MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF ALEXANDRIUM SPECIES (DINOPHYCEAE) FROM THE EAST COAST, USA

Erika N. Schwarz

A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the degree of Master of Science

Department of Biology and Marine Biology

University of North Carolina Wilmington

2011

Approved by

Advisory Committee

D. Wilson Freshwater R. Wayne Litaker

Alison R. Taylor Carmelo R. Tomas Chair

Accepted by

Dean, Graduate School

This thesis has

been formatted in accordance to the

author guidelines for the Journal of Phycology.

ii TABLE OF CONTENTS

ABSTRACT ...... iv

ACKNOWLEDGMENTS ...... v

DEDICATION ...... vi

LIST OF TABLES ...... vii

LIST OF FIGURES ...... viii

INTRODUCTION ...... 1

MATERIALS & METHODS ...... 8

RESULTS ...... 14

DISCUSSION ...... 16

LITERATURE CITED ...... 23

TABLES ...... 28

FIGURES ...... 37

APPENDIX 1 ...... 40

APPENDIX 2 ...... 57

APPENDIX 3 ...... 65

iii ABSTRACT

The dinoflagellates (Dinophyceae) are a group of highly diverse unicellular organisms that inhabit fresh, estuarine and saltwater environments. Some dinoflagellates produce toxins that have negative impacts resulting as both economic and public health risks. This study focuses on

Alexandrium peruvianum, a thecate, paralytic shellfish poisoning (PSP) toxin producing dinoflagellate. Alexandrium peruvianum was thought to be restricted to waters north of Cape

Cod, MA but this species was recently isolated from the New River, NC. The aim of this study was to thoroughly characterize A. peruvianum using both morphological and molecular techniques in order to distinguish it from the closely related A. ostenfeldii. Calcofluor White staining was used to visualize thecal plates to determine morphological differences between A. peruvianum and A. ostenfeldii. Significant differences were found in the first apical (1’), the sixth precingular (6’’) and the anterior sulcal (s.a.) plates between the two species. The differences observed were consistent with original descriptions of A. peruvianum and A. ostenfeldii. Utilizing 5.8S, 18S, 28S and internal transcribed spacer (ITS) sequences generated in this study in addition to those submitted to GenBank, phylogenetic analysis resolved four clades between the two species; Clades 1 and 2 were comprised of A. peruvianum and Clades 3 and 4 were comprised of A. ostenfeldii. Isolates from Clades 1 and 3, including A. peruvianum from

North Carolina, USA, Finland and A. ostenfeldii from Canada/Denmark/Scotland, respectively were used for comparison. Isolates from Clades 2 and 4, A. peruvianum from Spain and A. ostenfeldii from New Zealand, were not available for the morphological study. DNA sequence information for the Alexandrium isolates examined was used to develop a PCR-based method for detection and identification of A. peruvianum and A. ostenfeldii.

iv ACKNOWLEDGMENTS

First and foremost I would like to thank my advisor, Dr. Carmelo R. Tomas, who has believed in my abilities from the beginning. He has been a wonderful advisor and influence. He has shown me what it means to be a good advisor, professor and mentor and for that I am grateful. The only limiting factor is your mind and where it can take you when in his lab. In addition, the wonderful vegetables from his garden are always a welcome summertime surprise and lunches at Thai Spice will be missed. Thank you for everything!

I would like to send many thanks to my wonderful committee members. To Dr. R. Wayne

Litaker (NOAA) for always answering his phone to answer questions and offer advice or lend a helping hand when needed. Welcoming us into his lab and making the trek to Wilmington for our meetings are also things that are greatly appreciated. To Dr. D. Wilson Freshwater for his willingness to stop whatever he may be doing in order to answer questions without notice and to answer those questions in a way that is easy to understand. To Dr. Alison R. Taylor, not only for agreeing to serve as a committee member regardless of her busy schedule but for her wealth of knowledge and enthusiasm for science. Both traits make her someone to aspire to.

I would like to thank Mark W. Vandersea (NOAA) for sharing his knowledge and testing the A. ostenfeldii primers. Also, Dr. Anke Kremp for sharing cultures and associated sequences.

I would also like to thank my lab mates (past and present), Kristi Sommer, Lindsay Haus,

Cory Dashiell, Brooke Stuercke, Bob York, Michelle Stuart, Harris Muhlstein and Tara Haney who have always been a wealth of information and very supportive.

I would like to thank my best friend, Bart R. Frans, and family whose support throughout my educational endeavors has meant a great deal.

Lastly, I would also like to thank MARBIONC for funding this thesis project.

v DEDICATION

I would like to dedicate this thesis to my daughter, Athena N. Schwarz, whose presence has been the driving force for my motivation. My desire to go to college first started when she was born in hopes of building a better life for us. However, it quickly turned from a stepping- stone to get a decent job into a true passion. Because of Athena I was able to find my true interests when I took my first biology class as a requirement for my Associate’s degree. If it weren’t for her, I would have never given school another chance and for that I am eternally grateful.

vi LIST OF TABLES

Table Page

1. Recognized Alexandrium species and present knowledge of toxicity ...... 28

2. Isolates of A. peruvianum and A. ostenfeldii used in this study including isolate code, location of sample, isolation date, isolator, growth temperature and salinity ...... 29

3. Primers used in this study to generate partial SSU, ITS1, 5.8S, ITS2 and partial LSU sequences from A. peruvianum (AP0411-1) and A. ostenfeldii (CCMP1773) ...... 30

4. Alexandrium D1-D2 rDNA sequences obtained from GenBank and used in combination with those of this study for the D1-D2 alignment and phylogenetic analysis. A. insuetum and A. tamutum were the most closely related sister taxa and used as outgroup taxa ...... 31

5. Primers designed to distinguish between A. peruvianum (AP) and A. ostenfeldii (AO). Grey regions indicate the variable regions between the two species ...... 32

6. Genomic DNA used in cross-reactivity tests of A. peruvianum and A. ostenfeldii primer sets...... 33

7. Mean and standard deviation (sd) of plate measurements for A. peruvianum and A. ostenfeldii, number of samples (n) and significance (5% level) using a two-tailed student’s t-test. Measurements from AP0411-1, AOTVA4, AOF0933 were used to calculate A. peruvianum mean and sd and measurements from CCMP1773 and AONOR4 were used to calculate A. ostenfeldii mean and sd ...... 34

8. Results of cross-reactivity tests with A. peruvianum primer set APF3/APR3 ...... 35

9. Results of qPCR assays with A. ostenfeldii primer set AOF4/AOR3 ...... 36

vii LIST OF FIGURES Figure Page

1. Calcofluor White stained cells representing A. peruvianum showing the anterior sulcal (s.a.) plates, the ventral pore (v.p.), first apical plate (1’) and the 6th precingular plate (6’’); a) AP0411-1, b) B2-NR, c) C10-NR, d) D4-NR, e) AOTVA4 and f) AOF0933 ....37

2. Calcofluor White stained cells representing A. ostenfeldii isolates showing the s.a. plates, the ventral pore (v.p.), first apical plate (1’) and the 6th precingular plate (6’’); a) CCMP1773 and b) AONOR4 ...... 38

3. Phylogeny of A. peruvianum and A. ostenfeldii showing four distinct clades; 1) A. peruvianum from North Carolina, USA and Finland with the exception of 2 isolates identified as A. ostenfeldii, 2) A. peruvianum sequences from Spain isolates, 3) A. ostenfeldii isolates from Canada/Scotland/Denmark and 4) A. ostenfeldii isolates from New Zealand ...... 39

viii INTRODUCTION

Dinoflagellates (Dinophyceae) comprise a group of highly diverse unicellular organisms that live in freshwater, estuarine or marine environments and exhibit two dimorphic flagella during at least one life cycle stage. The term dinoflagellate originates from the Greek term dineo, which means “to whirl” (Graham et al. 2009). This term is descriptive of their characteristic whirling swimming motion driven by the combined action of the transverse and the longitudinal flagella (Dodge and Lee 2000). The cell surfaces of many dinoflagellates are covered with closely fitting cellulosic plates that help protect the cell (Steidinger and Tangen 1997). Species possessing these cellulosic plates are referred to as “thecate” dinoflagellates, whereas those lacking cellulosic plates are called “athecate”. All dinoflagellates are further characterized by tubular mitochondrial cristae and the presence of alveoli, flattened amphiesmal vesicles packed into a continuous layer beneath the plasmalemma. The amphiesmal vesicles are also common to several other major lineages of protists including ciliates and apicomplexans that are collectively referred to as ′Alveolates′.

Fossil evidence indicates that dinoflagellates are at least 240 million years old but some biological and biogeochemical evidence suggests origins dating back to 545 million years ago

(John et al. 2003). Over 2000 extant species and a similar number of fossil species were described. It is likely, however, that the species diversity of both extant and fossil species is considerably higher. Fifty percent or more of the extant species are non-photosynthetic and acquire their nutrition through heterotrophy, parasitism, osmotrophy or mixotrophy. The remaining species have secondarily acquired chloroplasts and are autotrophic or mixotrophic

(Graham et al. 2009). These autotrophic species are important primary producers and collectively contribute more oceanic primary productivity than any other eukaryotic group besides the diatoms (Graham et al. 2009). Despite this significant photosynthetic capacity, most autotrophic dinoflagellates retain their heterotrophic ability whereas a few species have evolved to become true parasites (Graham et al. 2009). Many but not all plastid-containing dinoflagellates are characterized by the presence of chlorophylls a and c and unique carotenoids.

Of these carotenoids a common accessory pigment, peridinin, has evolved in some dinoflagellates (Graham et al. 2009).

Dinoflagellates are classified as Chromalveolates, which implies that the last common ancestor of this group was a photosynthetic eukaryote with a secondary, red type plastid

(Cavalier-Smith 1999). This group includes the Cryptophyta, Haptophyta, Stramenopiles (or

Heterokontophyta) and the Alveolata (including dinoflagellates) (Delwiche 2007).

