAGT CATCGAATCT Phial amer AAT.GCGATA AGTAATGCGA ATTGCAGAAT TCCCGTGAGT CATCGAATCT 129

A8 AAT.GCGATA AGTAATGCGA ATTGCAGAAT T.CAGTGAGT CATCGAATCT Drechsler AAT.GCGATA AGTAGTGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT A6 AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATTT Kluyv ATT.GCGATA AGTATTGTGA ATTGCAGATT T.TCGTGAAT CATCGAATCT Ashbya ATT.GCGATA AGTATTGTGA ATTGCAGATT T.TCGTGAAT CATCGAATCT Candida AAT.GCGATA CGTAATATGA ATTGCAGATA T.TCGTGAAT CATCGAATCT Saccharomyces AAT.GCGATA CGTAATGTGA ATTGCAGAAT T.CCGTGAAT CATCGAATCT Endogone AAT.GCGATA CGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Bopul-l AAT.GCGATA CGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Glomus.mosseae AAT.GCGATA AGTAGTGTGA ATTGCATAAT T.TTGTGAAT CATCGAATCT Glomus.fasciculataum AAT.GCGATA AGTAGTGTGA ATTGCATAAT T.TTGTGAAT CATCGAATCT Glomus.momosporum AAT.GCGATA AGTAGTGTGA ATTGCATA.T T.TTGTGAAT CATCGAATCT Glomus.coronatum AAT.GCGATA AGTAGTGTGA ATTGCATAAT T.TTGTGAAT CATCGAATCT Glomus.claroideum ATTTGCGATA AGTAATGTGA ATTGCAGAAT T.ACGTGAAT CATCGAATCT Glomus.intratadices ATT.GCGATA AGTAATGTGA ATTGCAGAAT T.ACGTGAAT CATCGAATCT Glomus.etunicatum ATT.GCGATA AGTAATGTGA ATTGCAGAAT T.ACGTGAAT CATCGAATCT Z4 AGT.GCGATA AGTAATGTGA ATTGCAGTAT T.CCGTGAAT CATCNAATCT Z2 AGT.GCGATA AGTAATGTGA ATTGCAGAAT T.CCGTGAAT CATCGAATCT Zl AGT.GCGATA AGTAATGTGA ATTGCAGAAT T.CCGTGAAT CATCGAATTT Z3 AGT.GCGATA AGTAGTGTGA ATTGCAGAAT T.CCGTGGAT CATCGAATTT Giga.rosea AAT.GCGATA AGTAATATGA ATTGCAGAAT T.CCGTGAAT CATCAAATTT Giga.gigantea AAT.GCGATA AGTAATATGA ATTGCAGAAT T.CCGTGAAT CATCAAATTT Giga.margarita AAT.GCGATA AGTAATATGA ATTGCAGAAT T.CCGTGAAT CATCAAATTT Giga.albida AAT.GCGATA AGTAATATGA ATTGCAGAAT T.CCGTGAAT CATCAAATTT Scut.castanea AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CCGTGAAT CATCAAATTT Scut.heterogama AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CCGTGAAT CATCAAATTT Entrophospora AAT.GCGATA AATAATGTGA ATTGCAGAAT T.CCGTGAAT CATCGAGTTT Ceratobasidium AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Thanatephorus AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Trichaptum.laricinum AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Amanita AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGANTCT Tremella AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAATCT Cryptococ AAT.GCGATA AGTAATGTGA ATTGCAGAAT T.CAGTGAAT CATCGAGTCT Xenopus AGCTGCGAGA ATTAGTGTGA ATTGCAGGAC A.CATTGAT. CATCGACACT Homo AGCTGCGAGA ATTAATGTGA ATTGCAGGAC A.CATTGAT. CATCGACACT Arion AGCTGCGTGA ATTAATGTGA ATTGCAGAAC A.CATTGAA. CATCGACATC Dolichosaccus AACTGTGTGA ATTAATGTGA ACTGCATACT G.CTTTGAA. CATCGACATC

