Phylogenetics of Lymnaeidae (Mollusca: Gastropoda) with ...
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Eastern Illinois University e Keep Masters eses Student eses & Publications 2002 Phylogenetics of Lymnaeidae (Mollusca: Gastropoda) with Emphasis on the Evolution of Susceptibility to Fascioloides magna Infection Sarah Renee Joyce Eastern Illinois University is research is a product of the graduate program in Biological Sciences at Eastern Illinois University. Find out more about the program. is is brought to you for free and open access by the Student eses & Publications at e Keep. It has been accepted for inclusion in Masters eses by an authorized administrator of e Keep. For more information, please contact [email protected]. Recommended Citation Joyce, Sarah Renee, "Phylogenetics of Lymnaeidae (Mollusca: Gastropoda) with Emphasis on the Evolution of Susceptibility to Fascioloides magna Infection" (2002). Masters eses. 1496. hps://thekeep.eiu.edu/theses/1496
Transcript of Phylogenetics of Lymnaeidae (Mollusca: Gastropoda) with ...
Eastern Illinois UniversityThe Keep
Masters Theses Student Theses & Publications
2002
Phylogenetics of Lymnaeidae (Mollusca:Gastropoda) with Emphasis on the Evolution ofSusceptibility to Fascioloides magna InfectionSarah Renee JoyceEastern Illinois UniversityThis research is a product of the graduate program in Biological Sciences at Eastern Illinois University. Findout more about the program.
This is brought to you for free and open access by the Student Theses & Publications at The Keep. It has been accepted for inclusion in Masters Thesesby an authorized administrator of The Keep. For more information, please contact [email protected].
Recommended CitationJoyce, Sarah Renee, "Phylogenetics of Lymnaeidae (Mollusca: Gastropoda) with Emphasis on the Evolution of Susceptibility toFascioloides magna Infection" (2002). Masters Theses. 1496.https://thekeep.eiu.edu/theses/1496
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Phylogenetics of Lymnaeidae (Mollusca: Gastropoda) with Emphasis on the
Evolution of Susceptibility to Fascioloides magna Infection
(TITLE)
BY
Sarah Renee Joyce
THESIS
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
Master of Science
IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS
2002 YEAR
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Acknowledgements
I am grateful to:
My co-advisors Dr. Jeffrey Laursen and Dr. Mark Mort for supporting me both
professionally and personally throughout my graduate program, and challenging me to
honor myself and my goals. I especially thank both for blessing me with their continued
friendship.
Dr. Charles Pederson who served on my thesis committee, and always proved a
valuable resource for research endeavors and professional advice.
Department of Biology at Eastern Illinois University, especially Dr. Kipp Kruse
and Dr. Andrew Methven, for financial support, and promoting research in molecular
systematics.
Sigma Xi for funding me with a Grant-In-Aid-of-Research Award, as •veil as the
Graduate School at Eastern Illinois University for a Research/Creative Activity Award.
Park Managers and Conservation Officers at St. Croix State Park, MN, for their
hospitality, support, and enthusiasm regarding this study.
Scott Meiners, Bud Fischer, Gary Averbeck, Randy Delong, Jess Morgan, Amy
Wethington, Sam Loker, Aparna Palmer, Nick Owens, Michael Douglas, and Mark
Druffel for their assistance in systematic methods and taxon sampling.
Many family members and friends whose shoulders I have had the great privilege
of leaning on over the last two years, and who continue to inspire me every day with their
love, courage, and strength of character.
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Abstract
Snails in Lymnaeidae serve as intermediate hosts in the transmission of many
trematode species, including Fascioloides magna that is responsible for disease and death
in domestic livestock in North America. Previous classifications oflymnaeid snails have
relied primarily on morphological characters that exhibit high levels of homoplasy;
thereby, impeding a sound assessment ofrelationships in this group. The present study
provides a phylogenetic hypothesis for lymnaeid snails employing sequences from the
internal transcribed spacer (ITS) region of nrDNA, and addresses the evolution of
susceptibility to Fascioloides infection in lymnaeid snails. The final data set, comprising
ten species of lymnaeid snails and one species of Physidae, included 1368 characters, of
which 327 were parsimony-informative. Three major clades were recovered in neighbor
joining analyses that consisted of individuals of Stagnicola caperata, Fossaria s.s., and
Stagnicola s.s. + F. Bakerlymnea sp. Stagnicola caperata, the main host of F. magna in
Minnesota, revealed a closer alliance to Fossaria spp. than to other species of Stagnicola,
suggesting that its placement in the stagnicoline sub-genus Hinkleyia is suspect.
Members of Fossaria s.s., that have tricuspid first lateral teeth in the radula, were
monophyletic to the exclusion of F. Bakerlymnea, a well-supported member of the
stagnicoline clade. Therefore, our estimate of lymnaeid phylogeny supports the
taxonomic scheme proposed by Baker (1911) that suggests members of Bakerlymnea be
classified as stagnicolines based on their shared bicuspid dentition. Although a
stagnicoline clade was strongly supported, there was low resolution of species within the
clade. Log-determinent distances between species of Stagnicola s.s. were less than those
observed between individuals of Stagnicola caperata, indicating that a region with higher
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rates of evolution is necessary to determine relationships in this group. Susceptibility to
Fascioloides magna infection is widespread in North American lymnaeid snails based on
experimental infections. However, an examination of naturally infected intermediate
hosts suggests that host status may be due to high exposure rates that result from close
interactions between intermediate and definitive hosts.
l\'
Table of Contents
Title Page .................................................. i
Acknowledgements .......................................... ii
Abstract .................................................. iii
Table of Contents ........................................... v
Introduction ................................................ 1
Materials and Methods ....................................... 7
Results . ................................................... 10
Discussion ................................................. 13
Literature Cited ............................................ 20
Tables .................................................... 32
Figures ................................................... 35
Appendix 1 ............................................... 47
v
As buds give rise by growth to fresh buds, and these, if vigorous, branch out and overtop on all sides many a feebler branch, so by generation I believe it has been with the great Tree of Life, which fills with its dead and broken branches the crust of the earth, and covers the surface with its ever branching and beautiful ramifications.
Charles Darwin On the Origin of Species
Introduction
Lymnaeidae (Rafinesque, 1815) is a group of primarily aquatic snails that are
cosmopolitan in distribution, occurring in a variety of ecological habitats. The greatest
diversity of lymnaeid snails is found in the extensive freshwater ecosystems of the
northern United States and central Canada (Burch, 1982). Taxonomically, this group is
quite complex with between 57 to 100 species historically recognized in North America
alone (e.g., Baker, 1911; Hubendick, 1951; Clarke, 1973; Burch, 1982). Clearly,
classification of this group remains controversial despite considerable efforts. In
addition, lymnaeid snails serve as intermediate hosts for more than 70 trematode species
world-wide (Brown, 1978). Therefore, significance in investigating this group ranges
from taxonomic uncertainty to their role in transmitting parasitic disease.
On the American continents, lymnaeid snails serve as intermediate hosts for
fasciolid trematodes, a group of major medical and economic concern. Fascia/a
hepatica, the causative agent of fascioliasis in humans and other mammals, is emerging
as a medical concern in parts of Central and South America, as well as the Caribbean
(Mas-Coma et al., 1999a, 1999b). In North America, F. hepatica andFascio/oides
magna, the giant liver fluke, are causative agents of animal diseases in both wild and
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domesticated mammals. The definitive host of F. magna in North America is white
tailed deer, Odocoileus virginianus (Cheatum, 1951; Griffiths, 1962; Dutson et al., 1967;
Pursglove et al., 1977; Addison et al., 1988); however, domestic livestock sympatric with
deer are at risk to F. magna infection (Figure 1 ). Infection in cattle has resulted in
economic loss due to chronic disease and condemnation of livers at slaughter. In sheep
infection often results in death (Foreyt and Todd, 1976a, b). Fascioloides magna has a
disjunct range in North America (Figure 2), with isolated populations reported from the
Pacific Northwest, Rocky Mountains, Gulf Coast, and Great Lakes regions (Cheatum,
195l;Griffiths, 1962; Knapp and Shaw, 1963;Dutsonetal., 1967;Behrandetal., 1973;
Pursglove et al., 1977; Foreyt, 1981; Schillhorn Van Veen, 1987; Knapp et al., 1992).
This is likely due to the requirement for suitable snail intermediate hosts and cervid
definitive hosts to complete the life cycle (Foreyt, 1990). Stagnicola caperata, S. elodes
(also called S. palustris), Fossaria parva, F. modicella, and F. bulimoides are the only
documented natural hosts of Fascioloides magna in North America (Sinitsin, 1930, 1933;
Swales, 1935; Griffiths, 1959; Laursen, 1993). However, Stagnicola catascopium, S.
exilis, Lymnea stagnalis, Pseudosuccinea columella, F. obrussa rustica, and F.
ferruginea have been infected in laboratory studies (Krull, 1933a, 1933b, 1934; Dutson et
al., 1967; Griffiths, 1973; ). Due to the reliance of F. magna on lymnaeid snails for
transmission, confidence in lymnaeid taxonomy is imperative for correct identification of
intermediate hosts, and a robust phylogeny is necessary to evaluate host-parasite
relationships.
Previous classifications of lymnaeid snails have defined species based upon shell
morphology, reproductive morphology, and radular teeth formulas, along with some
emphasis on ecology. However, identification and classification has been compromised
by morphological and anatomical similarity between species of some genera (Bargues et
al., 2001), while other species exhibit high intraspecific variation in the presence of
different environmental conditions (Burch, 1982). Therefore, there is little consensus
among the available taxonomic keys. Different taxonomic schemes have recognized a
single genus (Walter, 1968), two genera (Hubendick, 1951; Clarke, 1973), and up to
seven genera (Baker, 1911; Burch, 1965, 1982) in the family. Proposed genera include
Lymnea, Bulimnea, Stagnicola, Fossaria, Radix, Psuedosuccinea, and Ace/la (Burch,
1982). Use of sub-groups within genera, of which many are considered artificial
(Hubendick, 1951; Walter, 1968), has contributed to the taxonomic confusion by putting
increased weight on characters like shell morphology that may exhibit extreme
homoplasy. Studies of immunology (Burch, 1968; Burch and Lindsay, 1968), karyology
(Burch, 1965; Inaba, 1969), cross-breeding (Burch and Ayers, 1973), and enzyme
electrophoresis (Rudolph and Burch, 1989) have either provided low systematic
resolution or disagreed with results from morphological examinations (Bargues et al.,
2001). Morphological examinations of reproductive organs have been useful in
differentiating some lymnaeid species, and may be more reliable than other
morphological characters for phylogeny reconstruction in this group (Hubendick, 1978;
Jackiewicz, 1988; Gloer and Meier-Brook, 1998).
Molecular data have been useful in evaluating relationships of European (Bargues
et al., 1997,2001; Bargues and Mas-Coma, 1997) and a limited number of North
American (Remigio and Blair, 1997a, 1997b; Remigio, in press) lymnaeid snails.
Sequences from 18S nrDNA have provided initial insights into lymnaeid relationships
3
(Bargues et al., 1997; Bargues and Mas-Coma, 1997). However, this region is highly
conserved, and may not be appropriate for species-level differentiation. Previous
analyses of 16S mtDNA (Remigio and Blair, 1997b; Remigio, in press) and the internal
transcribed spacer (ITS) from nrDNA (Remigio and Blair, 1997a; Bargues et al., 200 l)
have shown promise in resolving relationships among lymnaeid genera. For example,
sequences from 16S mtDNA revealed a paraphyletic relationship among stagnicoline
snails, and produced a topology that disputed previous hypotheses regarding the
evolution of chromosome number in Lymnaeidae (Remigio and Blair, 1997b ). In
addition, parsimony analyses ofITS sequences revealed that Stagnicola caperata did not
cluster with three additional species of Stagnicola s.s. (S. catascopium, S. emarginata,
and S. elodes), thus suggesting this taxon may deserve recognition as a distinct genus
(Remigio and Blair, l 997a). Stagnicola caperata has previously been classified as a
stagnicoline based on size, radular dentition, and, to a limited extent, shell morphology
(Burch, 1982). However, differences between S. caperata and other stagnicolines do
exist, most notably observed in the smaller size and pronounced periostracal ridges on
shells of S. caperata. In addition, S. caperata and species of Fossaria serve as the main
natural intermediate hosts for F. magna in the United States, which might indicate a close
alliance between these two groups. Determining the taxonomic position of Stagnicola
caperata will help to develop hypotheses regarding host susceptibility in Lymnaeidae.