Taxonomy of thecate dinoflagellates is primarily based on morphological characteristics such as number, shape, size, position and ornamentation of thecal plates (Fukuyo 1985,

Steidinger and Tangen 1997). Thecal plates are generally arranged in a series that can be systematically numbered. This system is known as the “Kofoidian system” of plate nomenclature

(Steidinger and Tangen 1997). A typical plate tabulation includes the apical pore plates (′), the anterior intercalary (a), precingular (′′), postcingular (′′′), posterior intercalary (p) and antapical

(′′′′) plates. The apical plates are those at the anterior end of the cell that come in contact with the apical pore complex (APC). Precingular and postcingular plates refer to those that come in contact with the cingulum on the apical side or the antapical side, respectively. Antapical plates are located at the antapex or posterior of the cell and may be in contact with the sulcal plates but not with cingular plates. Anterior and posterior intercalary plates are located between the precingular and the apical series or between the postcingular and the antapical series, respectively. Balech (1980) amended Kofoidan nomenclature to include the cingular (c) and

2 sulcal (s) plates and the apical pore complex (APC). The APC itself can include the apical pore

(po), canopy (cp) and ventral apical plate (X) (Steidinger and Tangen 1997). The Kofoidian system with Balech’s improvements gives a succinct plate formula that can be used in conjunction with other characteristics to identify thecate dinoflagellates at the ranks of order, family, genus and species (Steidinger and Tangen 1997).

In addition to a variety of nutritional modes and their importance as primary producers, approximately 60 dinoflagellate species also produce toxins that have cytolytic, hemolytic, hepatotoxic or neurotoxic activities depending on their chemical structures and conversion state of the toxin produced (Plumley 1997, Steidinger and Tangen 1997). These toxins are transferred through the food chain where they sometimes accumulate within particular organisms such as shellfish (La Du et al. 2002). They can adversely affect ecosystem function and may cause mass mortalities of aquatic organisms such as fish (La Du et al. 2002). Humans are most commonly affected by algal toxins via ingestion of contaminated shellfish, finfish or exposure to toxic aerosols (Franquelo 2009).

The genus Alexandrium Halim is of particular concern because 11 of the 32 described species produce potent neurotoxins collectively known as saxitoxins (Steidinger and Tangen

1997) (Table 1). These toxins act by blocking voltage-gated sodium channels thereby interfering with nerve conduction. Severity of human illness resulting from the consumption of shellfish feeding on Alexandrium cells depends on dosage and individual susceptibility. Symptoms include numbness, lack of coordination, paralysis and, in severe cases, death (Friedman and

Levin 2005). Collectively, this suite of symptoms is referred to as paralytic shellfish poisoning

(PSP) (Hansen 2003; Wang et al. 2007). Toxic Alexandrium blooms are a common occurrence in many parts of the world and often pose a dual economic/public health threat with losses

3 resulting from lost shellfish harvests, increased medical expenditures, and decreased tourism- related business associated with adverse media coverage. The threat posed by Alexandrium species was first recognized when people eating shellfish became ill during a large bloom of an unidentified dinoflagellate in Alexandria Harbour, Egypt in 1960 (Penna et al. 2008). From this bloom Halim (1960) first described the genus. Prior to the introduction of the genus

Alexandrium, similar species were commonly classified as Gonyaulax, Protogonyaulax and

Goniodoma.

Because some Alexandrium species are toxic while others are not, it is crucial that

Alexandriums be accurately identified. At the genus level this is straightforward.

Morphologically, Alexandrium cells are small (~20-50 µm), thecate, lack spines and have a characteristic shape (Sournia 1986, Steidinger and Tangen 1997). The displacement of the girdle, where one end of the cingulum enters the sulcus relative to where the cingulum enters the other side of the sulcus, is about 1-1.5 times the girdle width (Steidinger and Tangen 1997).

Accurate identification at the species level, however, is often challenging. The general plate formula for Alexandrium species is Po, cp, 4′, 0a, 6′′, 6c, 9 or 10s, 5′′′ and 2′′′′. These plates can be visualized most accurately using scanning electron microscopy (SEM). The plates can also be observed by staining cells with Calcofluor White (Fritz and Triemer 1985) that binds preferentially to the plate boundaries, allowing the size and shape of the various thecal plates to be clearly determined using epifluorescent microscopy.

Alexandrium species are separated by small differences in the size, shape, location and ornamentation of their thecal plates. Common types of ornamentation include pores, reticulae and vermiculae. Pores are channels through the theca and can be involved in a number of active processes such as pinocytosis and extrusion of mucocysts (Steidinger and Tangen 1997).

4 Ornamentation patterns, numbers and location of pores in particular are thought to be reliable characters for distinguishing species. Raised worm-like markings on the surface of the theca are collectively known as vermiculae, whereas, reticulae are irregular or straight lines on the surface of theca that form a mesh-like network (Steidinger and Tangen 1997). Unfortunately, many of these ultrastructural characteristics can be obscured by age of the cell and by the fact that the thecal plates lay beneath the plasmalemma in living cells (Steidinger and Tangen 1997). The vermiculae and reticulae are often the last structure to form thus, a younger cell may not exhibit representative structures of the entire genus or species. Older cell plates occasionally overlap and/or lose clarity regarding connections between certain plate types and structures. In the case of chain forming Alexandrium species, stressful conditions inhibit them from forming chains.

Therefore, the ability to form chains would not be suitable criteria for identification (Penna et al.

2008). When observing the cells through scanning electron microscopy (SEM), the fixation process may lyse or distort the cells causing artifacts. Due to these potential problems, identification based strictly on plate formulas and ornamentation is tedious and requires particular care to allow accurate identifications.

Morphological characteristics used to identify species are often subtle and a potential way to supplement information obtained from morphological analyses is to employ molecular identification techniques carefully tested using confirmed cultures. Such molecular approaches are becoming more precise and cost effective (Litaker et al. 2007). In addition, molecular identification techniques can be used to address a wide variety of questions including identification of culture isolates, distribution and abundance studies and population growth estimates (La Du et al. 2002). These techniques can also be incorporated into effective harmful algal bloom (HAB) monitoring systems (Anderson et al. 2005).

5 When selecting a particular gene for use in developing species-specific assays, it is important to choose one that is evolving at a rate that is consistent with speciation events among the species of interest. An ideal gene would exhibit within-species sequence variation, which is consistently less than that of the amount of sequence divergence among species. Commonly used regions in dinoflagellate molecular research include ribosomal DNA (rDNA) which encode structural rRNAs crucial for protein synthesis (Penna et al. 2008; Litaker et al. 2007; Wang et al.

2008). The rDNA is often used in helping distinguish closely related species. The rDNA region is transcribed as a single poylcistronic RNA that includes the small subunit (SSU, 18S) gene,

Internal Transcribed Spacer 1 (ITS1), 5.8S subunit gene, Internal Transcribed Spacer 2 (ITS2) and the large subunit (LSU, 28S) gene. The ITS1 and ITS2 regions are excised by a dedicated enzyme complex that liberates the SSU, 5.8S and LSU RNAs, which subsequently form the catalytic core of the ribosomes. The SSU (~1800 bp) and LSU (~3600 bp) genes both contain sub-regions that have different rates of sequence variation. Regions such as the D1-D3 portion of the LSU gene have evolved quickly enough that they can be used to distinguish species. On the whole, however, the SSU, LSU and 5.8S genes have evolved more slowly than the ITS regions because they are under strong stabilizing selection due to their critical role they play in translation (Litaker et al. 2007). Therefore the SSU, 5.8S, and LSU regions are more useful for determining phylogenetic relationships among more distantly related species. This is especially true for species which have only recently diverged or in the case where rates of substitution are lower than average. In these instances, it is the more rapidly diverging ITS regions that can be used to distinguish species. Once the ITS region of a given dinoflagellate has been established it can be utilized as a species-specific ′DNA barcode′ (Litaker et al. 2007).

DNA barcodes or markers can be designed from such regions to target a single species. A

6 species of local concern is Alexandrium peruvianum. This species produces saxitoxins causing

PSP and was once thought to have a southernmost distribution around Cape Cod, MA. In 2004 it was identified and isolated from New River, NC (Tomas, personal communication). Its presence in local waters poses a potential threat to the local shellfish industry and the health of animals and humans that commonly use the New River. To address this threat, a major goal of this project was to collect sequence data essential to design accurate species-specific markers for quick and reliable identification of A. peruvianum. Distinguishing between two or more closely related species is key prior to the design of a species-specific probe. Alexandrium ostenfeldii is the most closely related species to A. peruvianum and the two are known to co-occur in some regions. The successful development of a specific marker requires thorough characterization of both species. Alexandrium ostenfeldii was originally described from northern Iceland by Paulsen in 1904, however, this species was also reported in temperate waters of both hemispheres

(Tillmann et al. 2007). Spirolides, known as fast-acting toxins, were originally described from

A. ostenfeldii cells isolated from Nova Scotia (Cembella et al. 2000). This species was also found to produce PSP toxins (Tillmann et al. 2007).

Current controversy surrounding whether A. peruvianum and A. ostenfeldii should be maintained as two separate species exists because of close sequence homology of the ribosomal operon (18S–28S) and similar morphological characters (Kremp et al. 2010). An additional goal of this project was to analyze the morphology of the clones from the New River and to reconcile these data with the molecular results to ensure that the Alexandrium species isolated from the

New River were indeed A. peruvianum and not A. ostenfeldii. A reconciliation of the morphology and phylogenetic data is crucial in order to ensure that any subsequent species- specific molecular assays for these species are reliable. Utilizing species-specific assays to

7 develop qPCR probes is attractive in that it aids in; 1) identification, 2) quantifying DNA in environmental samples and 3) monitoring the environment and assessing the potential need for closure of shellfish beds in order to prevent human illness. This constitutes future work and further development of the species-specific PCR assays designed in this study.

MATERIALS & METHODS

Cultures

Cultures of A. peruvianum and other unidentified Alexandrium species established by isolating a single cell from subsurface (<1 m) water samples were maintained in L1 media

(Guillard and Hargraves 1993) under temperature/salinity conditions similar to that of the isolation sites (Table 2). Clones AP0411-1, B2-NR, C10-NR and D4-NR (CMS TAC) were used for molecular and morphological studies. Cultures of A. ostenfeldii were also used in this study and obtained as CCMP1773 from the Provasoli-Guillard National Center for Culture of Marine

Phytoplankton, West Boothbay Harbor, ME and AOTVA4, AONOR4 and AOF0933 from Dr.

