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Nematodes belonging to the genus of Belonolaimus – also known as sting nematodes – are
economically important ectoparasites of corn causing severe damage by trimming the lateral roots of corn
seedlings even if their number is as low as 1-10 per 100 CC of soil. Belonolaimus longicaudatus has a
wide host range including vegetables (e.g., beans, carrot, corn, crucifers, potato), fruits (e.g., citrus,
strawberry), agronomic crops (e.g., cotton, peanut, sorghum, soybean), turfgrasses (e.g., bermudagrass, St.
Augustinegrass, zoysiagrass) and forest crops (pine trees). Currently nine species of this genus (Table 1)
are recognized (Fortuner and Luc, 1987).
Table 1. The list of species belonging to the genus Belonolaimus.
№ Species of genus Belomolaimus Authors
1 Belonolaimus anama (Monteiro and Lordello, 1977) Fortuner and Luc, 1987
2 Belonolaimus euthychilus Rau, 1963
3 Belonolaimus gracilis Steiner, 1949
4 Belonolaimus jara (Monteiro and Lordello, 1977) Fortuner and Luc, 1987
5 Belonolaimus lineatus Roman, 1964
6 Belonolaimus lolii Siviour, 1978
7 Belonolaimus longicaudatus Rau, 1958
8 Belonolaimus maritimus Rau, 1963
9 Belonolaimus nortoni Rau, 1963
Sting nematodes are relatively large worms (between 1.0 – 3.0 mm). B.longicaudatus (Table 2)
possesses such characteristics as long, slender stylet of which cone constitutes 70-80% of the total stylet
length (Fig. 1, A), oesophageal glands overlapping beginning of intestine, female tail cylindroid with a
broadly rounded terminus, lateral fields (Fortuner and Luc, 1987). These worms are widely distributed in
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very sandy soils and are active when soils become warm, while in unfavourable conditions they migrate
deeper into the soil profile.
Table 2. The position of B.longicaudatus according to the classification.
Phylum Nematoda Potts, 1932
Class Chromadorea Inglis, 1983
Order Rhabditida Chitwood, 1933
Suborder Tylenchina Thorne, 1949
Infraorder Tylenchomorpha De Ley et Blaxter, 2002
Superfamily Tylenchoidea Orley, 1880
Family Belonolaimidae Whitehead, 1959
Genus Belonolaimus Steiner, 1949
Belonolaimus species with a single lateral line occur only in the USA where they are widely spread in
the Southeast and Midwest and occur sporadically in other regions. Belonolaimus species with four lateral
lines are known to occur in Australia, Puerto Rico, Venezuela, and Brazil and are considered by some
authors (Siddiqi, 2000) to constitute a separate genus, Ibipora (Monteiro and Lordello, 1977).
Figure 1. A) The anterior part of
Figure 1. B) Life cycle of
Figure 1. C) Attack of roots by
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It is common to find both sexes of sting nematodes in the soil as they reproduce sexually (Fig. 1, B).
After mating the female lays eggs in pairs in the soil until the food is available. Juveniles hatch out of eggs
after about five days and try to find a root of a plant in order to survive by feeding on it. Juveniles undergo
three moults before becoming adults. The total life cycle of Belonolaimus from egg to reproducing adult
takes about 18-24 days. While feeding the nematodes inject enzymes into root tissues and suck plant
juices out via their stylet (Fig. 1, C) killing the meristematic cells which leads to cessation of growing of
root‟s tip (Grosser et al., 2007). This will lead to abnormal formation of roots and consequently result in
dramatically decrease of harvest.
Due to their significant damage to different crops, measures based on rotation with alfalfa have been
applied and were successful, while chemical nematicides could only reduce the number of sting
nematodes (Rau, 1963). That is the main reason why these nematodes were of great interest for many
scientists (Rau, 1961; Rau, 1963; Abu-Gharbieh et al., 1970; Cherry et al., 1997; Koenning et al., 2006;
Han et al., 2006; Grosser et al., 2007; Grosser et al., 2007) and more thoroughly studies, both
morphological and molecular, should be conducted in order to understand their evolutionary origination as
well as establish the most efficient method of struggle against these economically important pests.