Phylogenetic models have provided interesting insights into the evolution of
symbioses among a diverse array of organisms (Moran et al., 1995; Herre et al., 1996;
Clark et al., 2000), including parasites and their hosts (Hafner and Page, 1995; Baker et
al., 1998; Hugot, 1998; DeJong et al., 2001; Xiao et al., 2001; Refardt et al., 2002).
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Molecular data, in particular, allow for robust analyses of host-parasite relationships by
providing independent estimates of phylogeny for both the host and parasite lineage(s)
(Page et al., 1998), while also reducing the phylogenetic noise that results from
morphological characters highly influenced by the association (Downes, 1990). In
addition, studies that have used molecular, morphological, and parasitiological data in
concert (e.g., Hugot, 1998) have been more successful in resolving host phylogeny.
In the absence of a robust host phylogeny, associations between host and parasite
can be useful in evaluating relationships. Xiao et al. (2001) used Hexamita infections of
fish in the family Xenocyprinae as a character for phylogenetic analysis, along with
segments from the mitochondrial genome, and found that host phylogeny based on
parasite fauna was congruent with that based on mtDNA sequences. Snail-trematode
interactions show equal promise for revealing patterns of parallel evolution due to high
host specificity exhibited by most digenean trematodes for their molluscan host (Roberts
and Janovy, 1996). For instance, mapping patterns of susceptibility to trematode
infection on snail phylogeny has been used in the development of hypotheses regarding
host and parasite biogeography, as well as predicting new potential host species due to
their genetic similarity to known host species (DeJong et al., 2001). Similar patterns
have been found between lymnaeid snails and fasciolid trematodes (Bargues and Mas
Coma, 1997), but these studies have not included North American snails or Fascioloides
magna. I hypothesize that inclusion of North American lymnaeid snails in the
phylogeny, in addition to mapping Fascioloides susceptibility, will provide additional
insights into the evolution of fasciolid susceptibility in lymnaeid snails, allowing for
inference of the ancestral character states and the origins of susceptibility.
5
My goals are to estimate phylogeny for lymnaeid snails in enzootic fluke regions
of Minnesota using internal transcribed spacer sequences, and use this phylogenetic
hypothesis as an evolutionary framework to examine the phylogenetic distribution of
susceptibility to Fascioloides magna infection in lymnaeid snails. In addition, the
evolution of radular dentition will be explored on this framework, as it is typically used to
define species of Fossaria.
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lVIethods
Sampling of Fascioloides magna and Lymnaeid Snails
Fascioloides magna adults and eggs were collected from white-tailed deer in
November of 1999, 2000, and 2001 during fire-arm hunts supervised by the MnDNR at
St. Croix State Park, MN. Fascioloides magna adults were collected by slicing the liver
in approximately 0.25 inch intervals, and removing worms from cysts within the organ.
Adult worms were deposited into warm, 0.9% saline solution or distilled water for
several hours to shed eggs. Eggs were concentrated by sedimentation, washed several
times with distilled water, and incubated at room temperature until fully embryonated (-3
weeks). They were then kept at 4°C to suspend development, so that small aliquots of
eggs could be hatched as needed for infection studies.
Lymnaeid snails were collected from eight sites in six counties in Minnesota.
Wetland habitats (sensu Eggers and Reed, 1987) sampled were emphemeral
ponds/ditches, marshes, prairie potholes, lakes, and streams. Specimens either were kept
alive in containers of water packed on ice or were fixed in 100% ETOH in the field.
Snails were identified based on shell morphology and radular dentition using available
taxonomic keys (e.g., Clarke, 1973; Burch, 1982). Eight putative species from three
genera within Lymnaeidae were identified (Table 1): one from Lymnea (stagnalis); four
from Stagnicola (caperata, catascopium, exilis, and elodes); and three from Fossaria
(Bakerlymnea sp., obrussa rustica, and parva). Snail colonies were established in
separate 10-gallon aquaria for each of five species of lymnaeid snails (S. caperata, S.
exilis, S. elodes, S. stagnalis and S. catascopium) for use in infection studies. A species
from the genus Physa, a proposed sister group to Lymnaeidae (Hubendick, 1947, 1978),
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was collected for use as an outgroup, and sequences for five additional lymnaeid snails
were retrieved from GenBank to compliment taxon sampling (Table 1).
Infection Study
Individuals of L. stagnalis, S. exilis, and two populations of S. caperata were
exposed to 3-6 miracidia in a 48-well plate for 3 hours in modification of Foreyt and
Todd (1978) and Laursen (1993). An infection tank was established for each species
where snails were housed and fed frozen lettuce ad libidum for 40 days to allow for larval
amplification. On day 40 post-infection, snails were crushed to determine infection
status. Individual species were coded as susceptible to infection if any individual of that
species contained redia or cercaria, or susceptibility based on experimental infection was
known from previous studies.
Phylogenetic Study
Whole DNA was extracted from either frozen or preserved foot tissue using a
HotSHOT protocol (Truett et al, 2000) modified for snails (J.A.T. Morgan, pers. comm.).
A small amount of tissue (- 3 mg) from each snail was dissected and pulverized in a 1.5
ml tube using sterile micro-grinders with 150 µL of an alkaline lysis reagent (25 mM
NaOH and 0.2 mM disodium EDTA at pH 12). Ground samples were incubated at 95°C
for one hour, then cooled to 25°C prior to the addition of 150 µL of neutralizing reagent
(40 mM Tris-HCl at pH 5). Presence of whole DNA was verified by electrophoreses on a
1 % agarose gel followed by staining with ethidium bromide. The complete ITS region
(ITSl- 5.8S - ITS2) of the nrDNA was amplified using the primers BDl and BD2 of
Degnan et al. (1990). Individual reactions contained 1 µL of template DNA in a 100 µL
reaction mixture with sterile double-distilled water, 0.2 mM of dNTP' s, 1 OX PCR buffer,
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5% DMSO, 0.25 ~tM primer concentrations, and one-half unit of TAQ polymerase.
Amplification used 40 cycles of 94°C for 45 seconds, 48°C for one minute, and 72°C for
four minutes. PCR products were purified using QIAquick~ PCR purification kit
following manufacturers instructions (QIAGEN Inc., Valencia, CA) and were
resuspended in 35-50 µL sterile double-distilled water. DNA fragments were sequenced
using external primers BDl and BD2 (Degnan et al., 1990), internal primers ERl and
ER2 (Remigio and Blair, l 997a), and a Dye Terminator Cycle Sequencing Kit
(Beckman-Coulter, Fullerton, CA), and visualized on a Beckman-Coulter CEQ 2000XL
automated sequencer. Sequences were edited with Sequencher (Gene Codes, Ann Arbor,
MI), and edited contigs were initially aligned using ClustalX (Thompson et al., 1997)
followed by visual adjustment. Distance and Parsimony analyses were conducted using
PAUP* (Swofford, 1998) on a G-4 Macintosh ( 400 mHz). The data set was analyzed
using 1000 replications of RANDOM TAXON addition with NNI branch-swapping,
which generated a pool of "starting" trees, the shortest of which were subsequently used
for more rigorous analyses employing TBR branch-swapping. The relative support for
the clades recovered by these analyses was assessed using fast bootstrapping (Mort et al,
2000). Susceptibility to infection was coded for terminal taxa, and phylogenetic
distribution of susceptibility was examined using MacClade (Maddison and Maddison,
1992).
9
Results
Sequence data
Sequences for sixteen individuals, comprising 11 species and four genera, were
included in the final data set (Appendix 1). The final alignment included 1368 sites, with
759 constant and 609 variable characters, of which 327 were parsimony-informative.
Mean base frequencies for individual nucleotides were 17% (A), 31 % (C), 29% (G), and
23% (T), with a G + C content of 60%. No significant shifts in nucleotide usage were
observed across taxa based on a chi-square test (x2 = 25.6, df = 45, P > 0.99).
Evolutionary distances computed using a log-determinant model (Lockhart, 1994) ranged
from 0.08 to 54% among ingroup taxa, with smallest distances observed among
stagnicoline snails, excluding S. caperata (Table 2). Pairwise sequence comparisons
revealed 27.8 transitions and 38. l transversions on average, and a mean transition to
transversion ratio of 0. 73: 1, suggesting standard models of evolution would apply
(Swofford et al., 1996). The average proportion of sites differing between sequences was
0.167.
Lymnaeid Phylogenetics
Parsimony analyses of the aligned data set recovered 250 minimum-length trees
of 900 steps (Figure 3). Homoplasy appeared to be low as assessed by the consistency
index (0.8778) and retention index (0.8503). Variation among tree topologies was
limited to the clade representing Stagnicola spp., in which low sequence divergence was
observed; mean sequence divergence between Stagnicola elodes, S. catascopium, S.
exilis, and S. emarginatawas 0.00571, based on pairwise sequence comparisons. Strict
consensus analyses of the minimum length trees recovered the same three major clades as
10
parsimony analyses (Figure 3) with strong bootstrap support: 1) individuals of S.
caperata (100%); 2) Stagnicola spp.-'- F. (Bakerlymnea) sp. (100%); and, 3) F. o. rustica
+ F. pan'a (100%). Placement of Fossaria truncatula and Lymnea stagnalis in the
consensus tree were suspect in light of previous hypotheses of lymnaeid taxonomy and
phylogeny (Burch, 1982; Remigio and Blair, 1997; Remigio, in press). In addition, this
topology did not show high support for relationships among Stagnicola s.s. An
examination of branch-lengths (Figure 3) suggested long-branch attraction could be a
factor, possibly causing parsimony analyses to be misleading (Felsenstein, 1978).
Greater sequence divergence was observed for S. caperata, F. truncatula, F. o. rustica, F.
parva, and L. stagnalis in contrast to nominal taxa in Stagnicola (i.e., S. catascopium, S.
elodes, S. exilis, and S. emarginata). These unequal rates of change among ingroup taxa
can cause tree topology to be incongruent with phylogenetic relationships (Felsenstein,
1978; Hendy and Penny, 1989).
Additional analyses were conducted to test topology from parsimony analyses
using methods less prone to long-branch phenomena. One method used was neighbor
joining analyses with log-determinent (Lockhart, 1994) distances; this method is better
suited for long-branch phenomena since it operates under a wider set of models and is
robust to base changes across taxa (Hillis et al., 1996). The resulting topology (Figure 4)
resolved three distinct clades with high bootstrap support: 1) Stagnicola spp. + F.
(Bakerlymnea) sp. (100%); 2) individuals of S. caperata (99%); and, 3) Fossaria spp.
(81 %). Neighbor-joining analyses also resolved a close alliance between S. caperata and
species of F ossaria, as opposed to other stagnicolines, but support for this relationship
was low(< 50%). Constraining the topology from neighbor-joining analyses, employing
11
a log-detertiment model, in parsimony analyses produced a tree with 13 additional steps,
which is less than a 2% difference from the initial tree produced by parsimony analyses.
Therefore, tree topology produced by neighbor-joining analyses was not appreciably
different than the maximum parsimony tree, and was accepted as the best estimate of
relationships among lymnaeid snails from Minnesota.