Anke Kremp, Finnish Environment Institute (SYKE), Helsinki, Finland. Cultures were grown in

ECG growth chambers on a 14:10 h light/dark cycle with 50-65 mol. photon quanta m2s-1.

Morphological methods

To ensure the use of comparable cells in the morphological analysis, samples in a similar growth stage were taken during log to late log phase growth at approximately 8AM each morning. Dinoflagellates divide late in the dark period and harvesting in the early morning

8 reduced the number of older cells having morphology and size changes. Once harvested, the cells were fixed using Lugol’s solution (Utermöhl 1958).

Cultures of A. peruvianum (CMS TAC AP0411-1) and A. ostenfeldii (CCMP1773) were examined morphologically along with a number of unidentified Alexandrium isolates obtained from the New River Estuary (B2-NR, C10-NR and D4-NR). In addition, isolates from

Scandinavia (AOTVA4, AONOR4 and AOF0933) were examined. One drop of Lugol’s fixed cells in addition to one drop of Calcofluor White Stain (Remel, Lenexa, KS USA) were placed on a slide and a coverslip added. In some cases cells were gently squashed by applying pressure to the coverslip using a pencil eraser but in many cases squashing was not necessary as natural separation of plates occurred with the addition of the cover slip. Cells were then examined on a

Zeiss Imager Z1 Epifluorescence microscope equipped with an AxioCam MRc5 camera. An excitation wavelength of 365 nm and an emission wavelength of 445 nm were used.

Images were taken at 40X and 100X and stored electronically. Plate measurements were calculated by using the outline or measure function in AxioVision 4.8 software. Areas were calculated using the outline tool in AxioVision and length and height measurements were calculated using the measure tool. The 1′ plate and ventral pore were each outlined and the software calculated the area of the outlined region. In addition, the s.a. plate and the 6′′ were measured using the measurement tool drawn along the tallest points and the widest points of both plates with AxioVision software calculating the length of each line. Ratios of width to height measurements for each the s.a. and 6′′ plates measured were calculated along with the ratio of the ventral pore area to 1′ plate area. Each measurement was then averaged and the standard deviation calculated. Independent groups two-tailed T-test was performed to test for significant

9 differences (http://www.dimensionresearch.com/resources/calculators/ttest.html) and to determine if the plate differences were consistent with those noted in Balech (1995).

Molecular Methods

DNA extraction - DNA was extracted using a 10% solution of Chelex. Approximately 1.5 mL of culture was transferred to a microfuge tube and spun for 3 minutes at 10,000 x g to pellet cells and supernatant was poured off. An aliquot of 0.3 mL of Chelex (10% w/v) was then added to the pelleted cells. The suspension was mixed on a vortex for 20 s, centrifuged for 3 min at

10,000 x g and incubated at 100˚ C for 20 minutes. Mixing, centrifugation and incubation was immediately repeated one more time and a final centrifuge step was added. The supernatant was then transferred into a clean 1.5 ml tube, taking care not to disturb the pellet, and stored in the

-20˚C freezer until use.

Polymerase Chain Reaction (PCR) Amplification - Partial dinoflagellate SSU, ITS1-ITS2 and the D1-D3 LSU regions were amplified using primers G22F and D2C (Table 3). PCR reactions were performed in 50 µL volumes with 10 µL of 5X Green GoTaq Reaction Buffer

(Promega, Madison, WI), 1 µL 10mM dNTP (Promega), 0.5 µL of 10 µM primers, 0.25 µL

GoTaq (Promega), 36.75 µL sterile distilled water and 1 µL DNA template. The PCR thermocycling program (Eppendorf Mastercycler Gradient, Westbury, NY) included an initial denaturing step at 94 °C for 4 min, followed by 40 cycles of DNA denaturation at 94 °C for 30 s, primer annealing at 56 °C for 45 s and fragment extension at 72 °C for 1.5 min. The final extension ran for an additional 7 min at 72 °C. PCR products were then purified using StrataPrep

PCR Purification Kit (Stratagene, La Jolla, CA) according to the manufacturer′s instructions.

10

Cloning - PCR products were used as the template in TOPO TA Cloning reactions

(K4500-01, Invitrogen, Carlsbad, CA, USA). Each reaction included 1 µL PCR template, 1 µL salt solution (1.2M NaCl, 0.06M MgCl2), 1 µL TOPO vector and the reaction was brought to a final volume of 6 µL with 3 µL of deionized water. This reaction was incubated at room temperature for a minimum of 5 min and then put on ice until use. Chemically competent E. coli cells were thawed on ice and 2 µL of the cloning reaction as per manufacturer’s instructions was gently swirled into the cells. This reaction was allowed to incubate on ice for 5–30 min. Cells were then heat shocked for 30 s in a 42˚ C water bath and immediately put back on ice and 250

µL of S.O.C. medium at room temperature was added and allowed to shake at 200 rpm in a 37˚

C incubator for 1 h. Transformed cells were then spread on 150 mm Petri dishes with LB Agar plus kanamycin and X-gal and incubated overnight at 37˚ C. White colonies containing plasmids were then selected and each placed into 3 mL of 2xYT media and grown overnight at 37˚ C with shaking (ca. 170rpm).

E. coli cells were lysed using reagents supplied by the manufacturer and plasmids were then pelleted in a centrifuge and cleaned using the Wizard Plus Minipreps DNA Purification

System (Promega, Madison, WI, USA) following the manufacturer’s instructions. Purified plasmids were then used as templates in PCR reactions using the vector primers M13F and

M13R (Table 3). PCR products yielding the correct sized fragment (~2000 bp) were purified using StrataPrep PCR Purification Kit (400773, Stratagene, La Jolla, CA) according to the manufacturer′s instructions.

11 Sequencing - Purified plasmid PCR products were used as templates in Big Dye (Applied

Biosystems, Foster City, CA, USA) cycle sequence reactions. Vector primers and internal primers were used for sequencing (Table 3). Sequencing reactions were run on a 3130xl Genetic

Analyzer (DNA Core Facility, Center for Marine Science, UNCW) and edited and aligned in

MacVector 12 (MacVector Inc., Cary, NC, USA).

Sequence Alignment – In general, most of the sequences in GenBank spanned only the 3’

SSU, the ITS/5.8S or the D1-D2 LSU region. By far the largest number of sequences were from the D1-D2 LSU region. Because there was little overlap between the sequences it was impossible to reliably align all the sequences at the same time. Consequently, separate sub alignments containing either the 3’ SSU, ITS/5.8S or the D1-D2 LSU sequences were built. Due to scarcity and high conservancy of the 3’ SSU data, it was excluded from further analysis. The

ITS/5.8S and D1-D2 LSU sequence data were aligned using the ClustalX algorithm with open and extended gap penalties of 8 and 5, respectively (Thompson et al. 1997). These alignments were used for phylogenetic analyses and for identifying unique DNA sequences, which could be used as a basis for developing species-specific assays.

Phylogenetic analysis

Phylogenetic analyses were undertaken to estimate if: 1) the sequences obtained from this study and GenBank fell into discrete genetic clades indicative of species level differences and 2) the degree to which sequences have been assigned the wrong species designation. In the latter case, if sequences with different species designations fall into the same clade it would indicate that at least some of those sequences had been ascribed to the wrong species.

12 The aligned sequences including those of A. peruvianum and A. ostenfeldii obtained from

GenBank (Table 4) were initially saved as a nexus file and MrModeltest version 3.7 used to estimate the most appropriate phylogenetic model (Nylander, 2004; Posada & Crandall, 1998).

The selected best fit model for each data set was a general time reversible model with invariant sites and gamma distribution (GTR +I+G). Phylogenetic relationships were determined using

MrBayes v3.0b41 (Ronquist and Huelsenbeck 2003). The program parameters were statefreqpr=dirichlet(1,1,1,1), nst=6, rates=invgamma, contype=halfcompatible, nswaps=2. The phylogenetic analyses employed two parallel analyses, each with 4 chains. Starting trees for each chain were selected randomly. In both parallel analyses there was one cold and three incrementally heated chains, where the heat of the ith chain is B = 1/[1 + (i - 1)T] and T = 0.01.

This allowed for more efficient swapping between chains. The analysis was run for 150,000 generations and trees were sampled from the cold chain every 100 generations. The first 1,500 generations, which constitute the “burn-in” phase, were excluded from the analysis.

Development of Species-Specific PCR Assays

The process of identifying unique species-specific sites in the ITS/5.8S and D1-D2 LSU regions was initiated by comparing only A. peruvianum and A. ostenfeldii. Because of their close sequence homology, this was the fastest way to eliminate all but the most promising sites. Once potential sites were identified, primers were designed for each species (Table 5) and tested for consistently strong amplification, and whether or not they would cross-react with genomic DNA isolated from other closely related Alexandrium species (Table 6).

13 RESULTS

Morphological Analysis

Cells from cultures AP0411-1, AOTVA4, AOF0933, B2-NR, C10-NR and D4-NR exhibited similar plate structures. The 1’ plates consistently exhibited a flat bottom side that came into contact with the top of the s.a. plate with the remaining sides angular in nature with a small ventral pore (Fig. 1). In addition, the 6’’ plate was often as tall as it was wide and the s.a. plate had an “A” shape also taller than wide. Cells from cultures AONOR4 and CCMP1773 consistently exhibited a more curved 1’ plate that came to a point where it met the s.a. plate and also contained a large ventral pore (Fig. 2). The s.a. and 6’’ plates of these cells were generally wider than tall (Fig. 2).

These morphological differences between the two groups were tested statistically using a two-tailed T-test. A significantly larger area of the 1’ plate in A. peruvianum cells (70.27 µm2) compared to A. ostenfeldii cells (33.41 µm2) was observed. The ratios of width to height measurements in the s.a. plate were significantly smaller in A. peruvianum cells (1.34) versus the

A. ostenfeldii cells (1.61) (Table 7). Also, the height of the 6’’ plate in A. peruvianum cells was significantly greater (12.02 µm) than that of A. ostenfeldii cells (9.23).

