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

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This thesis has

been formatted in accordance to the

author guidelines for the Journal of Phycology.

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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

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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.

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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.

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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.

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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

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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

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

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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

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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

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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).

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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).

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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

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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

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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

  9  

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

  10  

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.

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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.

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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.

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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).

  14  

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).

  15  

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.

  16  

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

  17  

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.

  18  

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

  19  

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.

  20  

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.

  21  

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

  22  

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.

  23  

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28  

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

  29  

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.

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

Proposed species Isolate ID Isolated from Isolation date Isolated by Temp. Salinity A. peruvianum a AP0411-1 New River, NC November 2004 Tomas, C.R. 22 15 Alexandrium sp. a 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

  30  

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

  31  

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

  32  

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

 

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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

  34  

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.

 

Sample

S.a. width (µm)

S.a. height (µm)

Ratio (w/h)

V. pore diameter

(µm)

V. pore area (vp)

(µm2)

1st apical area (1’)

(µm2) Ratio

(vp/1’) 6'' width

(µm) 6'' height

(µm) Ratio

(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

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

*Gel pictures included in Appendix 3.

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

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Table 9. Results of qPCR assays with A. ostenfeldii primer set

AOF4/AOR3*.

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

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 -

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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.  

  38  

   

 

 

 

 

 

 

 

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.    

 

  39  

   

 

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.      

  40  

APPENDIX 1

JF921179 A. peruvianum AP0411-1 Clone 1 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAACGATTTGCACGTCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACCA JF921180 A. peruvianum AP0411-1 Clone 2 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAACGATTTGCACGTCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACC

  41  

AGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCCTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCTGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAGCACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGCCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCGACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921181 A. peruvianum AP0411-1 Clone 3 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) TATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCCTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCGTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTC

  42  

ATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921182 A. peruvianum AP0411-1 Clone 4 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAACGATTTGCACGTCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACCCCTTGGTCCGTGTTTCAAGACGGGCCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCAT

  43  

GCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921183 A. peruvianum AP0411-1 Clone 5 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAACGATTTGCACGTCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAGCATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAA

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GAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921184 A. peruvianum AP0411-1 Clone 6 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GAACGATTTGCACGTCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGAAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCGGTTCCTTGTCCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAGCCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGGACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAGCTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921185 A. peruvianum AP0411-1 Clone 7 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCCGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCT

  45  

CCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATCTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGTAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGTGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921186 A. peruvianum AP0411-1 Clone 8 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) TATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAATACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGGCTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGTAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATT

  46  

TTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCGGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921187 A. peruvianum AP0411-1 Clone 9 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) TACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGCCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTTACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGTTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTC

  47  

GCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTTACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921188 A. peruvianum AP0411-1 Clone 10 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGATACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATCTCAAAATTACAATTCAAGGCCGGAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGGACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGGACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATC

  48  

AGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACCA JF921189 A. peruvianum AP0411-1 Clone 11 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) ATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAATACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATCTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGGGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCCACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921190 A. peruvianum AP0411-1 Clone 12 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) TACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCAC

  49  

GAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921191 A. peruvianum AP0411-1 Clone 13 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAA

  50  

ATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCGGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCACGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921192 A. peruvianum AP0411-1 Clone 14 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) AACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAACTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCCAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGGACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCCGCAATTCACAATGCATATCACATTTT

  51  

GCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921193 A. peruvianum AP0411-1 Clone 15 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACCCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACGTTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGGATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAACGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATG

  52  

CATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921194 A. peruvianum AP0411-1 Clone 16 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) TACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAGACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAAGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGAGTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921195 A. peruvianum AP0411-1 Clone 17 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) ATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCT

  53  

GCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGCCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACGGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCACCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCGCAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921196 A. peruvianum AP0411-1 Clone 18 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTG

  54  

CATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921197 A. peruvianum AP0411-1 Clone 19 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAACGATTTGCACCCCAGTATCGATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACCCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTT

  55  

CAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAAATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTTACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCCACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC JF921198 A. peruvianum AP0411-1 Clone 20 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GATACGAACCTCCATCAGAGTTTCCTCTGAATTCGTTCTGTTCAGGCATAGTTCACCATCTTTCGGGTCCTAATAGATATGCTCAAACTCAAACCTCTGTCTGCTAATCGGTCGGCTGCTGGTGCAAGTATCCCAGCCAATACTTTCATTGCGCAAAAAGGTTTGCCCACCCACGAACTTGCACATCTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCAAGCAGAAACATTTTGCCAGCAACAATGTGCAAGGGCTTGCAGTGCCCAGGGAGGAGAGCCCTCCCCTTTTGGCAAGAAGGCACACACAAGAACACCAGACACACGTATCAAATCACAAATCATTGCAAACACATGCATTCCAATACCCACAGGCAAATTACCATTCATGTGCAAGGGTAATGATTTGCAGAAATAGACGCTGACATTCAAGGTAAGAACCATTGCGCCCACAAGTGGACGCAACAATCTCACCAAGTAGATCAATTCTGTTTGCTTTCTGTTCAGCAAATGCAGACTCTTTTAATTCTCTTTTCAAAGTCCTTTTCATATTTCCCTCATGGTACTTGTTTGCTATCAGTCTCAATAATATATTTTCCTTTACATGGAATTTACCACTCACTTTGCATTCCAATGCCGAGGAGTGTGACTCGTCAAATATATACCGTGCACGGAGGACTGCAAATGACAAAAAGGAATCTCACCCTCATTGACGCTTTCTTTCAATAGGCTTGCACCTGCGCCTCCATTGGCAATACATTTCAAAATTACAATTCAAGGCCGAAGGCCCCAATTTTCAAGCTGAGCAGTTCCTTGTTCATTCGCAATTACTTAAGGAATCCCATTTGGTTCATTTTCCACCGCCTACTTATATGCTTAAATTCAGCAGGTTTACATGCTTCACTTCATGTTAAGTTTACGCGTGCAAGCTTCAGAAAACAAACACATTGCAACCAAATCCAGTGACAGAGGTTAGACCTAAGCAGACACGGTAAGGTTGCAAGGCGTTTGCAAGCGCGTGTGTCTAATGCGACAGCATTGACGCACACAGCTCACAATGTTGCTGAACCAGCAAGCCAGAGATTGGAAGTTAATTGACATTGAATCAAGCACACCTTCAAGCATATCCCAAAGGTGCAAATTACGTTCAAACATCTATTGGCTCACGGAATTCTGCAATTCACAATGCATATCACATTTTGCTGCATTCTTCATCATCAATTGAGCTAAGACATTCATTGCAAAAACACATTTCGCGATTAGGATTCAATCAAAGAACGAGCGTGTTACCAAGAATTGATGTTCAGGAGCACGGCAGCATGAAAGCGCTTGCTGCTGCAAGCCATGCCACCCACGAGCAGATGATGCAGAGCCCAAGGGTTGGGAACCACACCACAGCTCACAAAGTCGTGAAAGATTGTTGTTGTTAGGAATGTGCAAATGATCCTTCCGCAGGTTCACCTACAGAAACCTTGTTACGACTTCTCCTTCCTCTAAGTGATAAGGTTCATTAAACTTTCCACTGCAATGTTCAAGAACTGAACACTGCTGCAGTCCGAATTATTCACCGGATCACTCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAGCACAAGCTGATGACTCAAGCTT

  56  

ACTAGGAATTCCTCGTTTAAGATTAATAATTGCAATAATCAATCCCCATCACGATGCATTTTTTCAAGATTACCCAACCTTTCCAGGCAAGGCAACAAACTCGTTGAACACATCAGTGTAGCGCGCGTGCAGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCTTGCGTTATACACACAAAGTCCCTCTAAGAAGTTGCCTACATAACCGAAATTACGTGTAACTATTTAGCAGGTTAAGGTCTCGTTCGTTAACGGAATTAACCAGACAAATCACTCCACC