Traits for susceptibility based on experimental and natural infections, along with
radular dentition, were coded to examine their phylogenetic distribution (Table 3) using
topology from neighbor-joining analyses. Snails susceptible to Fascioloides magna
infection, as determined from infection studies in our laboratory and from a review of
literature, as well as species that are known intermediate hosts of F. magna were coded
with a value of one. Radular dentition, determined from slide mounts of the organ, was
also included (Table 3) since it has been used to define groups within the genus Fossaria
(Baker, 1911; Burch, 1982).
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Discussion
Phylogenetics of North American Lymnaeid Snails
Taxonomic certainty and a robust phylogenetic hypothesis for lymnaeid snails are
imperative for correctly identifying hosts of Fascioloides magna and interpreting host
parasite relationships in the group, respectively. The present study indicates that DNA
sequences of the internal transcribed spacer region will resolve relationships between
genera in Lymnaeidae, as first proposed by Remigio and Blair (l 997a). Clades
comprising Stagnicola caperata, Fossaria s.s, and Stagnicola s.s. + F. Bakerlymnea sp.
were strongly supported in both parsimony and distance analyses. However, until
additional sequences from the ITS region are included to overcome unequal rates of
change along different branches, distance methods that use multiple-hit corrections are
more reliable for assessing relationships in this group (Hendy and Penny, 1989; Hillis et
al., 1996). Particularly, distance methods employing a log-determinent model (Lockhart,
1994; Steel, 1994), which operates under a wider set of models and is robust to changes
in base composition across taxa, will be most successful (Hillis et al., 1996).
Neighbor-joining analyses found a closer alliance between Stagnicola caperata,
the primary intermediate host of F. magna in Minnesota, and members of the genus
Fossaria, in contrast to other members of Stagnicola. Although support for this
relationship was low (<50%), a recent study employing sequences from the 16S mtDNA
region revealed a similar alliance between S. caperata and fossariad snails (Remigio, in
press). This relationship disputes previous taxonomic schemes (Baker, 1928; Burch,
1982) which place S. caperata in the stagnicoline subgenus Hinkleyia. Also, S. caperata
exhibits extreme differences in shell morphology from other members of Stagnicola (e.g.,
13
smaller size and pronounced periostracal ridges); therefore, all available evidence
suggests that its placement in the subgenus Hinkleyia is discordant with both
morphological and molecular data.
Species of Fossaria have rarely been included in phylogenetic studies, mostly due
to the difficulty in locating and identifying members of this group. The present study
includes more species of Fossaria than have previously been evaluated. Fossaria
obrussa rustica, F. parva, and F. truncatula formed a well-supported clade (81%) and a
strong relationship was supported between F. o. rustica and F. parva (I 00%) in neighbor
joining analyses. Fossaria Bakerlymnea sp. was excluded from the clade of other species
of Fossaria, and instead clustered with the stagnicoline clade. Exclusion of F.
Bakerlymnea sp. from the Fossaria clade is also supported by differences in radular
dentition (see below). More than 40 species of Fossaria have been described, but it is
likely that many are synonyms (Burch, 1982). Extensive sampling of Fossaria species is
needed to resolve, with confidence, relationships among members of the genus, and to
evaluate taxonomy in this group effectively. Furthermore, continued use of molecular
data in Fossaria may provide markers useful for the identification of cryptic species in
the group, as this method has been successful in other snail (Jones et al., 1997, 1999) and
invertebrate taxa (Walton et al., 1999).
Species from Stagnicola s.s. (e.g., S. elodes, S. emarginata, S. catascopium, and
S. exilis) form a well-supported clade (100%), but relationships within the clade are
largely unresolved. In fact, the average percent nucleotide difference from pairwise
sequence comparisons (Table 2) were higher between individuals of S. caperata (3%)
14
than those observed between species comprising the Stagnicola s.s. clade (0.6%). One
clade within Stagnicola, consisting of S. exilis, S. catascopium (from Minnesota), and
F. Bakerlymnea sp., was moderately supported (74%); but, seems unreliable due to low
sequence divergence. Unlike other genera in Lymnaeidae, particularly Fossaria,
members of Stagnicola s.s. have distinctive shell characteristics that have allowed more
reliable identification of taxa. Previous descriptions of Stagnicola s.s. have
circumscribed species into two groups, elodes and emarginata/catascopium (Burch,
1982). Members of the elodes group, including S. exilis, typically have narrow,
elongated spires and inhabit stagnant waters such as ditches and marshes. Species in the
emarginata/catascopium group have more globose body whorls with shorter spires and
are found in open and/or flowing water systems (e.g., lakes and streams). However, shell
morphology among stagnicoline snails is greatly influenced by environmental conditions
(Burch, 1968; Burch and Lindsay, 1973; Patterson and Burch, 1978), which may explain
the discrepancies seen between highly diverse shell morphology and low sequence
divergence. Failure of both ITS nrDNA (this study) and 16S mtDNA sequences (Remigio
and Blair, 1997b; Remigio, in press) to resolve relationships in Stagnicola robustly
indicates that additional data are needed to determine species relationships. If this group
has undergone recent speciation events, then molecular studies that employ even more
rapidly evolving markers, and maybe hybridization studies, will be necessary to define
Stagnicola species. Also, studies that examine the effects oflimnological variables on
shell morphology may be necessary to determine how environmental factors are
influencing putative taxa.
15
The phylogenetic position of Lymnea stagnalis was unresolved in neighbor
joining analyses, reflecting the high sequence divergence between L. stagnalis and other
North American lymnaeids (e.g., 21.5 % on average from pairwise sequence
comparisons). Other studies employing molecular data (Barques et al., 2001; Remigio, in
press) consistently place N. American L. stagnalis as a sister to European Stagnicola
species, suggesting a recent introduction from Eurasia (Remigio and Blair, l 997b;
Remigio, in press). Like stagnicoline snails, L. stagnalis exhibits environmental
variations in shell morphology that have resulted in assignment of sub-species names.
With at least two type species recognized by both morphological and molecular data
(Clarke, 1973; Burch, 1982; Remigio, in press), molecular studies employing population
level markers will be necessary to evaluate relationships among the various conspecifics.
Fossariad Snails and the Evolution of Radular Dentition
Historically, taxonomists have recognized members of Fossaria based on their
small size and marked absence of the shell characteristics used to define other taxa.
Burch (1982) identified two subgenera within Fossaria, based upon differences in radular
dentition. Species of Fossaria s.s. (e.g., F obrussa rustica, F. parva, and F. truncatula)
have tricuspid teeth in the first lateral position, whereas F Bakerlymnea spp. (e.g., F.
bulimoides, F cubensis, and F dalli) are bicuspid. Bicuspid radular dentition is a trait
shared between the Bakerlymnea group and Stagnicola species, causing Baker (1928) to
classify bicuspid fossariad snails as the subgenus Nasonia in Stagnicola. Internal
transcribed spacer sequences support the relationship proposed by Baker, since
F. Bakerlymnea sp. was well-resolved within the stagnicoline clade (Figure 4). An
examination of the phylogenetic distribution of radular dentition suggests a single origin
16
for tricuspid, first lateral teeth that is limited to Fossaria s.s. (Figure 5). Based on our
estimate of phylogeny, radular dentition may provide more reliable phylogenetic signal
than shell morphology or ecology in this group. Noteworthy is that sequences from 16S
mtDNA (Remigio, in press) do not agree with our results, instead suggesting that
bicuspid and tricuspid Fossaria are congeners in accordance with Burch (1982). Both the
neighbor~oining tree presented here and the 16S phylogeny proposed by Remigio (in
press) included only one species from the Bakerlymnea group. Therefore, it is not yet
clear whether the Bakerlymnea group is monophyletic. Inclusion of additional species
from Bakerlymnea is required to test the monophyly of this tax on and ascertain its
position in the family. In addition, using a combination of molecular markers may be
necessary to elucidate the patterns of evolution of radular dentition; since ITS nrDNA
and 16S mtDNA have very different rates and modes of evolution (Hillis and Dixon,
1991) that may cause them to continue to be counteractive.
Evolution of Susceptibility to Fascioloides Infection
Experimental infections show that susceptibility to F. magna infection is
widespread among the taxa sampled in this study, as all lymneaid species tested show
some degree of susceptibility to infection. Stagnicola emarginata has not been tested,
therefore, no conclusions can be made regarding its susceptibility. Fossaria
Bakerlymnea sp. is not coded because it is not identified to species. However, it is likely
that both of these species would be susceptible to F. magna infection if tested, especially
F. Bakerlymnea sp., since members of this group include Fossaria bulimoides and F.
cubensis that are intermediate hosts found naturally infected in the United States.
17
Only a subset of species experimentally susceptible to F. magna infection serve as
natural intermediate hosts (Figure 6). These include S. caperata, S. elodes, and F. pan-a
in the United States, as well as F. truncatula in Europe. Each of these species is
commonly found in ephemeral waters, such as woodland ponds and ditches, where deer
may be more likely to forage. Therefore, host status among lymnaeid snails may be a
function of high exposure rates that result from close interactions between intermediate
and definitive hosts in endemic areas. Studies of cervid foraging behavior lend support to
different feeding strategies in enzootic and fluke-free regions of Minnesota (Laursen,
1993), providing one explanation for increased contact between intermediate and
definitive hosts. The northeast region of Minnesota that is endemic to F. magna typically
has low sodium concentrations in terrestrial vegetation and aquatic systems (Botkin et al.,
1973; Helgensen et al., 1973; Jordan et al., 1973; Olcott et al., 1978). Deer in these areas
make increased use of sodium-rich mineral licks, such as seeps or small springs (Weeks
and Kirkpatrick, 1976; Fraser and Hristienko, 1981). These ephemeral sites are also
suitable habitat for intermediate host species; therefore, frequent use by deer would
increase contact between intermediate and definitive hosts, and would help perpetuate the
fluke (Laursen, 1993 ).
Snails that were infected under laboratory conditions may be susceptible to
infection as a result of shared ancestry (i.e., genetic similarity), but not found as natural
hosts due to differences in habitat use. Susceptible species, such as S. catascopium are
typically found in large lakes and streams, while Stagnicola exilis and Lymnea stagnalis
are commonly found in marshes and prairie potholes. Larger aquatic habitats with deeper
water could make snail location by F. magna larvae more difficult, reducing the number
18
of infected intermediate hosts. This may prevent infection in numbers required for
continued transmission. In addition, a case can be made for moderate levels ofresistance
in some snail taxa. Stagnicola exilis and L. stagnalis are generally considered poor
intermediate hosts, as they are only readily infected at juvenile life stages (Campbell and
Todd, 1955a, 1955b;Griffiths, 1973;WuandKingscote, 1953, 1954). Thisisnottypical
of natural intermediate hosts, which remain susceptible throughout their lifetime. It is not
clear whether this change in susceptibility from juvenile to adult snail is due to
physiological differences or development of resistance prompted by historic exposure.
Either way, it is obvious that genetic and ecological variables need to be evaluated in
concert to better understand host susceptibility.