Molecular Analysis

Sequence data - Twenty rDNA sequences obtained from the A. peruvianum isolate

AP0411-1 (Accession no. JF921179-JF921198, Appendix 1) were aligned and found to be nearly identical (<0.0053 substitutions per site). In addition, eight sequences from A. ostenfeldii clone

CCMP1773 (Accession no. JF921171-JF921178, Appendix 2) were aligned with one another and found to also be highly similar (<0.0044 substitutions per site).

14

Phylogenetic Analysis

The phylogenetic analysis of the D1-D2 sequences showed that the A. peruvianum and A. ostenfeldii, fell into four distinct clades (Fig. 3). Clade 1 was composed of A. peruvianum sequences from isolates obtained from North Carolina, USA and Finland as well as two A. ostenfeldii sequences (AOTVA1 and AOTVA4) from Finland. Clade 2 consisted of A. peruvianum sequences from two isolates originating from Spain. Clades 3 and 4 were distinct and contained only sequences ascribed to A. ostenfeldii. Clade 3 contained isolates from

Canada/Scotland/Denmark and Clade 4 only isolates from New Zealand. While ITS data is less abundant than that of the D1-D2 region, an ITS/5.8S phylogeny also confirmed the geographic groupings to include clades formed by isolates of North Carolina, USA/Finland, Japan,

Spain/UK and Norway/Denmark (data not shown).

Assay development

Divergence rates within the ITS/5.8S and D1-D2 regions of A. peruvianum were 0.06 and

0.02 substitutions per site, respectively. Alignment of the D1-D2 LSU from A. peruvianum and

A. ostenfeldii indicated the presence of several potentially unique primer sites. However, when these sites were checked against an alignment of all Alexandrium D1-D2 sequences they were found to be present in other species as well.

The A. peruvianum and A. ostenfeldii ITS/5.8S alignment indicated the presence of two unique 4 bp segments in the ITS2 region. These unique sites were located at bp 406 and bp 481 of the alignment. When these sequences were compared to those found in the comprehensive

D1-D2 alignment, they appeared unique and potentially useful for probe development.

15 A series of species-specific primers designed to target these putatively unique regions of

A. peruvianum and A. ostenfeldii were used. The primary difference among these primers was that they were constructed to have the variable region occur in either the middle of the primer, or at the 5’ or 3’ end. The primer pair that produced the most consistent and robust amplification of

A. peruvianum was APF3 and APR3 located at bp position 405-425 and 479-499, respectively.

The APF3 and APR3 primer set failed to cross-react with A. ostenfeldii or the other genomic

DNA samples and is likely to be species-specific (Table 8). The resulting amplification product was 94 bp long (see gel picture, Appendix 3). The primer set that consistently amplified A. ostenfeldii was AOF4 and AOR3 located at bp position 395-414 and 473-493, respectively. The

AOF4 and AOR3 primer set did not cross-react with A. peruvianum or genomic DNA of other

Alexandrium species (Table 9).

Whether the A. peruvianum and A. ostenfeldii primer pairs would amplify genomic DNA from either Clade 2 or 4 isolates cannot be determined at this time due to the paucity of

Alexandrium ITS sequence data compared to that of the D1-D2 region for A. peruvianum and A. ostenfeldii. Our laboratory did not have access to isolates or genomic DNA from Clades 2 and 4 or sequence data needed to test cross-reactivity.

DISCUSSION

This study focused on utilizing a combination of morphology and molecular data in order to properly characterize A. peruvianum and to design a molecular assay that can unambiguously distinguish this species from other closely related Alexandrium species. This species produces saxitoxins and/or spirolides and has been recently found in waters of North Carolina (Tomas, unpublished data). However, A. peruvianum may be more widespread than previously thought

16 which makes the proper identification of this toxic species critical. Distinguishing Alexandrium species is not an easy task. Using brightfield microscopy, many Alexandrium species look identical. Plate number, structure and ornamentation are not apparent without the use of scanning electron microscopy (SEM) or fluorescent stains, such as Calcofluor. However, even with these tools preparation techniques can cause artifacts that obscure important characteristics. Due to these issues, using a combination of morphology and molecular data is key in properly characterizing and identifying Alexandrium species.

A. ostenfeldii is the most morphologically and quantitatively similar species to A. peruvianum. These species co-occur in certain parts of the world making it critically important to be able to distinguish between the two toxic species. There is some debate about whether these two species are two distinct species or whether they are part of a larger species complex. Studies performed by Kremp et al. (2010) placed tropical A. peruvianum apart from A. ostenfeldii and temperate A. peruvianum populations. The phylogenetic patterns they observed were not mirrored in the morphology or toxin composition analyzed in the same study. This controversy makes the proper characterization of both A. peruvianum and A. ostenfeldii all the more important.

Lectotype illustrations from Enrique Balech (1995) are an invaluable resource for the morphological characterization of A. peruvianum and A. ostenfeldii. These lectotypes are frequently used to aid in identifying Alexandrium species. There are often subtle differences in plate structures between Alexandrium species such as the position and size of the ventral pore or a curved versus angular 1’ plate. Because of these subtleties proper identification of Alexandrium species based on morphology takes a well-trained individual with a great deal of experience.

According to original descriptions by Balech and Tangen (1985) characteristics distinguishing A.

17 peruvianum from A. ostenfeldii are the angular nature of the 1’ plate, a smaller ventral pore and s.a. plate that is taller than wide. Morphological analyses in this study confirm those descriptions originally set forth by Balech and Tangen (1985).

Upon examination of the morphological characteristics using Calcofluor, the 1’ plate, ventral pore, 6’’ plate and s.a. plate yielded the best proxy to differentiate between these two species. Isolates AP0411-1, AOTVA4, AOF0933, B2-NR, C10-NR and D4-NR exhibited characteristic morphology of A. peruvianum. These characteristics included a 1’ plate that is angular in nature with a small ventral pore, a 6’’ plate that is often as tall as it is wide and an s.a. plate that is taller than wide. Isolates CCMP1773 and AONOR4 exhibited a 1’ plate with more curvature, a large ventral pore, a 6’’ plate that is wider than tall and an s.a. plate that is also wider than tall. Measurements of the 1’, ventral pore, 6’’ and s.a. revealed statistically significant differences (95% confidence level) in the area of the 1’ plate, height of the 6’’ and the ratio of width to height of the s.a. plate. Again, these differences agree with the descriptions by Balech and Tangen (1985) and support the idea that these are two separate species, A. peruvianum and

A. ostenfeldii.

This study has greatly increased A. peruvianum rDNA sequence data. Prior to this study there were only four A. peruvianum sequences available on GenBank and these were limited to the D1-D2 region of the LSU rDNA (i.e. FJ011436, FJ011437, FJ011438 and AM237340).

Twenty A. peruvianum sequences were generated in addition to eight A. ostenfeldii sequences and these data indicated the within genome variation among clones was relatively low and that no alternative form of the ribosomal gene was present as has been observed in A. fundyense

(Scholin et al. 1994). Not only were twenty A. peruvianum SSU – D1-D3 LSU sequences generated but according to the findings some A. peruvianum isolates appear to have been

18 misidentified and submitted to GenBank as A. ostenfeldii (FJ011432, FJ011433, FJ011439,

FJ011440).

Phylogenetic analysis of Alexandrium D1-D2 sequences from GenBank and those generated in this study were used to define the species boundaries of A. peruvianum and A. ostenfeldii. The D1-D2 region phylogenetic analysis in the present study revealed four clades;

Clade 1 - North Carolina, USA/Finland, Clade 2 – Spain, Clade 3 - Canada/Denmark/Scotland and Clade 4 - New Zealand. Clades 1 and 2 were comprised of mostly A. peruvianum with two exceptions that were ascribed to A. ostenfeldii (AOTVA1 and AOTVA4) whereas Clades 3 and 4 were comprised solely of A. ostenfeldii. While representatives from Clades 2 and 4 were not available for use, representatives of Clades 1 and 3 were available and analyzed morphologically.

Phylogenetic analysis supported multiple clades belonging to each species generating the following questions: 1) are there geographic clades within a species complex of A. peruvianum/A. ostenfeldii similar to that of the A. fundyense/A. tamarense/A. catenella species complex; 2) Do Clades 1 and 3 represent A. peruvianum and A. ostenfeldii, respectively and

Clade 2 is a population of A. peruvianum and Clade 4 is a population of A. ostenfeldii; 3) Are

Clade 1 A. peruvianum and Clade 3 A. ostenfeldii where Clades 2 and 4 represent new species?

Morphological and phylogenetic data from this study supports Clades 1 and 3 being A. peruvianum and A. ostenfeldii, respectively but to fully answer the above questions more information is necessary from Clades 2 and 4. In order to test the species complex idea, cross breeding of representative members of each clade would need to be performed in addition to toxin analysis. However, those studies were beyond the scope of this project.

19 A. peruvianum and A. ostenfeldii were clearly separated in this study both by phylogenetic and morphological analyses. This supports the notion that these are indeed two species with Clade 1 isolates belonging to A. peruvianum and Clade 3 isolates belonging to A. ostenfeldii. However, Clades 2 and 4 require further study in order to determine if they are composed of populations of A. peruvianum and A. ostenfeldii or if these are new species altogether. As seen in this study, the use of molecular data and morphological data in combination would be ideal in order to further analyze Clades 2 and 4.

After the differences between isolates were established it was possible to design species- specific molecular assays for A. peruvianum and A. ostenfeldii. The specificity of the A. peruvianum assay was tested with genomic DNA of other Alexandrium species. The assay reacted with all Clade 1 isolates and did not react with any Clade 3 isolates or other closely related Alexandrium spp. There is no way to know if the assay will react with Clade 2 isolates as they were not available for testing. Similarly, the A. ostenfeldii assay was tested against other

Alexandrium species and only reacted with Clade 3 isolates but none of the Clade 1 isolates or other DNA samples. To confirm this assay as specific additional testing is needed with representatives of Clades 2 and 4. In addition, representatives from these clades would need to be morphologically characterized and identified and probe tested before the assays designed here are labeled as all-inclusive A. peruvianum or A. ostenfeldii assays. The A. peruvianum and A. ostenfeldii specific markers designed in this study may only be Clade 1-specific for North

Carolina, USA/Finland and Clade 3-specific for Canada/Denmark/Scotland, respectively, as opposed to species-specific. Further testing of the marker on A. peruvianum isolates from Spain and surrounding regions and A. ostenfeldii isolates from New Zealand would aid in determining the specificity of the marker.