MATERIAL AND METHODS
A partial nucleotide sequence of unknown Belonolaimus genus was chosen for the project work for
further analyzing using different molecular and phylogeny programs. The first step was to find out the
species of this genus as well as the type of this given sequence in NCBI GenBank by BLAST of
nucleotides (Appendix, Figure 4).
According to NCBI GenBank, the studied gene was complete sequence of ITS1 region as well as
contained other partial parts in both ends. Two ITS regions of rDNA, which are located between 18S SSU
and 5.8S for ITS1 and 5.8S and 28S LSU for ITS2, are particularly well-suited for species and population
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level analyses because of appreciable nucleotide polymorphism (Campbell et al., 1995; Chilton et al.,
1995; Ferris et al., 1995). 18S and 28S genes of rDNA have a characteristic to evolve very slowly and can
be used to compare distant taxa where divergence occurred long ago. In comparison, two ITS regions have
higher evolution rates and consequently have been used for analysis of relatively recent evolutionary
events. Consequently, ITS regions are very important in comparison of closely related species (Subbotin
and Moens, 2006) and subspecies and play a role of a genetic marker in taxonomic studies (Cherry et al.,
1997). Moreover, rDNA sequences which encode for rRNA (only SSU, 5.8S and LSU are present in
mature rRNA after splicing) are present in abundant amount as it is common to find them from hundreds
to thousands of tandemly arranged repeats which are separated from each other by intergenic spacer
regions (IGS). Thus, several investigations based on molecular data were carried out in order to improve
the understanding of Belonolaimus’ systematics, phylogeny and distribution (Cherry et al., 1997; Gozel et
al., 2006; Han et al., 2006).
For phylogenetic analysis 13 species of nematodes were chosen, 9 out of which were considered as
in-group to the previously identified from the given sequence Belonolaimus species and they were
selected according to the genus they belong to (all of them were members of the Belonolaimus genus).
The rest 3 species were chosen from different families or at least different genera and were considered as
For analyzing the relationship of the chosen species several programs were run which are listed
BLAST (Basic Local Alignment Search Tool) is used for finding regions of local similarity
between given sequence and other sequences available in databases. The principle of the program is
based on comparison of nucleotide or protein sequences to sequence databases and calculates the
statistical significance of matches. Two of the most important parameters in BLAST are expect value
(represents the rate of found hit just by accident, meaning the smaller the E value – the less possibility
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that it was found just by chance, consequently, the more significant) and maximum identity (stands
for the maximum similarities shown in percentages, the higher the percentage – the more identical
sequences). BLAST can be used to infer functional and evolutionary relationships between sequences
as well as help to identify members of gene families.
Clustal is a widely used computer program for multiple sequence alignment (Thompson et al., 1997)
existing mainly in two variations: ClustalW and ClustalX. The former has a command line interface,
while the latter has a graphical user interface. Clustal gives a possibility to perform a global-multiple
sequence alignment by the progressive method. The process consists of three main steps: (1)
implementation of a pairwise alignment; (2) creation of a phylogenetic tree (or use a user-defined
tree); (3) usage of the phylogenetic tree to carry out a multiple alignment.
GenDoc is a software for carrying out editing processes of multiple sequence alignment as well as its
visualization and analysis. It is very convenient to use this program manual editing of sequence
alignment and prepare it for publication because of easy-to-use point as well as click user interface
with extensive keyboard mapping.
Forcon makes it easy to convert alignment files from one format into other, so converting formats
used by all popular software packages for sequence alignment and phylogenetic tree inference.