19
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Table 1: Lymnaeid snails sampled for infection and phylogenetic studies, with reference to sample location.
seecies Sameled Local it)'. Count!}'. Genbank Accession Number
Lymnea stagnalis Douglas County, MN USA ... Fossaria obrussa rustica Kanabec County, MN USA *
Fossaria parva Crow Wing County, MN USA * Fossaria truncatula Min ho Portugal AJ243018 (Mas-Coma et al., 2001 );
AJ243017 (Barges et al., 2001) Fossaria (Bakerlymnea) sp. Pine County, MN USA *
Stagnicola caperata 7 Mille Lacs County, MN USA * Stagnicola caperata 2 Mille Lacs County, MN USA * Stagnicola caperata 3 Manitoba Canada AFOl 3139 (Remigio and Blair, l 997a)
Stagnico/a el odes· 1 Pine County, MN USA * Stagnico/a elodes 2 Traverse County, MN USA * Stagnicola e/odes 3 Ann Arbor, Ml USA AFOl 3138 (Remigio and Blair, l 997a)
Stagnico/a exilis Mille Lacs County, MN USA * Stagnicola catascopium 1 Crow Wing County, MN USA * Stagnico/a catascopium 2 Ann Arbor, Ml USA AFOl 3143 (Remigio and Blair, l 997a)
Stagnico/a emarginata Ann Arbor, Ml USA AFOl 3141 (Remigio and Blair, l 997a) Phx._sa sp. Kanabec Count;t. MN USA *
* Genbank accession number available from authors
32
w
w
Tab
le 2
: E
volu
lion
my
dist
ance
s ac
cord
ing
to l
ogde
tenn
inen
t/pa
rali
near
mod
el (
show
n be
low
the
diag
onal
), a
nd p
erce
nt n
ucle
otid
e di
llcn
;ncc
(s
how
n ab
ove
the
diag
onal
) fr
om p
ai1w
ise
com
pari
son
of
pm1i
al I
TS
nrD
NA
seq
uenc
es o
f ly
mna
eid
::;na
ils s
ampl
ed.
LST
A
FRU
S FP
AR
SC
AP1
S
CA
P2
SCA
P3
FTR
U
SE
L03
SE
LO
l SE
XI
SCA
Tl
SC
AT
2 SE
MO
S
El.
02
L
ymn
ea
sta
gn
alis
L
STA
-
33
3
3
21
20
2
0
22
2
6
18
1
8
18
1
8
18
1
8
Fos
sari
a o
bru
ssa
msti
ca
FR
US
0.4
68
45
-
2 3
2
32
3
5
29
2
9
28
2
9
29
2
8
28
2
8
Fos
sari
a p
arv
a
FPA
R
0.4
85
52
0.0
19
70
3
3
33
3
6
31
29
2
9
30
2
9
29
2
9
29
S
tag
nic
o/a
ca
pe
rata
1
SC
AP
l 0
.25
18
5 0
.45
95
7 0
.48
67
4
1 4
17
1
3
12
1
2
12
12
1
2
12
Sta
gn
ico
la c
ap
era
ta 2
S
CA
P2
0.2
44
33
0.4
55
02
0.4
82
37
0.0
05
79
-
4 1
7
13
12
12
12
1
2
12
1
2
Sta
gn
ico
la c
ap
era
ta 3
SC
AP3
0
.26
83
6 0
.51
53
0 0
.54
55
3 0
.04
59
5 0
.04
17
1
-1
6
15
1
4
14
1
4
14
1
4
14
Fos
sari
a tr
un
catu
la
FTR
U
0.3
42
3 5
0.3
85
71
0
.42
51
5 0
.20
50
1
0.1
99
70
0.1
89
13
-
20
2
0
20
2
0
20
2
0
19
Sta
gn
ico
la e
/od
es
3 S
EL
03
0.2
19
86
0.3
86
98
0.4
04
48
0.1
51
27
0.1
49
65
0.1
76
97
0.2
60
07
0
1 1
1 1
1 S
tag
nic
ota
e/o
de
s 1
SE
LO
l 0
.21
72
3 0
.38
10
9 0
.39
83
4 0
.14
51
6 0
.14
35
3 0
.17
06
4 0
.25
06
3 0
.00
51
9
-1
0 0
0 0
Sta
gn
ico
la e
xilis
SE
XI
0.2
17
72
0.3
94
60
0.4
12
10
0.1
47
31
0
.14
56
9 0
.17
27
7 0
.25
66
5 0
.01
49
7 0
.00
96
3
-0
1 1
1 S
tag
nic
o/a
ca
tasc
op
itn
n
1 SC
A T
l 0
.21
01
2 0
.38
93
6 0
.40
69
0 0
.14
46
8 0
.14
30
8 0
.17
03
5 0
.25
48
2 0
.00
94
4 0
.00
41
5 0
.00
55
2
-1
0 1
Sta
gn
ico
/a c
ata
sco
piu
m 2
S
CA
T2
0.21
59
5 0
.3 7
93
9 0
.39
66
1
0.1
46
31
0
.14
46
7 0
.1 7
1 7
7 0
.25
09
3 0
.00
78
3 0
.00
26
2 0
.01
22
9 0
.00
68
1
-0
1 S
tag
nic
ola
em
arg
ina
ta
SEM
A
0.2
18
71
0
.38
46
3 0
.40
19
8 0
.14
64
6 0
.14
48
3 0
.17
19
9 0
.25
29
6 0
.00
62
8 0
.00
10
7 0
.01
07
1
0.0
05
23
0.0
03
69
-
0 S
tag
nic
ola
elo
de
s 2
SE
L02
0
.21
99
5 0
.38
11
3 0
.39
83
8 0
. 14
50
9 0
.14
34
5 0
.17
05
1
0.2
46
91
0
.00
80
1
0.0
02
81
0
.01
1 5
3 0
.00
59
4 0
.00
54
6 0
.00
39
1
-F
oss
ari
a (
Ba
kerl
ymn
ea
) sp
. FB
AK
0
.21
49
8 0
.38
78
3 0
.40
52
5 0
.14
52
1
0.1
43
61
0
.17
06
8 0
.25
26
2 0
.01
02
4 0
.00
49
8 0
.00
63
5 0
.00
08
8 0
.00
58
6 0
.00
60
5 0
.00
78
5
FBA
K
18
2
9
29
12
12
14
2
0
1 0 I 0 1 1 I -P
hysa
sp.
PH
YS
0.4
06
96
0.6
00
25
0.6
41
56
0.3
45
87
0.3
44
77
0.3
66
67
0.5
23
72
0.3
47
41
0
.34
84
2 0
.34
75
7 0
.34
67
7 0
.34
76
5 0
.34
71
5 0
.34
78
2 0
.34
47
0
PHY
S 3
0
39
41
2
7
27
2
8
36
2
7
27
'[
/
27
27
2
1
27
2
7
w
+:>.
Tab
le 3
: C
odes
use
d to
eva
luat
e ch
arac
ter
evol
utio
n in
lym
naei
d ta
xa.
Sus
cept
ible
sp
ecie
s an
d kn
own
inte
rmed
iate
hos
ts w
ere
code
d w
ith
a va
lue
of o
ne.
Rad
ular
de
ntit
ion
was
cod
ed a
s a
zero
for
bic
uspi
d, o
ne f
or t
ricu
spid
, an
d tw
o fo
r a
mul
ti-c
uspi
d fi
rst
late
ral
toot
h in
the
rad
ula.
S
peci
es w
ere
code
d w
ith
a da
sh i
f no
in
form
atio
n w
as a
vail
able
for
a p
arti
cula
r tr
ait.
Lym
ne
a s
tag
na
lis
Fo
ssa
ria
ob
russ
a r
ust
ica
F
oss
ari
a p
arv
a
Sta
gn
ico
/a c
ap
era
ta 1
S
tag
nic
o/a
ca
pe
rata
2
Sta
gn
ico
la c
ap
era
ta 3
F
oss
ari
a t
run
catu
la
Sta
gn
ico
/a e
lod
es
3 S
tag
nic
o/a
e/o
de
s 1
Sta
gn
ico
/a e
xilis
S
tag
nic
o/a
ca
tasc
op
ium
1
Sta
gn
ico
la c
ata
sco
piu
m 2
S
tag
nic
o/a
em
arg
ina
ta
Sta
gn
ico
la e
lod
es
2 F
oss
ari
a (
Ba
kerl
ymn
ea
) sp
. P
hysa
sp.
t:.x
pen
men
tal
Ho
st c
,d,e
,t,g
? ? 0
Nat
ura
l H
ost
h,•
,j
0 0 1 0 0 0 0 1 0 0
Kad
ula
r d
en
titi
on
a,b
0 0 0 0 1 0 0 0 0 0 0 0 0 2
Ref
eren
ces:
a)
Bur
ch,
19
82
; b)
Cla
rke,
19
73
; c)
Dut
son
et
al.,
19
67
; d)
Grif
fiths
, 1
95
9;
e) G
riff
iths,
19
73
; f)
Kru
ll, l
933
a; g
) K
rull,
l 9
33
b;
h) S
inits
on,
19
30
; I)
Sin
itson
, 1
93
3;
and,
j)
Sw
ales
, 1
93
5.
Figure 1: Life cycle of Fascioloides magna courtesy of Stromberg et al. (1983) that
describes each stage in the development of the parasite: a) eggs are shed in feces of
infected deer, b) miracidia hatch from eggs in an aquatic environment, c) miracidia
penetrate snails and undergoes larval amplification, d) cercaria emerge from snail, and, e)
encyst to become metacercaria, a stage infective to deer and domestic livestock.
35
"' "' 0
Ui Cl ..c: "C
03 c:
u Ill
"C ell ~
0
36
Figure 2: Map adapted from Griffiths (1962) documenting the occurrence of
Fascioloides magna in white-tailed deer in the United States.
37
Figure 3: One of 250 minimum-length trees produced by parsimony analyses employing
a heuristic search (100 replicates) and a tree-bisection-reconnection branch-swapping
algorithm. Trees were 900 steps, with a consistency index of 0.8778 and a retention
index of 0.8503. Bootstrap values (500 replicates) are shown above branches, while
branch-length values are below branches.
39
224 L stagnalis
100 5 S. caperata 1
85 20 100
58 1
S. caperata 2
64 120 22 S. caperata 3
62 F. truncatu!a
73 5 S. e!odes3
46 0 0 S. e!odes1
1 3 S.e!odes2
2 1 S. emarginata
100 2 S. catascopium 2
135
5 s . exilis
1 74 0 S. catascopium 1
2
0 F. Bakerfymnea sp.
100 1 F. o. rustica
14 F. parva
-15::. Physasp.
40
Figure 4: Neighbor-joining dendrogram inferred using the log-determinent/paralinear
model, with bootstrap values (500 replicates) shown above branches.
41
42
Figure 5: Topology from neighbor-joining analyses examining the evolution of radular
dentition. Tricuspid lymnaeid snails are located on dashed branches.
43
.-----------L stagnalis
• - - - F. (o.} rusfica 1--~ I I ___ F. pa!Ycl I I L - - - - - • F. fnrlcatu/a
s. capt:Jrata 1
s. caperata 2
S.~tata3
S. slodes1
S. slodss2
S. sfTl8fginata
s. catascopiJm 1
a slodssS
s. sxi/is
S. catascopiJm 2
F. Bakslfymnsa sp.
Physasp.
44
OJ --1-· ro n ro s:: .-+ (fJ ::r-0.
0..
Figure 6: Topology from neighbor-joining analyses coded for known intermediate
hosts of Fascioloides magna. Host species are shown in boxes.
45
.---------- L str:f}nafis
F. (o.) f1JStica
F.:pam
.....__-of F. tuncatuta•
$capeafa1
~eaB.6-
......__~s.~~
.-----1••"s.••.~•1u•••••••• •••:S.•IJkJ,dtis•4:·· y••·
Sema,gnata
.._ ___ s catascopiumi
-----~••S.•SkXles3>••• ....---- s. exilis
s. casscophm 2
F. Baketlymnea sp.
L---------- Ptffsa sp.
46
Appendix 1: Aligned data set of internal transcribed spacer sequences for lymnaeid
snails sampled that was used in parsimony and distance analyses.
47
&
lltJE
i<U'
.3
!Cre
ated
by
Se-
All
B
EGH
l D
ATA
; OIME~3IONS
NTA
X=1
6 tlC
HA
R=1
368;
FO
Rt1A
T tl
I SS
I tiG
=?