20 The combination of morphological, molecular and phylogenetic analyses was key in properly characterizing A. peruvianum and A. ostenfeldii and designing specific molecular assays that distinguish the two species. The morphological and molecular results indicated that only A. peruvianum is found in the New River Estuary, NC and that Clade 1 and Clade 3-specific PCR assays could be developed. The A. peruvianum Clade 1-specific molecular assay designed in this study will offer advantages for samples of A. peruvianum from Eastern USA. For areas near and within the Baltic Sea where A. peruvianum and A. ostenfeldii co-occur both Clade 1 and Clade 3- specific assays will be useful. These assays can be used to detect the presence of toxic A. peruvianum and A. ostenfeldii from cultured isolates or environmental samples and act as a compliment to morphology in order to properly identify these species.

Further work should be done in building a geographically diverse dataset of A. peruvianum and A. ostenfeldii sequences based on either the ITS/5.8S and/or D1-D2 LSU regions in order to allow comparisons on a larger scale. In addition, morphological analysis of representative isolates from each region should be initiated. Once these issues are addressed, specific molecular assays could be designed for each species or clade.

The development of a qPCR probe from the specific assays would be beneficial.

Quantitative polymerase chain reaction (qPCR), also referred to as real-time PCR, can be used in order to quantify initial DNA concentrations within samples. Unlike traditional PCR, qPCR quantifies DNA during the logarithmic amplification of the target (Gauthier et al. 2006).

Species-specific qPCR probes have proved useful in clinical laboratory diagnostics as well as diagnostics and detection of disease causing protists found to infect oysters (Gauthier et al.

2006). Clade 1 A. peruvianum and Clade 3 A. ostenfeldii qPCR probes would be beneficial in determining concentrations of DNA within environmental samples. This is important in

21 assessing cell concentrations within an area and can lead to a quick and reliable way to determine the potential need for closure of shellfish beds in order to prevent human illness. In regions where A. peruvianum and A. ostenfeldii co-occur, specific qPCR probes would be especially beneficial in teasing out which species is responsible for toxic blooms at any given time.

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27 Table 1. Recognized Alexandrium species and present knowledge of toxicity.

Species Toxic Alexandrium acatenella Yes Alexandrium affine No Alexandrium andersonii No Alexandrium balechii No Alexandrium camurascutulum No Alexandrium catenella Yes Alexandrium cohorticula Yes Alexandrium compressum No Alexandrium concavum No Alexandrium depressum No Alexandrium excavatum No Alexandrium foedum No Alexandrium fraterculum No Alexandrium fraterculus No Alexandrium fukuyoi No Alexandrium fundyense Yes Alexandrium hiranoi Yes Alexandrium insuetum No Alexandrium kutnerae No Alexandrium leei Yes Alexandrium margalefii No Alexandrium minutum Yes Alexandrium monilatum Yes Alexandrium ostenfeldii Yes Alexandrium peruvianum Yes Alexandrium pseudogonyaulax Yes Alexandrium satoanum No Alexandrium tamarense Yes Alexandrium tamiyavanichii Yes Alexandrium tamutum No Alexandrium taylorii No Alexandrium tropicale No

28 Table 2. Isolates of A. peruvianum and A. ostenfeldii used in this study including isolate code, location of sample, isolation date, isolator, growth temperature and salinity.

Proposed species Isolate ID Isolated from Isolation date Isolated by Temp. Salinity a A. peruvianum AP0411-1 New River, NC November 2004 Tomas, C.R. 22 15 a Alexandrium sp. B2-NR New River, NC April 2010 Tomas, C.R. 20 26 Alexandrium sp. a C10-NR New River, NC April 2010 Tomas, C.R. 20 26 Alexandrium sp. a D4-NR New River, NC April 2010 Tomas, C.R. 20 26 A. ostenfeldii b CCMP1773 Fjorden, Denmark May 1986 Hansen, PJ 15 30 A. ostenfeldii c AOTVA4 Ålond, Finland 2004 Kremp, A. 15 20 A. ostenfeldii c AOF0933 Ålond, Finland 2009 Kremp, A. 15 20

A. ostenfeldii c AONOR4 Oslofjorden, Norway Unknown Kremp, A. 15 30 a Maintained in the CMS TAC Culture Collection, UNCW, Wilmington, NC b Maintained in the CCMP Culture Collection, Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, ME c Maintained in the Finnish Environment Institute (SYKE), Marine Research Centre, Helsinki, Finland

29 Table 3. Primers used in this study to generate partial SSU, ITS1, 5.8S, ITS2 and partial LSU sequences from A. peruvianum (AP0411-1) and A. ostenfeldii (CCMP1773).

Primer name Sequence 5′ à 3′ Source Forward G22F TGGTGGAGTGATTTGTCTGG Litaker, R.W. et al. 2003 M13F GTAAAACGACGGCCAG Invitrogen, Carlsbad, CA, USA G18F CAATAACAGGTCTGTGATGC Litaker, R.W. et al. 2003

G45F CACTTAGAGGAAGGAGAAGT Vandersea, M.W. personal communication

Reverse

D2C CCTTGGTCCGTGTTTCAAGA Scholin et al. 1994

M13R CAGGAAACAGCTATGAC Invitrogen, Carlsbad, CA, USA G18R GCATCACAGACCTGTTATTG Litaker, R.W. et al. 2003 G45R ACTTCTCCTTCCTCTAAGTG Vandersea, M.W. personal communication ITSR2 TCCCTGTTCATTCGCCATTAC Litaker, R.W. et al. 2003

30 Table 4. Alexandrium D1-D2 rDNA sequences obtained from GenBank and used in combination with those of this study for the D1-D2 alignment and phylogenetic analysis.

Species Accession no. A. insuetum AF318233 A. insuetum AY962834 A. insuetum AB088249 A. insuetum AB088248 A. tamutum AJ535373 A. tamutum AJ535372 A. tamutum AY962863 A. tamutum AY268618 A. tamutum EU707459 A. tamutum AJ535354 A. tamutum AY268617 A. tamutum GQ120507 A. tamutum AY962865 A. tamutum AJ535366 A. tamutum AJ535367 A. tamutum AY962864 A. tamutum AY962862 A. tamutum AY268616 A. peruvianum FJ011437 A. peruvianum FJ011438 A. ostenfeldii FJ011437 A. ostenfeldii FJ011438 A. ostenfeldii AY962857 A. ostenfeldii AY268614 A. ostenfeldii AJ535358 A. ostenfeldii EU707483 A. ostenfeldii GQ120506 A. ostenfeldii GQ120505 A. ostenfeldii AY962858 A. ostenfeldii AJ535363 A. ostenfeldii AY268615 A. ostenfeldii AY268611 A. ostenfeldii AF033533 A. ostenfeldii AY962856 A. ostenfeldii AY268601 A. ostenfeldii AJ535357 A. ostenfeldii AY268603 31 Table 5. Primers designed to distinguish A. peruvianum (AP) and A. ostenfeldii (AO). Grey regions indicate the variable regions between the two species.

Primer Sequence (5' à 3') Forward APF1 TGTGTGCGTCAATGCTGTC APF2 CGTCAATGCTGTCGCATTAG APF3 CTGTCGCATTAGACACACGC AOF1 CTGTGTGTGTCAATGCGTGT AOF2 CTGTGTGCGTCAATGCTTTT AOF3 CGTGTGCATTCGACTCACG AOF4 TGTCAATGCGTGTGCATTCG AOF5 CGTTTGCATTAGACACATGCG AOF6 GTGTCAATGCGTTTGCATTAG AOF7 CTTTTGCATTAGACACACGCG AOF8 GCGTCAATGCTTTTGCATTAG Reverse APR1 TCCAGTGACAGAGGTTAGAC APR2 GCAACCAAATCCAGTGACAG APR3 CACATTGCAACCAAATCCAGT AOR1 GCACGTGACAGAGGTTAGA AOR2 ACCAATGCACATGACAGAGG AOR3 CATTGCAACCAATGCACATGA AOR4 GCAACCAAAGCACGTGACAG AOR5 CACATTGCAACCAAAGCACG

32 Table 6. Genomic DNA used in cross-reactivity tests of A. peruvianum and A. ostenfeldii primer sets. Species Culture ID A. affine CCMP 112 A. andersoni CCMP1718 A. catenella A. catenella A. hiranoi AH2215 A. hiranoi CCMP 2215 A. insuetum CCMP 2082 A. leei CCMP 2955 A. minutum CCMP 1888 A. monilatum A.monMiss A. monolatum A mono YK A. ostenfeldii AOTVA4 A. ostenfeldii AOF0933 A. ostenfeldii AONOR4 A. ostenfeldii CCMP1773 A. ostenfeldii CCMP 3248 A. peruvianum AP0411-1 A. tamarense CCMP 116 A. tamarense CCMP 1719 A. tamarense (clade 1) Dentist Dock A. tamarense (clade 2) CCMP1598 A. tamarense (clade 4) CCMP1771 Alexandrium sp. B2-NR Alexandrium sp. C10-NR Alexandrium sp. D4-NR

33 Table 7. Mean and standard deviation (sd) of plate measurements for A. peruvianum and A. ostenfeldii, number of samples (n) and significance (5% level) using a two-tailed student’s t-test. Measurements from AP0411-1, AOTVA4, AOF0933 were used to calculate A. peruvianum mean and sd and measurements from CCMP1773 and AONOR4 were used to calculate A. ostenfeldii mean and sd.