Forcon is able to convert files from CLUSTAL, EMBL, FASTA, GCG/MSF, Hennig86, MEGA,
NBRF/PIR, Parsimony Jackknifer, PAUP/NEXUS, PHYLIP and TREECON to any of mentioned
PAUP* (Phylogenetic Analysis Using Parsimony *and other methods) is a program for inferring
and interpreting phylogenetic trees. It analyzes molecular sequences data using maximum likelihood,
parsimony and distance methods. An extensive selection of analysis options and model choices are
included into PAUP*. Besides, it accommodates DNA, RNA, protein and general data types. The rich
array of options for dealing with phylogenetic trees including importing, combining, comparing,
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constraining, rooting and testing hypotheses makes this program very attractive to use (Wilgenbusch
and Swofford, 2003).
MrBayes is a program for the Bayesian inference of phylogeny, which is based on the posterior
probability distribution of trees. This probability is the probability of a tree conditioned on the
observations, executed using Bayes's theorem. Since it is not possible to calculate the posterior
probability distribution of trees analytically, the principle of MrBayes is based on usage of a
simulation technique called Markov chain Monte Carlo (or MCMC) to approximate the posterior
probabilities of trees.
TreeView is a simple, but very useful program for displaying and manipulating phylogenetic trees. It
gives a possibility to view the contents of such format tree file as NEXUS, PHYLIP, Hennig86,
Clustal and others. It is very convenient to run several tree files just in one TreeView and compare the
trees obtained from different programs as well as create publication quality trees.
The chosen 13 sequences from BLAST of nucleotides were saved as a “fasta” file format and multiple
sequence alignment was performed by the help of the program ClustalX v.1.8 which resulted in a new file
“PROJECT_seq.aln” (Appendix, Figure 5). For editing processes GenDoc 2.5 was used and a file
“PROJECT_GenDoc.rtf” was generated in which it was possible to find out the total length of sequences
as well as variation. Next, the previously obtained file was converted into Nexus format using the program
ForCon, in fact the “PROJECT.pau” file was generated. This program was several times edited by the
help of notepad and additional commands were typed in the end of the file (Appendix, Figure 6) for
performing phylogenetic analysis using such programs as PAUP* 4.0 and MrBayes. The program PAUP*
4.0 resulted in such files as “PROJECT_MP.tre” and “PROJECT_MP.txt” for maximum parsimony
method, “PROJECT_NJ.tre” and “PROJECT_NJ.txt” for minimum evolution (neighbor-joining) method,
“PROJECT_ML.tre” and “PROJECT_ML.txt” for maximum likelihood method, and
“PROJECT_distance.txt” for distance analysis. After, the program MrBayes was used in order to generate
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phylogenetic trees based on the posterior probability distribution of trees which resulted in files including
“PROJECT_BItree.mcmc”, “PROJECT_BItree.con” and several others. Finally, all obtained phylogenetic
trees were visualized by the help of the program TreeView.
The study of the given nucleotide sequence of Belonolaimus genus by BLAST in NCBI GeneBank
revealed partial sequence of 18S ribosomal RNA of Belonolaimus longicaudatus GV-2 (accession number
DQ494797) with complete internal transcribed spacer 1 (ITS1) region and partial 5.8S ribosomal RNA
gene the authors of which were Han, Jeyaprakash, Weingarther and Dickson (2006).
4 isolates from 3 species of the Belonolaimus genus and 6 isolates of B.longicaudatus with different
rates of similarities in ITS region were studied and used in phylogenetic analysis as in-group (indicated in
pink colour). Such species as Ditylenchus dipsaci (Tylenchina: Anguinidae), Hoplolaimus columbus
(Tylenchina: Hoplolaimidae) and Tylenchorynchus annulatus (Tylenchina: Belonolaimidae) were chosen
as out-group (indicated in blue colour). Despite the fact that all nematodes mentioned above are from the
same suborder Tylenchina, the latter three species belong to other families (except the last species) and
genera, which makes it possible to use them as out-group (Table 3).