GA
P=-
OA
TA
H'f'
E=o
tiA;
t1A
TR!i<
L.
stag
nali
s F
. o
. ru
sti
ca
F
. p
arl)
Q
s.
cap
era
ta
s.
cap
era
ta
2 s.
cap
era
ta
3 F.
tr
w1
catu
la
s.
elo
des
3 s.
el o
des
1 s.
ex
ilis
s.
ca
tasc
op
ium
s.
catascopiL~m
2 s.
em
arg
ina
ta
s.
elo
des
2 F.
b
. b
uli
mo
ides
P
hy
sa
sp.
L.
stag
nali
s F
. o
. rL~stica
F.
pa
rva
s.
ca
pera
ta
~
o,
cap
era
la
2 s.
ca
per
ata
3
F.
tru
ncalu
la
~
~-
elo
des
3 s.
el
od
es
1 s.
ex
ilis
s.
ca
tasc
op
ium
s.
ca
tasc
op
ium
2
s.
ema
rgin
ata
s.
elo
des
2 F
. b
. b
uli
mo
ides
P
hy
sa
sp.
L.
stag
nali
s F
. o
. ru
sli
ca
F.
pa
rva
ATT
--TTG
GTC
AG
TCTC
-AG
TCTC
AG
TCA
GTA
NC
TCC
ATG
-TC
CTT
--60-
TCC
CG
CG
GC
AG
GC
GTG
-CA
TGA
AG
CG
CTG
TC
AG
TTG
CTCC
TCG
TCTC
GTC
ACT
TGTC
AG
TCA
GTC
AG
TCA
GTC
TCG
TTG
&G
GCC
CCG
CGG
CAG
GCA
AD
JCA
TGA
AG
CGCT
GTC
A
GTT
GCT
CCTC
GTC
TCG
TCA
CTTG
TCA
GTC
AG
TCA
GTC
AG
TCTC
GTT
OO
GG
CCCC
GCG
GCA
GG
CAA
CGCA
TGA
AG
CGCT
GTC
A
GA
AA
AG
GG
AA
GA
AA
AC-
ATC
CTA
ACT
CAG
TCA
N-G
TCT-
-CA
GCT
C-G
G-C
CCCG
CGA
CAG
ACG
CG-C
ATG
AA
GCG
CCG
TC
AG
AA
AA
GG
GA
AG
AA
AA
C-A
TCCT
AA
GTC
AG
TCA
--GTC
T--C
AG
TT-G
GCC
CCCG
CGA
CAG
ACG
CG-C
ATG
AA
GCG
CCG
TC
ag
aaaag
gg
aag
aaaac-a
tccta
ag
tcag
tca--
gtc
t--c
ag
tt·-
gg
-ccccg
cg
acag
acg
cg
-catg
aag
cg
ccg
tc
aaccg
ag
cg
ttg
acla
ctt
lglt
gtc
tcag
tca--
gtc
ag
tcag
t·cag
g-c
cccg
cg
gcag
gcacg
-calg
aag
cg
ctg
tc
ag
cag
cta
ctg
lgtt
tttc
gtc
tcag
tcag
tctc
tgtc
cg
ctc
tct-
-gg
-ccccg
tgg
tcg
gcacg
-catg
aag
cg
tcg
cc
AGCAGCTACTGTGTTTTTCGTCTCAGTCAGTCTCTGTCCGCTC~:T--GG-CCCCGTGGTCGGCACG-CATGAAGCGTCGCC
NG
AC
GC
AC
CTG
TGTT
TT-C
GTC
TCA
GTC
AG
TCTC
TGTC
CG
CTc
;cT-
-GG
-CC
CC
GTG
GTC
GG
CA
CG
-CA
TGA
AG
CG
TCG
CC
A
GCA
GCT
ACT
GTG
TTTT
-CG
TCTC
AG
TCA
GTC
TCTG
TCCG
CTCT
CT--G
G-C
CCCG
TGG
TCG
GCA
CG-C
ATG
AA
GCG
TCG
CC
ag
cag
cta
ctg
tgtt
tl-c
glc
tcag
tcag
tctc
tgtc
cg
ct2
cct-
-gg
-ccccg
tgg
lcg
gcacg
-catg
aag
cg
lcg
cc
ag
cag
cta
ctg
tgtt
tt-c
gtc
tcag
tcag
tctc
tgtc
cg
cb
:tct-
-gg
-ccccg
tgg
tcg
gcacg
-catg
aag
cg
tcg
cc
AG
CAG
CCA
CTG
TGTT
TTTC
GTC
TCA
GTC
AG
TCTC
TGTC
CGT7
CTCT
--GG
-CCC
CGTG
GTC
GG
CACG
-CA
TGA
AG
CGTC
GCC
A
GC
AG
CTA
CTG
TGTT
TT-C
GTC
TCA
GTC
AG
TCTC
TGTC
CG
CfC
TCT-
-GG
-CC
CC
GTG
GTC
GG
CA
CG
-CA
TGA
AG
CG
TCG
CC
??
CCA
TGA
ACA
ATG
AA
ACG
TATC
AA
AA
CGTC
TCG
TCCG
ATG
TCTG
CCCG
GTC
GG
GA
CTT-
-GG
C--C
GCA
CGA
AG
CGCG
GTC
----
GC
GG
GG
C-T
GTG
AA
CG
C--
---T
ATG
TCTT
T-C
GG
GG
TAC
CTA
T---
CA
CTG
Ttltl
TCG
ATG
CG
AC
CC
AC
GG
TGA
CG
GC
GA
CAGC
GGGG
CCTG
C TA
TGTG
TCCG
CTTC
GTC
T TT
-CG
GG
GTA
CCTA
C---
TACT
GTC
CTCG
ATG
CGA
CCCA
C1JG
TGA
CGG
C G
ACA
GCG
GG
GCC
TGCT
ATG
TGTC
CGCT
TCG
TCTT
T-CG
GG
GTA
CCTA
C---T
ACT
GTC
CTCG
ATG
CGA
CCCA
CGG
TGA
CGG
C ---
-TC
GG
GG
C-T
GTG
AC
CG
C---
---A
TGTC
TTTG
CG
GG
GTA
CC
TATA
CTT
AC
TGTC
CTC
GA
TGC
GA
CC
CA
CG
GTG
GC
GG
C
----G
CG
GG
GC
-TG
TGA
CC
GC
------
ATG
TCTT
T-C
GG
GG
TAC
CTA
T-C
TTA
CTG
TCC
TCG
ATG
CG
AC
CC
AC
GG
TGG
CG
GC
--
--tc
gg
gg
c-t
gtg
accg
c--
----
atg
tctt
t-cg
gg
gta
ccta
t-ctl
actg
tcctc
gatg
cg
acccacg
gtg
gcg
gc
----
gcg
gg
gc-t
gtg
tccg
c--
---t
lcg
tctl
t-cg
gg
gta
ccta
t---
tactg
tcctc
galg
cg
acccacg
glg
acg
gc
----
gcg
gg
gc-t
gcg
accac--
----
atg
tctt
t-cg
gg
gta
ccla
t-cacaclg
lcclc
galg
cg
acccacg
gtg
acg
gc
----G
CG
GG
GC
-TG
CG
AC
CA
C---
---A
TGTC
TTT-
CG
GG
GTR
CC
TAT-
CA
CA
CTG
TCC
TCG
ATG
CG
RC
CC
RC
GG
TGA
CG
GC
---
-GC
GG
GG
C-T
GC
GA
CC
AC
------
ATG
TCTT
T-C
GG
GG
TRC
CTA
T-C
AC
AC
TGTC
CTC
GA
TGC
GA
CC
CR
CG
GTG
AC
GG
C
----G
CG
GG
GC
-TG
CG
RC
CA
C---
---A
TGTC
TTT-
CG
GG
GTA
CC
TAT-
CA
CA
CTG
TCC
TCG
ATG
CG
AC
CC
AC
GG
TGA
CG
GC
--
--g
cg
gg
gc-l
gcg
accac--
----
atg
tctl
t-cg
gg
gta
ccta
t-cacaclg
tcclc
gatg
cg
acccacg
gtg
acg
gc
----
gcg
gg
gc-t
gcg
accac--
----
alg
tctt
t-cg
gg
gla
ccta
t-cacactg
tcctc
gatg
cg
acccacg
glg
acg
gc
----G
CG
GG
GC
-TG
CG
RC
CA
C---
---A
TGTC
TTT-
CG
GG
GTR
CC
TRT-
CA
CA
CTG
TCC
TCG
RTG
CG
AC
CC
AC
GG
TGA
CG
GC
---
-GC
GG
GG
C-T
GC
GA
CC
AC
------
ATG
TCTT
T-C
GG
GG
TAC
CTR
T-C
AC
AC
TGTC
CTC
GA
TGC
GR
CC
CA
CG
GTG
AC
GG
C
TCG
ATC
GG
CTC
--G--R
CC
GC
TCC
CC
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GTT
T-C
GG
GG
TAC
CTA
G--T
GC
ATG
TCC
TCG
ATG
CG
AC
CC
AC
GG
TGA
CG
GC
TT
AG
AG
CC
CG
-TG
TG
CT
CG
CC
GG
G-T
CG
C--
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C--
----
GG
TT
CA
AR
GR
GT
GG
CC
GG
CC
TC
AC
G--
----
----
----
-TT
AG
AG
-CC
AG
TGTG
CTC
GC
CG
GG
-TC
GC
---G
AC
----
--G
GTT
CA
AA
GA
GTG
GC
CTG
C-T
C-G
GC
CTC
--C
TCG
GC
CTC
C
TTA
GA
G-C
CA
GTG
TGC
TCG
CC
GG
G-T
CG
C--
-GA
C--
----
GG
TTC
AA
AG
AG
TGG
CC
TGC
-TC
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CC
TC--
CTC
GG
CC
TCC
s.