S.a. S.a. V. pore V. pore 1st apical width height Ratio diameter area (vp) area (1’) Ratio 6'' width 6'' height Ratio Sample (µm) (µm) (w/h) (µm) (µm2) (µm2) (vp/1’) (µm) (µm) (6''w/h)

A. peruvianum mean 6.44 4.96 1.34 1.96 2.93 70.27 0.07 18.86 12.01 1.57 sd 1.16 1.18 0.26 0.61 1.06 28.63 0.12 3.14 0.99 0.22 n= 22 22 22 14 14 14 14 8 8 8

A. ostenfeldii 6.54 4.10 1.61 2.22 3.24 33.41 0.10 16.12 9.23 1.74 mean 1.00 0.42 0.30 0.11 0.23 4.50 0.01 3.10 1.01 0.17 sd 1.00 0.42 0.30 0.11 0.23 4.50 0.01 3.10 1.01 0.17 n= 7 7 7 3 3 3 3 3 3 3 t value 0.2046 1.8708 2.3095 0.7179 0.4920 2.1696 0.4219 1.2926 4.4018 1.1962 df 27 27 27 15 15 15 15 9 9 9 Confidence level 18.06 92.78 97.12 51.61 37.02 95.35 32.09 77.16 99.74 73.78 Significant at 5%? No No Yes No No Yes No No Yes No

34 Table 8. Results of cross-reactivity tests with primer set APF3/APR3*

Species Culture ID Amplified? A. peruvianum AP0411-1 Yes

A. ostenfeldii AOTVA4 Yes A. ostenfeldii AOF0933 Yes A. ostenfeldii AONOR4 No A. ostenfeldii CCMP1773 No

A. tamarense (clade 1) Dentist Dock No A. tamarense (clade 2) CCMP1598 No A. tamarense (clade 4) CCMP1771 No A. andersoni CCMP1718 No

A. monilatum A.monMiss No A. hiranoi AH2215 No A. catenella A. catenella No Alexandrium sp. B2-NR Yes

Alexandrium sp. C10-NR Yes Alexandrium sp. D4-NR Yes

*Gel pictures included in Appendix 3.

35 Table 9. Results of qPCR assays with A. ostenfeldii primer set AOF4/AOR3*.

Species Culture ID qPCR result A. ostenfeldii CCMP 3248 + A. ostenfeldii CCMP 1773 + A. ostenfeldii AONOR4 + A. tamarense CCMP 116 - A. tamarense CCMP 1771 - A. leei CCMP 2955 - A. insuetum CCMP 2082 - A. affine CCMP 112 - A. tamarense CCMP 1719 -

A. hiranoi CCMP 2215 - A. minutum CCMP 1888 - A. monilatum A mono YK -

*Tests performed by Mark W. Vandersea, National Ocean Service at the Center for Coastal Fisheries and Habitat Research in Beaufort, North Carolina.

36

Figure 1. Calcofluor White stained cells representing A. peruvianum showing the anterior sulcal (s.a.) plates, the ventral pore (v.p.), first apical plate (1’) and the 6th precingular plate (6’’); a) AP0411-1, b) B2-NR, c) C10-NR, d) D4-NR, e) AOTVA4 and f) AOF0933.

37

Figure 2. Calcofluor White stained cells representing A. ostenfeldii Isolates showing the s.a. plates, the ventral pore (v.p.), first apical plate (1’) and the 6th precingular plate (6’’); a) CCMP1773 and b) AONOR4.

38

Figure 3. Phylogeny of A. peruvianum and A. ostenfeldii showing four distinct clades; 1) A. peruvianum from North Carolina, USA and Finland with the exception of 2 isolates identified as A. ostenfeldii, 2) A. peruvianum sequences from Spain isolates, 3) A. ostenfeldii isolates from Canada/Scotland/Denmark and 4) A. ostenfeldii isolates from New Zealand.

39 APPENDIX 1

JF921179 A. peruvianum AP0411-1 Clone 1 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAACGATTTGCACGTCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTC GTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAA ACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATT GCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGT GTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGC AGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACC AGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAG GCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCA AGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAAT TCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTT TCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACA TGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATA TACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTT CTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCA AGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTA AGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTT TACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATT GCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGG CGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTT GCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTT CAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCT GCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGAC ATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTAC CAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCAT GCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCA CAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTC ACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACT TTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATC ACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCA CAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATA ATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCA ACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGC ATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAA GAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTT CGTTAACGGAATTAACCAGACAAATCACTCCACCA

JF921180 A. peruvianum AP0411-1 Clone 2 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAACGATTTGCACGTCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTC GTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAA ACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATT GCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGT GTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGC AGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACC

40 AGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAG GCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCA AGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAAT TCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCCTTTCAAAGTCCTTT TCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACA TGGAATTTACCACCCACTTTGCATTCCAATGCTGAGGAGTGTGACTCGTCAAATATA TACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTT CTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCA AGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTA AGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTT TACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAGCACATT GCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGG CGTTTGCAAGCGCGTGTGCCTAATGCGACAGCATTGACGCACACAGCTCACAATGTT GCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTT CAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCT GCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGAC ATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTAC CAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCAT GCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCA CAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTC ACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACT TTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATC ACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCA CAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATA ATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCGACCTTTCCAGGCAAGGCA ACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGC ATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAA GAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTT CGTTAACGGAATTAACCAGACAAATCACTCCACC

JF921181 A. peruvianum AP0411-1 Clone 3 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) TATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTC ACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGT CGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCA CCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGC AGAAACATTTTGCCAGCAACAATGTGCAAGGGCCTGCAGTGCCCAGGGAGGAGAGC CCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCA CAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGC AAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCC CACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAG CAAATGCAGACTCGTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTA CTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACT TTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACT GCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCAC CTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAAT TTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTC

41 ATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATG TTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGA CAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGT GTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCC AGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAG GTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCAT ATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAAC ACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTC AGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAA ATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGA TTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTT GTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTT CAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGA GCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCA AGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCAC GATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAAC ACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTA TTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACAT AACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTA ACCAGACAAATCACTCCACC

JF921182 A. peruvianum AP0411-1 Clone 4 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAACGATTTGCACGTCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTC GTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAA ACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATT GCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACCCCTTGGTCCGT GTTTCAAGACGGGCCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGC AGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACC AGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAG GCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCA AGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAAT TCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTT TCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACA TGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATA TACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTT CTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCA AGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTA AGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTT TACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATT GCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGG CGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTT GCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTT CAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCT GCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGAC ATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTAC CAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCAT

42 GCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCA CAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTC ACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACT TTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATC ACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCA CAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATA ATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCA ACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGC ATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAA GAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTT CGTTAACGGAATTAACCAGACAAATCACTCCACC

JF921183 A. peruvianum AP0411-1 Clone 5 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAACGATTTGCACGTCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTC GTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAA ACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATT GCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGT GTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGC AGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACC AGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAG GCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCA AGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAAT TCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTT TCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACA TGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATA TACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTT CTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCA AGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTA AGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTT TACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATT GCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGG CGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTT GCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTT CAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCT GCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGAC ATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTAC CAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCAT GCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCA CAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTC ACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACT TTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATC ACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCA CAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATA ATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCA ACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAGCATCTAAGGGC ATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAA

43 GAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTT CGTTAACGGAATTAACCAGACAAATCACTCCACC

JF921184 A. peruvianum AP0411-1 Clone 6 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GAACGATTTGCACGTCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTCG TTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAA CCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTG CGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTG TTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCA GTGCCCAGGGAGAAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCA GACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGG CAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAA GGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATT CTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTT CATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACAT GGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATAT ACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTT CTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCA AGGCCGAAGGCCCCAATTTTCAAGCTGAGCGGTTCCTTGTCCATTCGCAATTACTTA AGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTT TACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATT GCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGG CGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTT GCTGAGCCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTT CAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCT GCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGAC ATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTAC CAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCAT GCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCA CAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTC ACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACT TTCCACTGCAATGTTCAAGGACTGAACACTGCTGCAGTCCGAATTATTCACCGGATC ACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCA CAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATA ATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCA ACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGC ATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAA GAAGTTGCCCACATAACCGAAATTACGTGTAGCTATTTAGCAGGTTAAGGTCTCGTT CGTTAACGGAATTAACCAGACAAATCACTCCACC

JF921185 A. peruvianum AP0411-1 Clone 7 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCA TCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCCGCTAATCGGTCGG CTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCC ACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGA AACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCT

44 CCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAA ATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAG GGTAATGATCTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCAC AAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAA ATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTG TTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGC ATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGTA AATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTG TGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTT CAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATT TTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTA AGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAG AGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTC TAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGA GATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGC AAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCA CATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATT TCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGA GCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGA TGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGT TGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTA CGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAG AACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGA CGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCT TACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATG CATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACAT CAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGC CTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACC GAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCA GACAAATCACTCCACC

JF921186 A. peruvianum AP0411-1 Clone 8 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) TATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTC ACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGT CGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCA CCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGC AGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGC CCTCCCCTTTTGGCAAGAAGGCACACACAAGAATACCAGACACACGTATCAAATCA CAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGC AAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCC CACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAG CAAATGCAGGCTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTAC TTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTT GCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTG CAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACC TGCGCCTCCATTGGTAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATT

45 TTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCA TTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGT TAAGTTTACGCGTGCAAGCTTCGGAAAACAAACACATTGCAACCAAATCCAGTGAC AGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTG TCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCA GAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGT GCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATAT CACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACAC ATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAG GAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAAT GATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATT GTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGT TACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCA AGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGC GACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAG CTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGA TGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACAC ATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTG CCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAAC CGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACC AGACAAATCACTCCACC

JF921187 A. peruvianum AP0411-1 Clone 9 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) TACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATC TTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGCCGGCT GCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCAC GAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAA CATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCC CCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAAT CATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGG TAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAA GTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAAT GCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTT TGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCAT TCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAA TGACAAAAAGGAATCTTACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCG CCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCA AGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTT CCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAG TTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAG GTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTA ATGCGACAGCATTGACGCACACAGCTCACAATGTTGTTGAACCAGCAAGCCAGAGA TTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAA ATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACA TTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTC

46 GCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGC ACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATG CAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTG TTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTTACCTACAGAAACCTTGTTACG ACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAA CTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACG GGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTA CTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCA TTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCA GTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTC AAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAA ATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGAC AAATCACTCCACC