101 150 Isoetes TTGAACGCAA CTTGCGCCCG GGG.CT .. TG TCCGAGGGCA TGTCTGCGTG Selaginella TTGAACGCAA CTTGCGCCCG AGG.CT .. TG TCCGAGGGCA TGCCTGCTTG Marsilea TTGAACGCAA .TTG.GCCCG AGG.CT .. CG TCCGAGGGCA CGTCTGCCTG Osmunda TTGAACGCAA GTTG.GCCCG CGG.CT .. CG TCCAAGGGCA TGCCTGCCTG Taxus TTGAACGCAA GTTGGCCCGG AG .. CT .. CG GCCGAGGGCA CGTCTGCTTG Pinus TTGAACGCAA TTTGCGCCCG AGGCCT .. CG GTCGAGGGCA CGTCTGTCTG Brassica TTGAACGCAA GTTGCGCCCC AAGCCTTCTG GCCGAGGGCA CGTCTGCCTG Gossypium TTGAACGCAA GTTGCGCCCC AAGCCATTAG GCCGAGGGCA CGTCTGCCTG Oryza TTGAACGCAA GTTGCGCCCG AGGCCATCCG GCCGAGGGCA CGCCTGCCTG Cistella TTGAACGCAC ATTGCGCCCC TTGGTATT .. CCGGGGGGCA TGCCTGTTCG Al TTGAACGCAC ATTGCGCCCC TTGGTATT .. CCGGGGGGCA TGCCTGTTCG Scler TTGAACGCAC ATTGCGCCCC TTGGTATT .. CCGGGGGGCA TGCCTGTTCG All TTGAACGCAC ATTGCGCCCC TTGGTATT .. CCGGGGGGCA TGCCTGTTCG Rutstroe TTGAACGCAC ATTGCGCCCC TTGGCATT .. CCGGGGGGCA TGCCTGTTCG A9 TTGAACGCAC ATTGCGCCCC TTGGTATT .. CCGGGGGGCA TGCCTCTTCC Phial greg TTGAACGCAC ATTGCGCCCT CTGGTATT .. CCGGGGGGCA TGCCTGTTCG A2 TTGAACGCAC ATTGCGCCCT CTGGTATT .. CCGGGGGGCA TGCCTGTTCG Bopu2-7 TTGAACGCAC ATTGNGCCCT CTGGTATT .. CCGGGGGGCA TGCCTGTTCG AlO TTGAACGCAC ATTGCGCCCG CCAGTATT .. CTGGCGGGCA TGCCTGTCCG Trichod.vi TTGAACGCAC ATTGCGCCCG CCAGTATT .. CTGGCGGGCA TGCCTGTCCG Cerato TTGAACGCAC ATTGCGCCTG GCAGTATT .. CTGCCAGGCA TGCCTGTCCG Ophios TTGAACGCAC ATTGGGCCCG CCAGTATT .. CTGGCGGGCA TGCCTGTCCG Claviceps TTGAACGCAC ATTGCGCCCG CCAGTATT .. CTGGCGGGCA TGCCTGTTCG Neurospora TTGAACGCAC ATTGCGCTCG CCAGTATT .. CTGGCGAGCA TGCCTGTTCG Nect TTGAACGCAC ATTGCGCCCG CCAGTATT .. CTGGCGGGCA TGCCTGTTCG 130

Colleto TTGAACGCAC ATTGCGCCCG CCAGCATT .. CTGGCGAGCA TGCCTGTTCG Glomerel TTGAACGCAC ATTGCGCCCG CCAGCATT.C CTGGCGGGCA TGCCTGTTCG Gaeuman TTGAACGCAC ATTGCGCCCG CCGGTATT .. CCGGCGGGCA TGCCTGTTCG Thermo TTGAACGCAC ATTGCGCCCT CTGGTATT .. CCGGGGGGCA TGCCTGTCCG Monascus TTGAACGCAC ATTGCGCCCC CTGGTATT .. CCGGGGGGCA TGCCTGTCCG Morchella TTGAACGCAC ATTGCGCCCC CTGGTATT .. CCGGGGGGCA TGCCTGTTCG A7 TTGAACGCAC ATTGCGCCCC CTGGTATT .. CCGGGGGGCA TGCCTGTTCN Gelatinipulvinella TTGAACGCAC ATTGCGCCCC CTGGTATT .. CCGGGGGGCA TGCCTGTTCG Dactylaria TTGAACGCAC ATTGCGCCCC