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CC
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TGTG
CTC
GC
CG
GG
-TC
GC
---G
AC
------
GG
TAC
AA
FDA
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CG
GC
-TC
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GC
TC-A
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TA
TTA
GA
GC
CC
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GTG
CTC
GC
CG
GG
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GC
---G
AC
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t
tcig
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g t-
g tg
c: tc
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---
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t tc
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ag tg
9 c:
c:9•
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c:-g
ac:-
tc:-
---
--ag
e t -
-ttagagcccgt-gtgctcgccggg-tcgc---gac------ggttca~agagtggccggc-tt-ggc-tc------agcl-
t ta
gag
cccg
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tgc
tc:•
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gg-
tcg
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gm::-
---
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g t
tco:
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gg
ccg
ac--
aaag
c-
tc-
---
----
---
-TTAGAGCCCGA-GTGCTCGCCGGG-TCGC---GAC------GGTTC~AAGAGTGGCCGAC--AAAGC-TC-----------
TT
AG
RG
CC
CG
R-G
TG
CT
CG
CC
GG
G-T
CG
C--
-GR
C--
----
GG
TT
CA
AA
GA
GT
GG
CC
GR
C--
AA
AG
C-T
C--
----
----
-TT
AG
AG
CCCG
A-G
TGCT
CGCC
GG
G-T
CGC-
--G
AC
----
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GTl
CAA
AG
AG
TGG
CCG
AC-
, -A
AA
GC
-TC
----
----
----
t l•
:iga
gc:c
cga-
g tg
c tc
gcc
gg
g-lc
gc--
-gac--
----
gg
ttcc
mag
ag tg
9cc:
gm::
--aa
•:ig
c-le
---
----
---
--t t
ag•:
igcc
cga-
g lg
c tc
gcc
gg
g-
tcg
c---
gac--
----
99
! tca
aag
ag lo
;ig c
c9•:
ic--
cma9
c-tc
---
---
---
---
TT
AG
AG
CC
CG
A-G
TG
CT
CG
CC
GG
G-T
CG
C--
-GA
C--
----
GG
fTC
AA
AG
AG
TG
GC
CG
AC
--A
AA
GC
-TC
----
----
---
TT
AG
AG
CC
CG
A-G
TG
CT
CG
CC
GG
G-T
CG
C--
-GA
C--
----
G6T
TC
AA
AG
AG
TG
GC
CG
AC
--A
AA
GC
-TC
----
----
---
-TA
GA
GC
------
TGC
GA
A-C
GG
GC
TCG
CC
GG
GTC
GC
GC
TAG
JTTC
AA
AG
AG
TGG
CTC
GG
-TC
CG
GTG
TTT-
CG
CC
------
----
----
----
----
----
----
----
----
----
----
----
GT
CG
GC
GA
CC
GC
CC
CG
CA
C-T
CG
TC
-CT
CG
TC
CA
CG
TC
CA
GCCC
CAGC
TCTC
GGTG
GGTC
GGTC
GGAC
GAG
AG
----
TCA
A-G
CAG
GCG
ACC
GCC
CCG
TTG
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:HC-
CACA
CACG
TCCG
CA
GCC
CCA
GCT
CTCG
GTG
GG
TCG
GTC
GG
ACG
AG
TG---
-TCA
A-G
CAG
GCG
ACC
GCC
CCG
TTG
-TCG
TC-C
ACA
CACG
TCCG
C
AG
----
--C
TCTC
CTC
GA
GC
CA
GA
----
GT-
CC
GTG
--TC
A--
GTC
GG
CG
AC
CG
CC
CC
CTG
AC
CC
GTC
----
-GTC
G-A
GA
C
AG
----
--C
TCTC
CTC
GA
GC
CA
GA
----
GT-
CC
GTG
--TC
A--
GTC
GG
CG
AC
CG
CC
CC
CTG
AC
CC
GTC
----
-GTC
G--
GA
ca
get -
cag
e tc
agc
tctc
ccc-g
a--
---
-gcc
-ag
agtc
c9a9
lc•J
atcg
gcg
•:ic
cgcc
ccc
tg•:
icc:
-c:g
tc•;i
tcg
--g
a
c9
----
----
----
----
----
a9
a--
----
----
----
----
-gtc
ag
ccg
gcg
ac:c
9cccc--
----
--g
ccg
tcg
---
-aaccc-a
cctc
---9
tg--
tgag
t---
-gtg
cct-
----
----
9tc
gg
c:g
cic
cg
ccecg
att
-ttg
tc-c
gala
aet-
c9
a
-AA
CC
C-A
CC
TC--
-GTG
--TG
AG
T---
-GTG
CC
T---
----
--G
TCG
GC
GA
CC
GC
CC
CG
ATT
-TTG
TC-C
GA
TAA
CT-
CG
A
-AA
CC
C-A
CC
TC--
-GA
G--
TGA
GT-
--TG
TGC
CT-
----
----
GTC
GG
CG
AC
CG
CC
CC
GA
TT-T
TGTC
-CG
ATA
AC
T-C
GA
-A
AC
CC
-AC
CTC
---G
AG
--TG
AG
T---
TGTG
CC
T---
----
--G
TCG
GC
GA
CC
GC
CC
CG
ATT
-TTG
TC-C
GA
TAA
CT-
CG
A
-aaecc-a
cclc
---g
tg--
tgag
t---
-gtg
ec:t
----
----
-gtc
:gg
cg
ac:c
gcc:c
cg
att
-ttg
tc:-
cg
ala
act-
cg
a
-aaeec-a
cctc
---g
tg--
tga9
t---
-9tg
cct-
----
----
gtc
:gg
cg
accg
ccc:c
:gatt
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tc-e
gata
act-
cg
a
-AA
CC
C-A
CC
TC--
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AG
T---
-GTG
CC
T---
----
--G
TCG
GC
GA
CC
GC
CC
CG
ATT
-TTG
TC-C
GA
TAA
CT-
CG
A
-AA
CC
C-A
CC
TC--
-GA
G--
TGA
GT-
--TG
TGC
CT-
----
----
GTC
GG
CG
AC
CG
CC
CC
GA
TT-T
TGTC
-CG
ATA
AC
T-C
GA
--
----
----
----
---G
GG
CC
G--
----
----
--G
GC
GA
CC
GC
CC
CG
CT
A--
TG
TC
TC
GA
AA
GT
GC
C--
GTC
CC
AC
AA
AC
TGTC
AG
AA
G-T
CTA
AC
GG
GA
GG
TTA
CG
TCC
GG
GG
TAC
CTA
TGC
CC
TG--C
AC
GC
G-T
-GC
-TTG
C---
--G
----
----
----
AA
AG
GG
AG
GT
T-T
AT
CA
AA
----
---C
CG
GG
GT
RC
CT
AT
GC
CC
TG
CC
CG
CG
CT
C--
GC
---T
CT
C--
-G
----
----
----
AA
AG
GG
AG
GT
TG
TA
TC
AA
A--
----
-CC
GG
GG
TA
CC
TA
TG
CC
CT
GC
CC
GC
GC
TC
--G
C--
-TC
TC
---
AA
ATG
CG
CA
CA
CA
AA
AG
AG
GA
GG
AG
GTT
CG
T----
----C
CG
GG
GTA
CC
TATG
CC
CTG
CC
A---
C---
-GC
-ATG
CTC
---
AA
ATG
CA
CA
CA
CA
AA
AG
AG
GA
GG
AG
GTT
CG
T----
----C
CG
GG
GTA
CC
TATG
CC
CTG
CC
A---
C---
-GC
-ATG
CTC
---
aci1
:itgc
:cic
aclla
aaaa
•;ia
ggag
ga9•
;i t l
eg l-
----
---c
cgg
gg
tacc
ta tg
ee e
tgec
ci--
-e--
--g
e-a
tge
le-
--
--c
aacia
cia
cic
--ag
gag
9tt
a9
t---
----
-c:c
gg
gg
tacela
t9eeet9
cc:-
-eg
eg
cte
gc:t
ct-
--eg
e-
c--
----
----
----
-ag
gag
gtt
----
eg
t---
----
-ce9
gg
gta
ccta
lgecet9
ccl9
elc
gcll
ge-t
tgctc
gctt
C
----
----
----
---A
GG
AG
GT
T--
--C
GT
----
----
CC
GG
GG
TA
CC
TA
TG
CC
CT
GC
CT
GC
TC
GC
TT
GC
-TT
GC
TC
GC
TT
C
----
----
----
---A
GG
AG
GT
T--
--C
GT
----
----
CC
GG
GG
TA
CC
TA
TG
CC
CT
GC
CT
GC
TC
GC
TT
GC
-TT
GC
TC
GC
TA
C
----
----
----
---A
GG
-GG
TT
----
CG
T--
----
--C
CG
GG
GT
AC
CT
AT
GC
CC
TG
CC
TG
CT
CG
CT
TG
C-T
TG
CT
CG
CT
A
c--
----
----
----
-ag
gag
glt
----
cg
t---
----
-cc:g
9g
gta
ccta
lgcect9
cctg
clc
gctt
gc-t
tgctc
gctt
c--
----
----
----
-a9
ga9
9tt
----
cg
t---
----
-ce9
gg
gta
ccta
lgccc:t
gectg
ctc
gctl
gc-t
tgc:t
e9
ctt
C
----
----
----
---A
GG
AG
GT
T--
--C
GT
----
----
CC
GG
GG
TR
CC
TA
TG
CC
CT
GC
CT
GC
TC
GC
TT
GC
-TT
GC
TC
GC
TT
C
----
----
----
---A
GG
AG
GT
T--
--C
GT
----
----
CC
GG
GG
TA
CC
TA
TG
CC
CT
GC
CT
GC
TC
GC
TT
GC
-TT
GC
TC
GC
TA
Ph
yso
sp
.
L.
sto
gn
ali
s F
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stic
o
F. par~va
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era
ta
s.
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pera
tQ
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co
pera
ta
3 F
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w1
cotu
lo
s.
elo
des
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des
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m
2 c ~. e~Jrginato
s.
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des
2 F
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uli
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ides
Ph
yso
sp
.
~
L.
sto
gn
ali
s 0
F.
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ico
F
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pero
to
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ta
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co
pero
ta
3 F
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Ltt
1ca
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s.
elo
des
3 s.
elo
des
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ilis
o
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s.
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lasc
op
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2
s.
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gin
ato
~-
elo
des
2 F
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uli
mo
ides
P
hy
so
sp.
L.
stag
nali
s F
. o
. rL~stica
F.
pat~va
o.
capet~ata
s.
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era
ta
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ca
pera
ta
3 F
. tr
un
catu
la
s.
elo
des
3 s.
el
od
es
l
----
----
--G
OT
---
----
-CA
AG
GG
GT
AC
CT
GC
GT
CT
CG
TC
CC
GC
GA
GG
GA
----
----
----
------
-----A
TGC
AG
GG
TGG
TAG
CTC
CA
GC
TTA
GC
TAG
CTC
GTC
-AC
TT-T
GG
CC
GC
GA
GG
TTC
AA
AG
AG
TC-G
GC
C-G
T G
CG
CTG
GC
AG
CA
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GG
T-G
GTA
GC
TCC
ATC
A---
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CTC
GA
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TT-T
GG
CC
GC
GA
GG
TTC
AA
AG
AG
AC
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CC
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GC
GC
TGG
CA
GC
AT-
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GG
TAG
CTC
CA
TCA
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GC
TCG
AT-
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GC
CG
CG
AG
GTT
CTA
AA
GA
GA
-CG
AC
CG
T GDJ-TGCCTGCA---GGGC-GGTAGCTCCAGCA--------TCGTTTACTTAT~CCGCGAGGTTCAAAGAGTC-GACC-GC
GC
G-T
GC
CTG
CA
---G
GG
C-G
GTA
GC
TCC
AG
C--
----
---T
CG
TTTA
CTT
-TG
GC
CG
CG
AG
GTT
CA
AA
GA
GTC
-GA
CC
-GC
g
cg
-tg
cctg
ca--
-gg
gc-g
glo
gctc
cag
c--
----
---l
cg
ttta
ctt
-tg
gccg
cg
ag
gll
caaag
ag
lc-g
occ-g
c
gcc--
g--
-gca--
-ag
gc-g
gta
gctc
co
gc--
----
---t
cg
ct-
o-t
t-lg
gccg
cg
ag
gtt
co
ao
gag
ac-g
occ-g
t g
cc-t
gcctg
ca--
-gg
gt-
gg
tag
ctc
co
gc--
----
---t
cg
tt--
ctt
-tg
gccg
cg
og
glt
cao
ag
og
tc-g
gcc-g
l GCC-TGCCTGCA---GGGT-GGTAGCTCCAGC---------TCGTT--CT~TGGCCGCGAGGTTCAAAGAGTC-GGCC-GT
GC
C-T
GC
CTG
CA
---G
GG
T-G
GTA
GC
TCC
AG
C--
----
---T
CG
TT--
CTT
-TG
GC
CG
CG
AG
GTT
CR
AA
GA
GTC
-GG
CC
-GT
GC
C-T
GC
CTG
CA
---G
GG
T-G
GTA
GC
TCC
AG
C--
----
---T
CG
TT--
CTT
-TG
GC
CG
CG
AG
GTT
CA
AA
GA
GTC
-GG
CC
-GT
gcc-l
gcctg
ca--
-gg
gl-
gg
lag
ctc
cag
c--
----
---l
cg
lt--
clt
-tg
gccg
cg
ag
glt
caaag
ag
tc-g
gcc-g
t g
cc-t
gcctg
ca--
-gag
t-g
gla
gclc
cag
c--
----
---t
cg
tt--
ctt
-lg
gccg
cg
ag
gtt
caaag
ag
tc-g
gcc-g
t G
CC
-TG
CC
TGC
A--
-GG
GT-
GG
TAG
CTC
CA
GC
----
----
-TC
GTT
--C
TT-T
GG
CC
GC
GA
GG
TTC
AA
AG
AG
TC-G
GC
C-G
T G
CC
-TG
CC
TGC
A--
-GG
GT-
GG
TAG
CTC
CA
GC
----
----
-TC
GTT
--C
TT-T
GG
CC
GC
GA
GG
TTC
AA
AG
AG
TC-G
GC
C-G
T ---
--TG
GG
GC
GG
GA
GC
TCC
AG
CTT
CC
ATA
GC
A---
------
--GG
CC
GC
GA
GG
TTC
AA
AG
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AA
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AA
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AA
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ta-c
cag
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aaclt
gctc
lctc
cg
lgg
gcaacg
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gccg
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gg
cctc
cla
aaa--
----
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ta--
----
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ctt
gctc
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gctt
cg
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gccg
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gg
cclc
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co
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AC
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AA
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CC
AC
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CTC
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CTT
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CG
CC
CC
GG
GC
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AA
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AA
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CC
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AA
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actt
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gctt
cg
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gccg
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gg
cctc
cta
aaa--
----
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tttc
caaa--
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ccac-a
clt
gctc
tc--
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gctt
cg
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gccg
ccccg
gg
cclc
cta
aaa--
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lllc
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CC
AC
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TTG
CTC
TC--
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CTT
CG
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CC
CC
GG
GC
CTC
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AA
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CA
AA
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CC
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CC
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AA
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AA
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GT
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A-G
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s F.