JF921188 A. peruvianum AP0411-1 Clone 10 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCA TCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGC TGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCA CGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAA ACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTC CCCTTTTGGCAAGAAGGCACACACAAGAACACCAGATACACGTATCAAATCACAAA TCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGG GTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACA AGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAA TGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGT TTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCA TTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAA ATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGC GCCTCCATTGGCAATACATCTCAAAATTACAATTCAAGGCCGGAGGCCCCAATTTTC AAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTT TCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAA GTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGA GGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCT AATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGGACCAGCAAGCCAGAG ATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCA AATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCAC ATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTT CGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAG CACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGAT GCAGAGCCCAAGGGTTGGGGACCACACCACAGCTCACAAAGTCGTGAAAGATTGTT GTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTAC GACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGA ACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGAC GGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTT ACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGC ATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATC

47 AGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCT CAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGA AATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGA CAAATCACTCCACCA

JF921189 A. peruvianum AP0411-1 Clone 11 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) ATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCAT CTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCT GCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCAC GAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAA CATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCC CCTTTTGGCAAGAAGGCACACACAAGAATACCAGACACACGTATCAAATCACAAAT CATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGG TAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAA GTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAAT GCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTT TGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCAT TCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAA TGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCG CCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCA AGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTT CCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAG TTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAG GTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTA ATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGA TTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAA ATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACA TCTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTC GCGATTAGGATTCAATCAAAGAACGGGCGTGTTACCAAGAATTGATGTTCAGGAGC ACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATG CAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTG TTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCCACAGAAACCTTGTTACG ACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAA CTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACG GGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTA CTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCA TTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCA GTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTC AAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAA ATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGAC AAATCACTCCACC

JF921190 A. peruvianum AP0411-1 Clone 12 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) TACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATC TTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCT GCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCAC

48 GAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAA CATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCC CCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAAT CATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGG TAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAA GTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAAT GCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTT TGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCAT TCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAA TGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCG CCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCA AGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTT CCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAG TTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAG GTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTA ATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGA TTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAA ATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACA TTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTC GCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGC ACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATG CAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTG TTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACG ACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAA CTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACG GGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTA CTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCA TTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCA GTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTC AAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAA ATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGAC AAATCACTCCACC

JF921191 A. peruvianum AP0411-1 Clone 13 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCA TCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGC TGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCA CGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAA ACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTC CCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAA TCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGG GTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACA AGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAA TGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGT TTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCA TTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAA

49 ATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGC GCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTC AAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTT TCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAA GTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGA GGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCT AATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAG ATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCA AATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCAC ATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTT CGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAG CACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGAT GCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTT GTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTAC GACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGA ACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGAC GGGCGGTGTGTACAAAGGGCGGGGACGTAATCAGCACAAGCTGATGACTCAAGCTT ACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGC ATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATC AGTGTAGCACGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCT CAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGA AATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGA CAAATCACTCCACC

JF921192 A. peruvianum AP0411-1 Clone 14 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) AACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTC GGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTG GTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAAC TTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATT TTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTT TTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATT GCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAAT GATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGG ACGCAACAATCTCACCAAGTAGATCAACTCTGTTTGCTTTCTGTTCAGCAAATGCAG ACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCT ATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCA ATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGAC AAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTC CATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCT GAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCAC CGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTA CGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTA GACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCCAATGC GACAGCATTGACGCACACAGCTCACAATGTTGCTGGACCAGCAAGCCAGAGATTGG AAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTA CGTTCAAACATCTATTGGCTCACGGAATTCCGCAATTCACAATGCATATCACATTTT

50 GCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCG ATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACG GCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAG AGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTG TTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACT TCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTG AACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGG CGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACT AGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATT TTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGT GTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCA AACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAA TTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACA AATCACTCCACC

JF921193 A. peruvianum AP0411-1 Clone 15 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCA TCTTTCGGGTCCTAATAGATATGCTCAAACCCAAACCTCTGTCTGCTAATCGGTCGG CTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCC ACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGA AACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCT CCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAA ATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAG GGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCAC AAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAA ATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTG TTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGC ATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCA AATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTG CGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTT CAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATT TTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTA AGTTTACGCGTGCAAGCTTCAGAAAACAAACACGTTGCAACCAAATCCAGTGACAG AGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTC TAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGA GATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGC AAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCA CATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATT TCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGA GCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGA TGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGT TGTTGTTAGGGATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTA CGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAACGTTCAAG AACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGA CGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCT TACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATG

51 CATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACAT CAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGC CTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACC GAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCA GACAAATCACTCCACC

JF921194 A. peruvianum AP0411-1 Clone 16 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) TACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATC TTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCT GCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCAC GAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAGA CATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAAGGAGGAGAGCCCTCC CCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAAT CATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGG TAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAA GTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAAT GCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTT TGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCAT TCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAA TGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCG CCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCA AGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTT CCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAG TTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAG GTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTA ATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGA TTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAA ATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACA TTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTC GCGAGTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGC ACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATG CAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTG TTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACG ACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAA CTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACG GGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTA CTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCA TTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCA GTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTC AAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAA ATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGAC AAATCACTCCACC

JF921195 A. peruvianum AP0411-1 Clone 17 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) ATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCAT CTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCT

52 GCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCAC GAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAA CATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCC CCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAAT CATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGG TAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAA GTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAAT GCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTT TGCTATCAGCCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCAT TCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAA TGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCG CCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCA AGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTT CCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAG TTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACGGAG GTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTA ATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGA TTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAA ATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACA TTTTGCTGCATTCTTCACCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTC GCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGC ACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATG CAGAGCCCAAGGGTTGGGAACCACACCACAGCTCGCAAAGTCGTGAAAGATTGTTG TTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACG ACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAA CTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACG GGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTA CTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCA TTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCA GTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTC AAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAA ATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGAC AAATCACTCCACC

JF921196 A. peruvianum AP0411-1 Clone 18 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACC ATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCG GCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACC CACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAG AAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCC TCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACA AATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAA GGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCA CAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCA AATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTT GTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTG

53 CATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGC AAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCT GCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTT TCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCAT TTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTT AAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACA GAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGT CTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAG AGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTG CAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATC ACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACA TTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGG AGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATG ATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTG TTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTT ACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAA GAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCG ACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGC TTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGAT GCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACA TCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGC CTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACC GAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCA GACAAATCACTCCACC

JF921197 A. peruvianum AP0411-1 Clone 19 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAACGATTTGCACCCCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTC GTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAA ACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATT GCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGT GTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGC AGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACC AGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAG GCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCA AGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAAT TCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTT TCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACA TGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATA TACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTT CTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCA AGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTA AGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTT TACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATT GCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGG CGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTT GCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTT

54 CAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCT GCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGAC ATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTAC CAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCAT GCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCA CAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTC ACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACT TTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATC ACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCA CAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATA ATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCA ACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGC ATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAA GAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTT CGTTAACGGAATTAACCAGACAAATCACTCCACC

JF921198 A. peruvianum AP0411-1 Clone 20 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCA TCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGC TGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCA CGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAA ACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTC CCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAA TCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGG GTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACA AGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAA TGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGT TTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACTCACTTTGCA TTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAA ATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGC GCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTC AAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTT TCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAA GTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGA GGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCT AATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAG ATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCA AATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCAC ATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTT CGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAG CACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAGATGAT GCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTT GTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTAC GACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGA ACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGAC GGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTT

55 ACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGC ATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATC AGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCT CAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCTACATAACCGA AATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGA CAAATCACTCCACC

56 APPENDIX 2

JF921171 A. ostenfeldii CCMP1773 Clone 1 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CAAAAGGGGGGGAAAAAAACCCCGTTCTTTCCAGGGGGAAAGGGCCCCCACTACCG TAAACCCTTCACCCCTAATCAAATTTTTTGGGGGTCGAGGGGCCCGTAAAGCACTAA ATGGGAACCCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAA CGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCA AGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTA CAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTG CGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATT AAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTG AATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCG GCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGG TTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGT TATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACTGATGT GTTCAACGAGTTTGTTGCCTTGCCTGGATAGGTTGGGTAATCTTGAAAAAATGCATC GTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCT TGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTC TTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAA CATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAAC AAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAA TCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTT GCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAA CATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTT TGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAATGTGATATGCA TTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTG GGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGC TGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGC TTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCAT TGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAG CATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATG GGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGC CTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGC CTATTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCC GTGCACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCAAAGTGGGTG GTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCACACAAGTACCAT GAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGA ACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGTCCACTTGTGGGTG CAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATAT GAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTG ATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCT CCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCY TCTTRAAACACGGACCAAGGAAGGGCGAATTCCAGCACACTGGCGGCCGT

57 JF921172 A. ostenfeldii CCMP1773 Clone 2 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GGGGGGAAAAAACCCCGTTCTATTCAGGGGCGGATGGGCCCCCCTTACGTTGAAAC CCTTCCCCCCTAAATCAAAGTTTTTTTGGGGGTCGAGGGTGCGGTAAAAGCACTAAA TCGGAAACCCTAAAGGGAGCCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAA CGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCA AGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTA CAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTG CGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATT AAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTG AATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCG GCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGG TTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGT TATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACTGATGT GTTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGAAAAAATGCATC GTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCT TGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTC CTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAA CATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAAC AAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAA TCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTT GCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAA CATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTT TGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAATGTGATATGCA TTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTG GGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGC TGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGC TTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCAT TGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAG CATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATG GGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGC CTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGC CTATTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCC GTGCACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCAAAGTGGGTG GTAAATTCCATGTAAAGGAAAATATATTATTGAGGCTGATAGCAAACAAGTACCAT GAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGA ACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGTCCACTTGTGGGTG CAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATAT GAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTG ATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCT CCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCG TCTTGAAACACGGACCAAAGGGCGAATTCCAGCACACTGGCGGCCGTT

JF921173 A. ostenfeldii CCMP1773 Clone 3 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAAAAAAAACCCGTTCTTTTTCAAGGGGCGGAAGGGCCCCCCTTACCTGGGAACC CTTCCACCCTTAATTCAAATTTTTTTGGGGGTGGAGGGTGCCGTAAAACCACTAAAT