  57  

APPENDIX 2

JF921171 A. ostenfeldii CCMP1773 Clone 1 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CAAAAGGGGGGGAAAAAAACCCCGTTCTTTCCAGGGGGAAAGGGCCCCCACTACCGTAAACCCTTCACCCCTAATCAAATTTTTTGGGGGTCGAGGGGCCCGTAAAGCACTAAATGGGAACCCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGTTATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACTGATGTGTTCAACGAGTTTGTTGCCTTGCCTGGATAGGTTGGGTAATCTTGAAAAAATGCATCGTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCTTGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCTTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAACATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAACAAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAATCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTTGCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAACATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTTTGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAATGTGATATGCATTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTGGGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGCTGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGCTTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCATTGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAGCATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATGGGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGCCTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGCCTATTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCCGTGCACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCAAAGTGGGTGGTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCACACAAGTACCATGAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGAACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGTCCACTTGTGGGTGCAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATATGAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTGATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCTCCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCYTCTTRAAACACGGACCAAGGAAGGGCGAATTCCAGCACACTGGCGGCCGT

  58  

JF921172 A. ostenfeldii CCMP1773 Clone 2 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GGGGGGAAAAAACCCCGTTCTATTCAGGGGCGGATGGGCCCCCCTTACGTTGAAACCCTTCCCCCCTAAATCAAAGTTTTTTTGGGGGTCGAGGGTGCGGTAAAAGCACTAAATCGGAAACCCTAAAGGGAGCCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGTTATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACTGATGTGTTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGAAAAAATGCATCGTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCTTGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAACATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAACAAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAATCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTTGCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAACATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTTTGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAATGTGATATGCATTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTGGGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGCTGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGCTTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCATTGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAGCATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATGGGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGCCTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGCCTATTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCCGTGCACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCAAAGTGGGTGGTAAATTCCATGTAAAGGAAAATATATTATTGAGGCTGATAGCAAACAAGTACCATGAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGAACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGTCCACTTGTGGGTGCAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATATGAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTGATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCTCCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCGTCTTGAAACACGGACCAAAGGGCGAATTCCAGCACACTGGCGGCCGTT JF921173 A. ostenfeldii CCMP1773 Clone 3 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CGAAAAAAAACCCGTTCTTTTTCAAGGGGCGGAAGGGCCCCCCTTACCTGGGAACCCTTCCACCCTTAATTCAAATTTTTTTGGGGGTGGAGGGTGCCGTAAAACCACTAAAT

  59  

TGGGAACCCTAAAGGGGAGCCCCCCGATTTAGAGCTGGACGGGGAAGGCCGGCGAACGTGCGGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGTTATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACTGATGTGTTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGAAAAAATGCATCGTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCTTGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAACATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAACAAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAATCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTTGCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAACATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTTTGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAATGTGATATGCATTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTGGGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGCTGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGCTTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCATTGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAGCATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATGGGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGCCTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGCCTATTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCCGTGCACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCAAAGTGGGTGGTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAACAAGTACCATGAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGAACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGTGTCCACTTGTGGGTGCAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATATGAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTGATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCTCCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCYTCTTRAAACASGGACCAAGGAAGGGCGAATTCCAGCACACTGGCGGCCGTACTAGTGG JF921174 A. ostenfeldii CCMP1773 Clone 4 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GTTCAAAAGGGGGGGAAAAAAACCCCGTTTATTTCAAGGGGCGAATGGCCCCCCTTTCGGGGAACCCTTCCACCCCTAAATCCAGGTTTTTTGGGGGTTCGAGGGGGCCGTAAAAGCACTAAAATCGGAACCCTAAAAGGGAGCCCCCGATTTAGACCTGGCGGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAG

  60  

GGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGTTATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACCGATGTGTTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGAAAAAATGCATCGTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCTTGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAACATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAACAAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAATCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTTGCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAACATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTTTGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAATGTGATATGCATTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTGGGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGCTGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGCTTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCATTGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAGCATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATGGGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGCCTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGCCTATTGAAAGAAAGCGTCATTGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCCGTGCACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCAAAGTGGGTGGTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAACAAGTACCATGAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGAACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGCCCACTTGTGGGTGCAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATATGAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTGATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCTCCCTGGGCACWGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCGTYTTGAARCACGGACCAAGGAAGGGCGAATTCCAG JF921175 A. ostenfeldii CCMP1773 Clone 5 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) AAGGGGGGGAAAAAAAACCCGTTTTTTTCCCAGGGGCGAATGGGCCCCCCTAACGTGAAACCCTTCCACCCTAATCCAAGTTTTTTGGGGGTCGAGGGGCCCGTAAAGCACTAATTTCGAAACCTAAAGGGGAGCCCCCGATTTAGAGCTTGACGGGAAAAGCCGGCGAACGTGGCGAGAAAGGAAGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATT

  61  

AAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGTTATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGCAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACTGATGTGTTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTCGGGTAATCTTGAAAAAATGCATCGTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCTTGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAACATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAACAAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAATCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTTGCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAACATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTTTGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAATGTGATATGCATTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTGGGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGCTGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGCTTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCATTGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAGCATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATGGGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGCCTTCGGCCTTGAATTGTAAATTTTTGAAAATGTATTGCCAATGGAGGCGCAGGTGCAAGCCTATTGAAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCCGTGCRCGGTATATATTTGACGAGTCWCACTCCTTGGCATTGGAATGCAAAGTGGGTGGTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAACAAGTRCCATGAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGAACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGTCCACTTGTGGGTGCAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATATGAATTGTAATTTGCCCGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTGATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCTCCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTG JF921176 A. ostenfeldii CCMP1773 Clone 6 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CCCCGTTTTATTCACGGGGGGGGATGGGCCCCCCCTTTCCGGGGAAACCCTTCCCCCCCTAAATCCAAGTTTTTTTGGGGGGTGGAGGGGGCCCGTAAAAGCACTTAAATTGGAAACCCCTAAAGGGAAGCCCCCGAATTAAGAGCTTGACGGGGAAAGGCCGGGGAACCGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGTTATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACTGATGTGATCAACGAGTTTGTTGCCTTGC

  62  

CTGGAAAGGTTGGGTAATCTTGAAAAAATGCATCGTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCTTGAGTCATCAGCTTGTGCTGATTAACAGGCTGCCCTTTGTACACACCGCTCTGGGCTGCACGCGCGCTACACTGATGTGATCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGAAAAAATGCATCGTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCTTGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAACATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAACAAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAATCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTCGGGCTCTGCATCATTTGCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAACATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTTTGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAAATGTGATATGCATTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTGGGATATGCTTGAAGGTGTGCTTGATTCAGTGTCAATTAACTTCCAATATCTGGCTTGCTGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGTGTGTGCATTCGACTCACGCGCTTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCATTGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAGCATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATGGGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGCCTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGCCTATTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCCGTGCACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCAAAGTGGGTGGTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAACAAGTACCATGAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGAACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGTCCACTTGTGGGTGCAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATATGAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGCTTGCAATGATTTGTGATTTGATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCTCCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCGTYTTRAAACACGGACCAAGGAAGGGCGAATTCCAGCACACTGGCGGCCG JF921177 A. ostenfeldii CCMP1773 Clone 7 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) GGGAAAAAAACCCCGTTTTTTCCAGGGGGAGATGGGGCCCCCTTTCCGGAAACCCATCCCCCCTTAATCCAATTTTTTTGGGGGTTGGGGGGGCCGTAAAAGCCATTAATTCGGAACCCTAAAAGGGAGCCCCCCGATTTAGAACTTGACGGGGGAAAACCGGCGAACGTGGCGAGAAAAGAAGGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGCCGCGGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGTTATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGGGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACTGATGTG

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TTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGAAAAAATGCATCGTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCTTGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAACATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAACAAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAATCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTTGCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAACATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTTTGCAATGAATGTCTTAGCTCAATTGATGATGAGGAATGCAGCAAAATGTGATATGCATTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTGGGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGCTGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGCTTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCATTGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAGCATGTAAACCTGCTGAATTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATGGGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGCCTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGCCTATTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCCGTGCACGGTATATATTTGACGAGCCACACTCCTTGGCATTGGAATGCAAAGTGGGTGGTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAACAAGTACCATGAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGAACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGTGTCCACTTGTGGGTGCAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATATGAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTGATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCTCCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCGTCTWGAAACRCGGACCAAGGAAGGGCGAATTCCAGCACACTGGCGGCCGTACTAGTGG JF921178 A. ostenfeldii CCMP1773 Clone 8 – 18S, ITS1, 5.8S, ITS2, 28S (D1-D3) CCAGGGGGGGGAGTGGGCCCCCCATATACGGGGAAACCCATTCCCCCCCTTTAATCAAAATTTTTTTGGGGGGTGCGAGGGGGCCGGGTAAAAGCCCTTTAATTTGGGAACCCTAAAAAGGGAGCCCCCCGGATTTTAGAGCCTTGACGGGGAAAACCCGGGGAACCGGGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGGAGCGGGCGTATGGGCGCTGGCAAATGTAGCCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCCTTATGCGCCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTTACACGTAATTTCGGTTATGTGGGCAACTTCTTAGAGGGACTTTGTGTGTATAACGCAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGCTCTGGGCTGCACGCGCGCTACACTGATGTGTTCAACGAGTTTGTTGCCTTGCCTGGAAAGGTTGGGTAATCTTGAAAAAATGCATCGTGATGGGGATTGATTATTGCAATTATTAATCTTAAACGAGGAATTCCTAGTAAGCTTGAGTCATCAGCTTGTGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTC

  64  

CTACCGATTGAGTGATCCGGTGAATAATTCGGACTGCAGCAGTGTTCAGTTCTTGAACATTGCAGTGGAAAGTTTAATGAACCTTATCACTTAGAGGAAGGAGAAGTCGTAACAAGGTTTCTGTAGGTGAACCTGCGGAAGGATCATTTGCACATCCCTAACCACATCAATCTTTCACGACTTTGTGAGCTGTGGTGTGATTCCCAAGCCTTGGGCTCTGCATCATTTGCTCGTGGGTGGCATGGCTTGCAGCAGCAAGTGCTTTCATGCTGCCGTGCTCCCGAACATTATTTCTTGGCTACACGTCGTTCTTTGATTGAATCCTAATCGCGAAATGTGTTTTTGCAATGAATGTCTTAGCTCAATTGATGATGAAGAATGCAGCAAGATGTGATATGCATTGTGAATTGCAGAATTCCGTGAGCCAATAGATGTTTGAACGCAATTTGCACCTTTGGGATATGCTTGAAGGTGTGCTTGATTCAATGTCAATTAACTTCCAATATCTGGCTTGCTGGTTCAGCAACATTGTGAGCTGTGTGTGTCAATGCGTGTGCATTCGACTCACGCGCTTGCAAACGCCTTGCATCCTTATCGTGTCTGCTTAGGTTGAACCTCTGTCATGTGCATTGGTTGCAATGTGTTTGTGTCTGAAGCTTGCACGCGTAAACTTAACATGAAGTGAAGCATGTAAACCTGCTGAATTTAAGCATATAAGTAGGCGGTGGAAAATGAACCAAATGGGATTCCTTAAGTAATTGCGAATGAACAAGGAACTGCTCAGCTTGAAAATTGGGGCCTTCGGCCTTGAATTGTAATTTTGAAATGTATTGCCAATGGAGGCGCAGGTGCAAGCCTATTGAAAGAAAGCGTCAATGAGGGTGAGATTCCTTTTTGTCATTTGCAGTCCTCCGTGCACGGTATATATTTGACGAGTCACACTCCTTGGCATTGGAATGCAAAGTGGGTGGTAAATTCCATGTAAAGGAAAATATATTATTGAGACTGATAGCAAACAAGTACCATGAGGGAAATATGAAAAGGACTTTGAAAAGAGAATTAAAAGAGTCTGCATTTGCTGAACAGAAAGCAAACAGAATTGATCTACTTGGTGAGATTGTTGCGTCCACTCGTGGGTGCAATGGTTCTTGCCTTGAATGTCAGCGTCTATTTCTGCAAATTATTACCCTTGCATATGAATTGTAATTTGCCTGTGGGCATTGGAATGCATGTGTTTGCAATGATTTGTGATTTGATACATGTGTCTGGTGTTCGCGTGTGTGCCTTCTTGCTAACACGGGGAGGGCTCTCCTCCCTGGGCACTGCATGCCCTTGCACATTGTTGCTGGCAAAATGTTTCTGCTTGACCCGTCTTGAAACRSGGACCAAGGAAGGGCGAATTCCAGCACACTGGCGGCCG

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APPENDIX 3

               

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.

   

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18