TTGGTATT .. CCGAGGGGCA TGCCTATTCG A3 TTGAACGCAC ATTGCGCCCC TTGGTATT .. CCGAGGGGCA TGCCTATTCG Sphaero TTGAACGCAC ATTGCGCCCC CCGGCATT .. CCGAGGGGCA TGCCTGTTCG Phyllac TTGAACGCAC ATTGCGCCCC TTGGTATT .. CCGAGGGGSA TGCCTGTTCG AS TTGAACGCAC ATTGCGCCCC TTGGTATT .. CCGAGGGGCA TGCCTGTTCG A4 TTGAACGCAC ATTGCGCCCA TTAGTATT .. CTATTGGGCA TGCCTGTTCG Phoma TTGAACGCAC ATTGCGCCCC TTGGTATT .. CCATGGGGCA TGCCTGTTCG A12 TTGAACGCAC ATTGCGCCCT TTGGTATT .. CCGAAGGGCA TGCCTGTTCG Phial amer TTGAACGCAC ATTGCGCCCT TTGGTATT .. CCAAAGGGCA TGCCTGTCCG AS TTGAACGCAC ATTGCGCCCT TTGGTATT .. CCGAAGGGCA TGCCTGTTCG Drechsler TTGAACGCAC ATTGCGCCCT TTGGTATT .. CCAAAGGGCA TGCCTGTTCG A6 TTGAACGCAT ATTGCGCCCT TTGGCATT .. CCGAAGGGCA TACCTGTTCG Kluyv TTGAACGCAC ATTGCGCCCT TTGGTATT .. CCAGGGGGCA TGCCTGTTTG Ashbya TTGAACGCAC ATTGCGTCCT CTGGTATT .. CCAGGGGGCA TGCCTGTTTG Candida TTGAACGCAC ATTGCGCCCT CTGGTATT .. CCGGAGGGCA TGCCTGTTTG Saccharomyces TTGAACGCAC ATTGCGCCCC TTGGTATT .. CCAGGGGGCA TGCCTGTTTG Endogone TTGAACGCAT ATTGCACTCT CTGGTACT .. CCGGGGAGTA TGCCTGTTTC Bopul-l TTGAACGCAC ATTGNACTCC TTGGTATT .. CCGAGGAGTA TGCCTGTTTC Glomus.mosseae TTGAACGCAA ATTGCACTCC CTGGTATT .. CCGGGGAGTA TGCCTGTTTG Glomus.fasciculataum TTGAACGCAA ATTGCACTCC CTGGTATT .. CCGGGGAGTA TGCCTGTTTG Glomus.momosporum TTGAACGCAA ATTGCACTCC CTGGTATT .. CCGGGGAGTA TGCCTGTTTG Glomus.coronatum TTGAACGCAA ATTGCACTCT CTGGTATT .. CCGGGGAGTA TGCCTGTTTG Glomus.claroideum TTGAACGCAT ATTGCACTCT CTGGTAAT .. CCGGGGAGTA TGCCTGTTTG Glomus.intratadices TTGAACGCAT ATTGCACTCT CTGGTAAT .. CCGGGGAGTA TGCCTGTTTG Glomus.etunicatum TTGAACGCAT ATTGCACTCT CTGGTAAT .. CCGGGGAGTA TGCCTGTTTG Z4 TTGAACGCAA ATTGCACTTT CTGGTATT .. CCGGAAAGTA TGCCNGTTTG Z2 TTGAACGCAA ATTGCACTCT CTGGTATT .. CCGGAAAGTA TGCCTGTTTG Zl TTGAACGCAA ATTGCACTCT CTGGTACT .. CCGGANAGTA TGCCTGTTTG Z3 TTGAACGCAA ATTGCACTCT CTGGTATT .. CCGGAGAGTA TGCCTGTTTG Giga.rosea TTGAACGCAA ATTGCACTTC TTGGTATT .. CTGAGAAGTA CACATGCTTG Giga.gigantea TTGAACGCAA ATTGCACTTC TTGGTATT .. CTGAGAAGTA CACATGCTTG Giga.margarita TTGAACGCAA ATTGCACTTC TTGGTATT .. CCGAGGAGTA CACATGCTTG Giga.albida TTGAACGCAA ATTGCACTTC TTGGTATT .. CCAAGGAGTA CACATGCTTG Scut.castanea TTGAACGCAA ATTGCACTTT TCGGTATT .. CCGAGAAGTA CACCTGCTTG Scut.heterogama TTGAACGCAA ATTGCACTCC TTGGTATT .. CCGAGGAGTA CACCTGCTTG Entrophospora TTGAACGCAC ATTGCACTCC TTGGTATT .. CCGAGGAGTA CGCCTGTTCG Ceratobasidium TTGAACGCAC CTTGCGCTCC TTGGTATT .. CCTTGGAGCA TGCCTGTTTG Thanatephorus TTGAACGCAC CTTGCGCTCC TTGGTATT .. CCTTGGAGCA TGCCTGTTTG Trichaptum.laricinum TTGAACGCAC CTTGCGCTCC TTGGTATT .. CCGAGGAGCA TGCCTGTTTG Amanita TNGANCGCAC NTTGGCCTCC TTGGTATT .. CCGAGGAGCA TGCCTGTTTG Tremella TTGAACGCAT CTTGCGCCCT TTGGTATT .. CCGAAGGGCA TGCCTGTTTG Cryptococ TTGAACGCAA CTTGCGCCCT TTGGTATT .. CCGAAGGGCA TGCCTGTTTG Xenopus TCGAACGCAC CTTGCGGCCC CGGGTTCCT. CCCGGGGCCA CGCCTGTCTG Homo TCGAACGCAC . TTGCGGCCC CGGGTTCCT . CCCGGGGCTA CGCCTGTCTG Arion TTGAACGCAT ATGGCGGCCT CGGGTCCAT. CCCGGGGCCA CGCCCGTCTG Dolichosaccus TTGAACGCAC ATTGCG.CCA TGGGTT.AG. CCCATGGC.A CGCCTGTCCG

151 160 Isoetes AGCGTC .... Selaginella AGCGTC .... Marsilea AGCGTC .... Osmunda AGCGTC .... Taxus GGCGTC .... Pinus GGCGTC .... Brassica GGTGTC .... Gossypium GGTGTC .... 131

Oryza GGCGTC ... . Cistella AGCGTC ... . Al AGCGTC ... . Scler AGCGTC ... . All AGCGTC ... . Rutstroe AGCGTC ... . A9 AGCGTC ... . Phial greg AGCGTC ... . A2 AGCGTC ... . Bopu2-7 AGCGTC ... . AID AGCGTC ... . Trichod.vi AGCGTC ... . Cerato AGCGTC ... . Ophios AGCGTC ... . Claviceps AGCGTC ... . Neurospora AGCGTC ... . Nect AGCGTC ... . Colleto AGCGTC ... . Glomerel AGCGTC ... . Gaeuman AGCGTC ... . Thermo AGCGTC ... . Monascus AGCGTC ... . Morchella AGCGTC ... . A7 AACGTC ... . Gelatinipulvinella AGCGTC ... . Dactylaria AGCGTC ... . A3 AGCGTC ... . Sphaero AGCGTC ... . Phyllac AGCGTC ... . AS AGCGTC ... . A4 AGCGTC ... . Phoma AGCGTC ... . A12 AGCGTC ... . Phial amer AGCGTC ... . AB AGCGTC ... . Drechsler AGCGTC ... . A6 AGCGTC ... . Kluyv AGCGTC ... . Ashbya AGCGTC ... . Candida AGCGTC ... . Saccharomyces AGCGTC ... . Endogone AGTATC ... . Bopul-l AGTATC ... . Glomus.mosseae AGGGTC ... . Glomus.fasciculataum AGGGTC ... . Glomus.momosporum AGGGTC ... . Glomus.coronatum AGGGTC ... . Glomus.claroideum AGGGTC ... . Glomus.intratadices AGGGTC ... . Glomus.etunicatum AGGGTC ... . Z4 AGGGTC ... . Z2 AGGGTC ... . Zl AGGGTC ... . Z3 AGGGTC ... . Giga.rosea AGGGTC ... . Giga.gigantea AGGGTC ... . Giga.margarita AGGGTC ... . Giga.albida AGGGTC ... . Scut.castanea AGGGTC ... . Scut.heterogama AGGGTC ... . Entrophospora AGCGTC ... . Ceratobasidium AGTATC ... . Thanatephorus AGTATC ... . Trichaptum.laricinum AGTGTC ... . 132

Amanita AGTGTC ... . Tremella AGTGTC ... . Cryptococ AGAGTC ... . Xenopus AGGGTC ... . Homo AGCGTC ... . Arion AGGGTC ... . Dolichosaccus AGGGTC ... .