58 TGGGAACCCTAAAGGGGAGCCCCCCGATTTAGAGCTGGACGGGGAAGGCCGGCGA ACGTGCGGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGC AAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCT ACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGT GCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGAT TAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTG AATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCG GCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGG TTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGT TATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACTGATGT GTTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGAAAAAATGCATC GTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCT TGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTC CTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAA CATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAAC AAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAA TCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTT GCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAA CATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTT TGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAATGTGATATGCA TTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTG GGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGC TGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGC TTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCAT TGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAG CATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATG GGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGC CTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGC CTATTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCC GTGCACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCAAAGTGGGTG GTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAACAAGTACCAT GAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGA ACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGTGTCCACTTGTGGGTG CAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATAT GAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTG ATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCT CCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCY TCTTRAAACASGGACCAAGGAAGGGCGAATTCCAGCACACTGGCGGCCGTACTAGT GG

JF921174 A. ostenfeldii CCMP1773 Clone 4 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GTTCAAAAGGGGGGGAAAAAAACCCCGTTTATTTCAAGGGGCGAATGGCCCCCCTT TCGGGGAACCCTTCCACCCCTAAATCCAGGTTTTTTGGGGGTTCGAGGGGGCCGTAA AAGCACTAAAATCGGAACCCTAAAAGGGAGCCCCCGATTTAGACCTGGCGGGGGAA AGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAG

59 GGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTA ATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAG GGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCT GCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAAC GACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGC ATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGT GATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACAC GTAATTTCGGTTATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGA AGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGC TACACCGATGTGTTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGA AAAAATGCATCGTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATT CCTAGTAAGCTTGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACC GCCCGTCGCTCCTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTT CAGTTCTTGAACATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAG AAGTCGTAACAAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTA ACCACATCAATCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTC TGCATCATTTGCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCG TGCTCCCGAACATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGA AATGTGTTTTTGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAAT GTGATATGCATTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATT TGCACCTTTGGGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATA TCTGGCTTGCTGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCG ACTCACGCGCTTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTG TCATGTGCATTGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACAT GAAGTGAAGCATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATG AACCAAATGGGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAA AATTGGGGCCTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCA GGTGCAAGCCTATTGAAAGAAAGCGTCATTGAGGGTGAGATTCCTTTTTGTCATTTG CAGTCCTCCGTGCACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCA AAGTGGGTGGTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAAC AAGTACCATGAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGC ATTTGCTGAACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGCCCAC TTGTGGGTGCAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACC CTTGCATATGAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATT TGTGATTTGATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAG GGCTCTCCTCCCTGGGCACWGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCT GCTTGACCCGTYTTGAARCACGGACCAAGGAAGGGCGAATTCCAG

JF921175 A. ostenfeldii CCMP1773 Clone 5 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) AAGGGGGGGAAAAAAAACCCGTTTTTTTCCCAGGGGCGAATGGGCCCCCCTAACGT GAAACCCTTCCACCCTAATCCAAGTTTTTTGGGGGTCGAGGGGCCCGTAAAGCACTA ATTTCGAAACCTAAAGGGGAGCCCCCGATTTAGAGCTTGACGGGAAAAGCCGGCGA ACGTGGCGAGAAAGGAAGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCA AGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTA CAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTG CGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATT

60 AAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTG AATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCG GCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGG TTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGT TATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGCAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACTGATGT GTTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTCGGGTAATCTTGAAAAAATGCATC GTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCT TGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTC CTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAA CATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAAC AAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAA TCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTT GCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAA CATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTT TGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAATGTGATATGCA TTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTG GGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGC TGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGC TTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCAT TGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAG CATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATG GGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGC CTTCGGCCTTGAATTGTAAATTTTTGAAAATGTATTGCCAATGGAGGCGCAGGTGCA AGCCTATTGAAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTC CTCCGTGCRCGGTATATATTTGACGAGTCWCACTCCTTGGCATTGGAATGCAAAGTG GGTGGTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAACAAGTR CCATGAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTG CTGAACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGTCCACTTGTG GGTGCAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGC ATATGAATTGTAATTTGCCCGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTG ATTTGATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGC TCTCCTCCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTT G

JF921176 A. ostenfeldii CCMP1773 Clone 6 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CCCCGTTTTATTCACGGGGGGGGATGGGCCCCCCCTTTCCGGGGAAACCCTTCCCCC CCTAAATCCAAGTTTTTTTGGGGGGTGGAGGGGGCCCGTAAAAGCACTTAAATTGGA AACCCCTAAAGGGAAGCCCCCGAATTAAGAGCTTGACGGGGAAAGGCCGGGGAAC CGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCA AGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTA CAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTG CGGGCCTCTTCGCTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGAC CTTAACCTGCTAAATAGTTACACGTAATTTCGGTTATGTGGGCAACTTCTTAGAGGG ACTTTGTGTGTATAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTA GATGTTCTGGGCTGCACGCGCGCTACACTGATGTGATCAACGAGTTTGTTGCCTTGC

61 CTGGAAAGGTTGGGTAATCTTGAAAAAATGCATCGTGATGGGGATTGATTATTGCAA TTATTAATCTTAAACGAGGAATTCCTAGTAAGCTTGAGTCATCAGCTTGTGCTGATT AACAGGCTGCCCTTTGTACACACCGCTCTGGGCTGCACGCGCGCTACACTGATGTGA TCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGAAAAAATGCATCGT GATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCTTG AGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCT ACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAACA TTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAACAA GGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAATC TTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTCGGGCTCTGCATCATTTGC TCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAACA TTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTTTG CAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAATGTGATATGCATT GTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTGGG ATATGCTTGAAGGTGTGCTTGATTCAGTGTCAATTAACTTCCAATATCTGGCTTGCTG GTTCAGCAACATTGTGAGCTGTGTGTGTCAATGTGTGTGCATTCGACTCACGCGCTT GCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCATTG GTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAGCA TGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATGGG ATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGCCTT CGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGCCTA TTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCCGTG CACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCAAAGTGGGTGGTA AATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAACAAGTACCATGAG GGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGAACA GAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGTCCACTTGTGGGTGCAA TGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATATGAA TTGTAATTTGCCTGTGGGCATTGGAATGCATGTGCTTGCAATGATTTGTGATTTGATA CATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCTCCC TGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCGTYT TRAAACACGGACCAAGGAAGGGCGAATTCCAGCACACTGGCGGCCG

JF921177 A. ostenfeldii CCMP1773 Clone 7 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GGGAAAAAAACCCCGTTTTTTCCAGGGGGAGATGGGGCCCCCTTTCCGGAAACCCA TCCCCCCTTAATCCAATTTTTTTGGGGGTTGGGGGGGCCGTAAAAGCCATTAATTCG GAACCCTAAAAGGGAGCCCCCCGATTTAGAACTTGACGGGGGAAAACCGGCGAACG TGGCGAGAAAAGAAGGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGCCGCGGGCAA GTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTAC AGGGCGCGTCCATTTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGC GGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTA AGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGA ATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCGG CCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGGT TAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGTT ATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGGGGCAA TAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACTGATGTG

62 TTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGAAAAAATGCATCG TGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCTT GAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCC TACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAAC ATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAACA AGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAAT CTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTTG CTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAAC ATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTTT GCAATGAATGTCTTAGCTCAATTGATGATGAGGAATGCAGCAAAATGTGATATGCAT TGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTGG GATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGCT GGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGCT TGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCATT GGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAGC ATGTAAACCTGCTGAATTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATGGG ATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGCCTT CGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGCCTA TTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCCGTG CACGGTATATATTTGACGAGCCACACTCCTTGGCATTGGAATGCAAAGTGGGTGGTA AATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAACAAGTACCATGAG GGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGAACA GAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGTGTCCACTTGTGGGTGCAA TGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATATGAA TTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTGATA CATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCTCCC TGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCGTCT WGAAACRCGGACCAAGGAAGGGCGAATTCCAGCACACTGGCGGCCGTACTAGTGG

JF921178 A. ostenfeldii CCMP1773 Clone 8 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CCAGGGGGGGGAGTGGGCCCCCCATATACGGGGAAACCCATTCCCCCCCTTTAATC AAAATTTTTTTGGGGGGTGCGAGGGGGCCGGGTAAAAGCCCTTTAATTTGGGAACCC TAAAAAGGGAGCCCCCCGGATTTTAGAGCCTTGACGGGGAAAACCCGGGGAACCGG GGCGAGAAAGGAAGGGAAGAAAGCGAAAGGGAGCGGGCGTATGGGCGCTGGCAAA TGTAGCCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCCTTATGCGCCCGCTA CAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTG CGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATT AAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTG AATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCG GCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGG TTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGT TATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGATGCTCTGGGCTGCACGCGCGCTACACTGATGT GTTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGAAAAAATGCATC GTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCT TGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTC

63 CTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAA CATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAAC AAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAA TCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTT GCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAA CATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTT TGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAGATGTGATATGCA TTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTG GGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGC TGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGC TTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCAT TGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAG CATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATG GGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGC CTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGC CTATTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCC GTGCACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCAAAGTGGGTG GTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAACAAGTACCAT GAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGA ACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGTCCACTCGTGGGTG CAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATAT GAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTG ATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCT CCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCG TCTTGAAACRSGGACCAAGGAAGGGCGAATTCCAGCACACTGGCGGCCG

64 APPENDIX 3

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18

Gel pictures of cross reactivity tests; 1) A. peruvianum (AP0411-1), 2) A. tamarense (clade 1), 3) A. hiranoi, 4) A. ostenfeldii, 5) A. monilatum, 6) A. minutum, 7) A. tamarense (clade 4), 8) A. tamarense (clade 2), 9) A. andersoni, 10) Negative control, 11) A. peruvianum, 12) A. ostenfeldii (CCMP1773), 13) B2-NR, 14) C10-NR, 15) D4-NR, 16) AOF0933, 17) AONOR4, 18) Negative control.

65