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eral
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s.
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erat
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s.
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3 F.
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ides
GG
CCA
CGCC
CGTC
TGA
GG
GTC
GG
CTA
GTC
ACA
AA
GCA
ATC
GTG
TCC-
---TG
TAG
CTCT
CGCA
AA
ACT
GG
-AG
CCG
TCTC
-C
GG
CCA
CGCC
CGTC
TGA
GG
GTC
GG
CTA
GTC
ACA
AA
GCA
ATC
GTG
TCC-
---TG
TAG
CTCT
CGCA
AA
ACT
GG
-AG
CCG
TCTC
-C
ggcc
:acg
cc:c
g tc
tgag
gg
tcg
gc
tog
tc:a
co:m
agca
a tc
:g lg
tcc--
--t_g
tag
c tc
tcg
cacm
oc
tgg
-a9
ccg
tc tc
:-c
????
????
????
????
????
??g
c to
g tc
aecm
ag
ea t
lco;
i tg
tee--
-t
tgeo
Jge
tc tc
o;ic
aoaa
oecg
o]Q
o;JC
C t_
t_--
--g
g
gceacg
cccg
tctg
og
gg
lcg
gcla
gtc
aeaaag
caalc
gtg
tec--
-tt9
co
9ctc
tcg
ca9
gaceg
9-a
gectt
----
c
GG
CCA
CGCC
CGTC
TGA
GG
GTC
GG
CTA
GTC
ACA
AA
GCA
ATC
GTG
TCC-
--TTG
CAG
CTCT
CGCA
GG
ACC
GG
-AG
CCTT
----C
G
GCC
ACG
CCCG
TCTG
AG
GG
TCG
GCT
AG
TCA
CAA
AG
CAA
TCG
TGTC
C---T
TGCA
GCT
CTCG
CAG
GA
CCG
G-A
GCC
TT---
-C
GG
CCA
CGCC
CGTC
TGA
GG
GTC
GG
CTA
GTC
ACA
AA
GCA
ATC
GTG
TCC-
--TTG
CAG
CTCT
CGCA
GG
ACC
GG
-AG
CCTT
----C
g
gec
acg
cccg
le tg
agg
g tc
g9
c ta
g tc
oca
a o
gca
o le
g tg
tee:
---
t t•;
icag
c tc
tcgc
aggo
]cco
;i9-
og
cc t
t --
--c
•;
igcc
ac9c
ccg
tc lo
;io9g
g tc
gg
e la
gtc
acao
og
caa
lc9
lg tc
e--
-t.
tgca
g c
tc tc
•;ic
a•;i
9acc
g9-0
9cc
t_ t_
--
--c
: G
GCC
ACG
CCCG
TCTG
AG
GG
TCG
GCT
AG
TCA
CAA
AG
CAA
TCG
TGTC
C---T
TGCA
GCT
CTCG
CAG
GA
CCO
G-A
GCC
TT---
-C
GG
CCA
CGCC
CGTC
TGA
GG
GTC
GG
CTA
GTC
ACA
AA
GCA
ATC
GTG
TCC-
--TTG
CAG
CTCT
CGCA
GG
ACC
GG
-AG
CCTT
----C
G
GC
CA
CG
TCC
GTC
TGA
GG
GTC
GG
CG
AC
T---
AA
ATC
AA
TCG
AG
CTC
GTC
TGTT
TTTC
CA
CG
----
----
----
----
----
-
GG
----
----
TTC
CTC
CC
ATC
AA
CC
AC
CC
AC
C--
GC
TCG
T---
TGG
GTG
GA
AG
GA
AG
AG
GG
GG
GG
GG
GA
C--
----
----
TG
?GC
CG
TGG
CC
TTG
CG
GC
GG
CG
CC
AG
TCTC
TCC
TCA
AC
GC
TCG
AT-
GG
TGG
GG
OG
------
------
AA
CA
CA
AC
GC
GC
TGTT
?G
CC
GTG
GC
CTT
GC
GG
CG
GG
CG
CA
GTC
TCTC
CTC
A-C
GC
TCG
AT-
GG
TGG
GG
GG
------
-----G
AA
CA
-AA
CG
CG
CTG
TT
CC
CC
CTG
GC
A--
----
-CA
CA
CC
GTC
TCC
GA
CTT
GC
TCG
T---
T-G
GA
GA
CG
CG
AC
GC
T-G
GG
G-G
AG
TA--
----
----
-C
CC
CC
TGG
CA
----
---C
AC
AC
CG
TCTC
CG
AC
TTG
CTC
GT-
--T-
GG
AG
AC
GC
GA
CG
CT-
GG
GIJ
-GA
GTA
----
----
---
ccc--
tgg
co
----
---c
ocaccg
tctc
c9
actt
gctc
gt-
--t-
gg
a9
acg
cg
oc9
ct-
gg
9g
9g
a9
to--
----
----
-cg
gcg
tgag
clc
t---
-cacg
ctg
-ctc
gg
c--
-galg
gt-
--t-
gg
ala
cg
c--
----
----
----
----
----
----
--
cg
ccg
tgg
actc
tco
tlcacag
cg
cctc
cg
actt
gctc
gtc
g--
-gg
gt-
----
--g
ctt
gg
gg
-gaccc--
----
---g
tc
CG
CC
GTG
GA
CTC
TCA
TTC
AC
AG
CG
CC
TCC
GA
CTT
OC
TCG
TCG
---G
GG
T----
---G
CTT
OG
GG
-GA
CC
C---
------
GTC
C
GC
CG
TGG
AC
TCTT
ATT
CA
CA
GC
GC
CTC
CG
AC
TTG
CTC
GTC
G---
GG
GG
G---
---tJC
TTG
GG
G-G
AC
CC
------
---G
TC
CG
CC
GTG
GA
CTC
TTA
TTC
AC
AG
CG
CC
TCC
GA
CTT
GC
TCG
TCG
---G
GG
GG
------
GC
TTG
GG
G-G
AC
CC
------
---G
TC
cgcc
g tg
gac
tc te
at
tcac
agcg
cc: t
ccg
ac t
tgc
tcg
tcg
---
ggg
t---
----
gc t
t•;i
ggg-
gacc
:c--
---
----
g tc
cg
ccg
tgg
actc
tcatt
cacag
cg
cc:t
c:c
gactt
gcte
gtc
:g--
-gg
gt-
----
--g
ctt
gg
gg
-gaccc--
----
---g
tc
CG
CC
GTG
GA
CTC
TCA
TTC
AC
AG
CG
CC
TCC
GA
CTT
GC
TCG
TCG
---G
GG
T----
---G
CTT
GG
GG
-GA
CC
C---
------
GTC
C
GC
CG
TGG
AC
TCTT
ATT
CA
CA
GC
GC
CTC
CG
AC
TTG
CTC
GTC
G---
GG
GG
G---
---G
CTT
GG
GG
-GA
CC
C---
------
GTC
A
CA
C--
----
----
TC
AA
TCG
G-C
AC
CG
GC
TGG
AC
AC
GC
TCTG
GA
CC
TTC
GC
GG
-----T
TTG
C---
-GC
TGTC
--GC
TGC
AC
TATT
CG
T----
---
GCT
CGG
-CG
ATG
GCT
GG
ATA
CGCC
TTG
GA
CCCT
CGCG
G---
CCTT
AG
CCG
TTG
CTG
CCTT
GCT
TTTT
TTTT
GG
CTCT
CGG
CA
GCT
CGG
-CG
ATG
OCT
GIJA
TACG
CCTT
GG
ACC
CTCG
CGG
---CC
TTA
GCC
GTC
GCT
GCC
TTG
CTTT
TTTT
TTG
GCT
CTCO
GCA
---
-GG
-CA
CCG
GTT
GG
ACA
CGCC
CTG
GA
CCCT
CGCG
G---
CCT-
ATA
CCG
TCG
TCIJ
CTG
CCG
CTA
GCC
CTTG
CGG
TTG
GTG
---
-GG
-CA
CCG
GTT
GG
ACA
CGCC
CTG
GA
CCCT
CGCG
G---
CCT-
ATA
CCG
TCG
TCG
CTG
CCG
CTA
GCC
CTTG
CGG
TTG
GTG
--
--gg
-cac
c•;i
g t t
gg
acac
gcc
c tg
gac:
cc tc
•;ic
gg--
-cc
t-a ta
ccg
b::g
t cg
ccgc
cgc:
c: t
tccc tg
•;J t
gg
l tg
g tg
--
----
----
c:c
:tg
gaccctc
gcg
g--
-cc:a
-aag
ctg
lcg
ttg
c:-
----
----
--ctg
ctc
g--
----
gtl
cgg
-cac
c:g
gtc
gg
oca
cgcc
c:tg
gac
cctc
gc:
gg
---
cct-
acaccg
tc:g
ctg
ctt
----
----
----
-----
----
G
TT
CG
G-C
AC
CG
IJT
CG
GA
CA
CG
CC
CT
GG
AC
CC
TC
GC
GG
---C
CT
-AC
AC
CG
TC
GC
TG
CT
T--
----
----
----
----
---
GTT
CG
G-C
AC
CG
IJTC
GG
AC
AC
GC
CC
TGG
AC
CC
TCG
CG
G--
-CC
T-A
CA
CC
GTC
GC
TGC
TT--
----
----
----
G
TT
CG
O-C
AC
CG
GT
CG
GA
CA
CG
CC
CT
GG
AC
CC
TC
GC
GG
---C
CT
-AC
AC
CO
TC
GC
TG
CT
T--
----
----
----
----
---
gtl
cg
g-c
accg
gtc
gg
ac:a
cg
ccctg
go
ccctc
gcg
g--
-cct-
acaccg
tcg
clg
ctt
----
----
----
----
----
-g
ttcg
g-c
accg
gtc
gg
acacg
c:c
ctg
9accctc
gcg
g--
-cct-
ac:a
c:c
gtc
gctg
ctt
----
----
----
----
----
-G
TT
CG
G-C
AC
CG
GT
CG
GA
CA
CG
CC
CT
GG
AC
CC
TC
GC
GG
---C
CT
-AC
AC
CG
TC
GC
TG
CT
T--
----
----
----
----
---
GT
TC
GG
-CA
CC
GG
TC
GG
AC
AC
GC
CC
TG
GA
CC
CT
CG
CG
G--
-CC
T-A
CA
CC
GT
CG
CT
GC
TT
----
----
----
----
----
--
Ph
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sp
.
L.
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GTC
GG
GC
TGG
AA
AG
CG
GG
CTC
GC
CC
TGG
AC
CG
TCG
AG
GTT
TCC
T----
----C
GC
TCC
------
----
--A
C
GCG
GTG
ATG
GCA
AC-
---G
GCC
CCG
TGG
TCTT
AA
GT-
ACA
AA
CCG
CGCC
GTT
GTC
CGTT
-CA
TCTC
GTA
ACG
TCTT
-CG
ACG
G
CGG
CGA
CGG
TGA
C----
GG
CCCC
GTG
GTC
TTA
AG
TGC-
-AA
GCC
GC-
-GCC
G---
--ATG
TCCG
TTTC
ACC
TCG
TAA
CGTC
G
CGG
CGA
CGG
TGA
C----
GG
CCCC
GTG
GTC
TTA
AG
TGC-
-AA
GCC
GC-
-GCC
G---
--ATG
TCCG
TTTC
ACC
TCG
TAA
CGTC
G
CGG
TGA
TAG
TGG
TGG
-TG
GCC
CCG
TGG
TCTT
AA
GC-
GCA
AG
CCG
CGCC
GTT
GTC
CGTT
-CA
TCTC
GTA
ACG
TCTT
-CG
ACG
G
CGG
TGA
TAG
TGG
TGG
-TG
GCC
CCG
TGG
TCTT
AA
GC-
GCA
AG
CCG
CGCC
GTT
GTC
CGTT
-CA
TCTC
GTA
ACt
3TCT
T-CG
ACt
3 g
cgg
tga b
:ig l
gg
tgg
g tg
gccccg
tg•;J
le: t
taag
c-g
caag
ccg
cg
cc9
t l.g
tccg
t t-
ca tc
tcg
taacg
tc t
t-cg
acg
g
qig
cg
acg
g tg
•:ic
---
-gg
tc:c
cg tg
g le
: t t
a •J
gc-
gca
•:ig
ccg
cgcc
g l
l•;J t
ccg
t t -
cq
tc tc
9 tm
ic9
tc t.
t -eo
;ic1c
•;J
gc9
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;iac
cig
l•;i
gt-
---g
gcc
ccg
tgg
let
tcia
gc-
gcc
iog
ccg
cgcc
9 l
tgtc
cg
t t-
co
le le
o;il
cioc
g le
t l-
c9m
::•;
i G
CGG
TGA
CAG
TGG
T----
t3G
CCCC
GTG
GTC
TTA
AG
C-G
CAA
GCC
GCG
CCl3
TTG
TCCG
TT-C
ATC
TCG
TAA
CGTC
TT-C
GA
CG
GCG
GTG
ACA
GTG
GT-
---G
GCC
CCG
TGG
TCTT
AA
GC-
GCA
AG
CCG
CGCf
GTT
GTC
CGTT
-CA
TCTC
GTA
ACG
TCTT
-CG
ACG
G
CGG
TGA
CAG
TGG
T----
GG
CCCC
GTG
GTC
TTA
AG
C-G
CAA
GCC
GCG
CCG
TTG
TCCG
TT-C
ATC
TCG
TAA
CGTC
TT-C
GA
CG
gc•;J
•;J t
gacag
tgg
t--
--g
•;ic
cccg
tgg
tc t
laag
c-g
caag
ccg
c9
ccg
t t9
tcc9
t t-
c•J le
leg
l•:io
:icg
tc l
t-c9
•:ic
g
gcg
9tg
•:ic
ag tg
gt-
---,
;ig
ccccg
tgg
tct
taag
c-gc
a•:i
•;ic
cgc•
;icc
gt tg
tccg
t t-
catc
tc•;
it•:
iacg
tc: t
t-c
gac
•;i
GCG
GTG
ACA
GTG
GT-
---G
GCC
CCG
TGG
TCTT
AA
GC-
GCA
AG
CCG
CGCC
GTT
GTC
CGTT
-CA
TCTC
GTA
ACG
TCTT
-CG
ACG
G
CGG
TGA
CAG
TGG
T----
GG
CCCC
GTG
GTC
TTA
AG
C-G
CAA
GCC
GCG
CCG
TTG
TCCG
TT-C
ATC
TCG
TRA
CGTC
TT-C
GA
CG
TCG
GG
CG
G--
----
GG
AT-
-CC
CC
GTG
GTT
TCA
AG
T-C
CA
AG
CTG
CG
CC
GTC
GTC
CT-
---A
TGTC
CT-
----
----
CtJ
ATG
CTG
CC
CTG
CTC
ATG
GC
GG
C---
CTG
TCC
GTT
TTC
TCTA
------
-CC
GC
C---
-AG
GC
AG
GA
CC
CG
GC
TCG
-CTA
CTT
IJA
TT
TT-C
GA
CGCT
GCC
CTG
CTCT
TGG
TGG
CGG
CCTG
TCCG
TTTT
OTC
TCCG
CC-G
CCA
GG
CAG
GA
CCCG
GCT
CG---
-CTT
TATA
TT
TCG
ACG
CTG
CCCT
GCT
CTTG
GTG
GCG
GCC
TGTC
CGTT
TTG
TCTC
CGCC
-GCC
AO
GCA
GG
ACC
CGG
CTCO
----C
TTTA
TA
CTG
CC
CTG
CTC
TTG
GC
GG
CG
GTC
TGTC
CA
TTTT
CTC
TA---
----C
CG
CC
----A
GG
CA
GG
AC
CC
GG
CTC
G---
-CTA
ATC
C
TGC
CC
TGC
TCTT
GG
CG
GC
GG
TCTG
TCC
ATT
TTC
TCTA
------
-CC
GC
C---
-AG
GC
RG
GA
CC
CG
GC
TCG
----C
TAA
TC
ctg
cc:c
tgctc
:ttg
gcg
gtg
gtc
tglc
catl
ltclc
la--
----
-ccg
cc--
--ag
gcag
go
cccg
gclc
g--
--clo
ctc
clg
ccclg
ctc
ttg
gc:g
gc--
-c:t
gtc
cg
tttt
ctc
lo--
----
-ccg
cc--
--ag
gco
gg
occcg
gclc
:g-c
ltactt
tat
c lo
;icc:
c tg
ctc
l b
;ig
cgg
c:--
-ctg
tccc
it t
t_ tc
tctc
i---
----
ccg
cc---
-a•;
igca
g1;i
accc
ggc
tcg
-c tact
l t..:
i 1.
CTG
CC
CTG
CTC
TTG
GC
GG
C---
CTG
TCC
ATT
TTC
TCTA
------
-CC
GC
C---
-AG
GC
AG
GA
CC
CG
GC
TCG
GC
TAC
TTTA
TC
TGC
CC
TGC
TCTT
GG
CG
GC
---C
TGTC
CA
TTTT
CTC
TA--
----
-cco
cc--
--A
GG
CA
GG
AC
CC
GG
CTC
G-C
TAC
TTTA
TC
TGC
CC
TGC
TCTT
GG
CG
GC
---C
TGTC
CA
TTTT
CTC
TA---
----C
CG
CC
----A
GG
CA
GG
AC
CC
GG
CTC
G-C
TAC
TTTA
Tctg
ccctg
ctc
ttg
gcg
gc--
-ctg
tcco
tttt
ctc
ta--
----
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cc--
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gcag
gacccg
gctc
g-c
lcic
tlta
t.
ctg
ccctg
c:t
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9g
cg
9c--
-ctg
lcco
tttt
c:t
cta
----
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cg
cc--
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gca9
go
cccg
9ctc
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lactt
tat
CTG
CC
CTG
CTC
TTG
GC
GG
C---
CTG
TCC
ATT
TTC
TCTA
------
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GC
C---
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GC
AG
GA
CC
CG
GC
TCG
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CTT
TAT
CTG
CC
CTG
CTC
TTG
GC
GG
C---
CTG
TCC
ATT
TTC
TCTA
------
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GC
C---
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GC
AG
GA
CC
CG
GC
TCG
-CTA
CTT
TAT
CT
GC
----
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TG
CG
CG
G--
----
----
----
TT
TC
A--
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GC
CT
GG
CA
GG
AC
----
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CG
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TA
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AR
TT
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TG
GA
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CG
GG
CC
TG
CA
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CC
TC
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CA
TC
G-T
CC
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TT
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CC
----
----
----
----
---
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GG
CGTA
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GCC
TGCA
AG
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GCA
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CAG
CAG
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T-G
GG
TGG
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AG
CGA
ACG
AA
CRCC
G
------
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GG
CGTA
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GG
GCC
TGCA
AG
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GCA
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CAG
CAG
CTCG
T-G
GG
TGG
AG
AG
CGA
ACG
AA
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G
----
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CG
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CC
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CA
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CC
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TG
GC
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TT
AC
CG
C--
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T-A
GG
GT
GG
AG
A--
----
---
----
----
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GA
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TC
GG
GC
CT
GC
A-G
TC
C--
AT
GG
CG
--T
TA
CC
GC
---T
CT
-AG
GG
TG
GA
GA
----
----
----
----
----
----
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gt-
ctc
g9
9cclg
ca-g
lcc--
alg
gc9
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atl
9c--
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ag
gg
t9g
ag
t---
----
----
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t_t_
.;it
tatc
gt-
g9
cg
tlctc
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ca-g
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lcg
co
gc--
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c:g
t-g
gg
tgg
ag
a--
----
----
---
gg
l---
----
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al-
cacg
99
cctg
ca-g
tcc--
atg
gca--
tcg
clg
c--
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ag
g9
c9
9ag
o--
----
----
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CG
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CG
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CR
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CC
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GC
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TC
GC
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GT
GG
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----
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GG
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GG
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GG
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CC
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GC
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T-A
GG
GT
GG
AG
A--
----
----
---
gg
t---
----
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ac-c
acg
gg
cclg
ca-g
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atg
gca--
lcg
ctg
c--
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ag
gg
tgg
ag
a--
----
----
---
gg
t---
----
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at-
cacg
gg
cctg
ca-g
lcc--
atg
gca--
tcg
ctg
c--
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ag
gg
lgg
ag
a--
----
----
---
GG
T--
----
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CG
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CG
GG
CC
TG
CA
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CC
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TG
GC
A--
TC
GC
TG
C--
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T-R
GG
GT
GG
RG
R--
----
----
---
GG
T--
----
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CG
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GG
CC
TG
CR
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CC
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TG
GC
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GC
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T-R
GG
GT
GG
AG
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GG
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RC
CG
RA
GR
RC
GTR
R--C
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AC
GA
GTG
TGTT
GTC
GG
TCG
GC
GC
CTG
TC---
CA
C
RC
CG
RR
GA
RC
TTA
R--C
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AC
GR
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TGTT
GTC
GG
TCG
GC
GC
CTG
TC---
CA
t A
TGA
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GCT
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TTTC
GRG
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GCG
CCRG
CCTT
TCA
C A
TGA
GG
GCT
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TTTC
GRG
GA
C-G
R----
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GG
CIJC
CAG
CCTT
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gg
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alc
gg
cg
ccag
cclt
lcac
acaag
gg
gcl-
cta
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acg
c--
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lcg
gcg
cccg
tcg
ttg
aa
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gg
gg
ctc
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alc
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cg
cccg
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tcac
RTC
GG
GG
CTC
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GR
GG
RC
CG
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CC
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TCG
GC
GC
CC
GTC
TGTC
AC
A
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GG
GC
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RG
GR
CC
GA
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AC
CTG
ATC
GG
CG
CC
CG
TCTG
TCA
C
ATC
GG
GG
CTC
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GA
GG
AC
CG
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CC
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TCG
GC
GC
CC
GTC
TGTC
AC
atc
gg
gg
clc
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gaccg
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alc
gg
cg
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gg
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gg
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cccg
tctg
tca
c
RTC
GG
GG
CTC
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T--C
GR
GG
AC
CG
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CC
TGA
TCG
GC
GC
CC
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TGTC
AC
A
TCG
GG
GC
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CC
GR
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CG
CC
CG
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