Such alignment characteristics as the total length of studied sequences as well as variations in length
are given below (Table 3). According to the data, the total length and variation of the specimen of our
choice is the same, 685 bp in fact.
Table 3. The list of species used for the project work.
№ Accession Species E value Max identity
1 DQ494797 Belonolaimus longicaudatus 0.0 100 685 685
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2 DQ672373 Belonolaimus longicaudatus 0.0 97 1191 1170
3 DQ672380 Belonolaimus longicaudatus 0.0 95 1182 1161
4 DQ672377 Belonolaimus longicaudatus 0.0 94 1184 1163
5 U89696 Belonolaimus longicaudatus 0.0 90 704 683
6 DQ672384 Belonolaimus longicaudatus 0.0 85 1176 1155
7 DQ672385 Belonolaimus gracilis 6e-105 94 1094 1073
8 DQ672386 Belonolaimus gracilis 0.0 87 1169 1148
9 DQ672382 Belonolaimus euthychilus 6e-105 94 1081 1060
Belonolaimus sp. “Manteo North
6e-105 94 590 590
11 GQ469496 Ditylenchus dipsaci 6e-61 91 967 946
12 DQ309584 Hoplolaimus columbus 1e-62 89 1269 1248
13 EF030983 Tylenchorhynchus annulatus 1e-62 87 1198 1177
Distance matrix characteristics are shown below (Figure 2). The total character differences are
illustrated below diagonal, while the mean character differences are given above diagonal. According to
the table, Belonolaimus longicaudatus of our choice (DQ494797) has differences only in 13 nucleotides
comparing with Belonolaimus longicaudatus DQ672373. The maximum differences in nucleotide
sequence are with Hoplolaimus columbus DQ309584, 251 nucleotides in fact. If consider the mean
character differences, the lowest percentage is 1,906% between Belonolaimus longicaudatus DQ494797
and Belonolaimus longicaudatus DQ672373, whereas the highest percentage is 40,426% between
Belonolaimus longicaudatus DQ494797 and Ditylenchus dispaci GQ469496. Indeed the species which
have the maximum differences in nucleotides and the highest percentage of dissimilarity are out-group
species as they differ from the specimen of our choice a lot. On the other hand, Belonolaimus
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longicaudatus DQ672373 is very similar to the one we are studying in the current work (DQ494797) and
that is the reason why they both occur in the same clade in all phylogenetic trees (discussed below).
Figure 2. Distance matrix.
Trees obtained from 4 phylogenetic methods of analysis have a bit different topology except MP and
ML cases. According to NJ-phylogram (Figure 7, 8) the basal clade (since it is unresolved situation)
consists of 2 branches, one is EF030983, while the other one divides into GQ469496 and DQ309584. The
latter basal clade consists of only out-group species and is highly supported, 100% in fact. The in-group
species are divided into 2 clades: one of which is highly supported (100%) and composed of DQ672385,
DQ672382 and DQ494803. Despite the fact that the second clade is supported only by 77%, it includes all
B.longicaudatus and only one B.gracilis (DQ672386) with BS=98%. The position of studied sequence
sample is in one branch with DQ672373. This clade and its sister clade as well as the branch forming
these two clades are all supported by 100%.
In MP-cladogram (Figure 7, 9) the basal clades are similar as in NJ-phylogram and one of them is
supported by 100%. Then it forms one clade with only one DQ672386 which in its turn divides into two
clades: the first one consisting of only B.longicaudatus species (BS=88%) and the second one comprised
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of other species of the same genus (BS=100%) as well as only one B.longicaudatus DQ672384. If
considering the first sister clade, it is very well supported: it has one branch supported by 98% which
divides into two clades both of which are supported by 100%. The position of the studied B.longicaudatus
is exactly the same as in NJ-phylogram.
The phylogenetic tree of ML (Figure 7, 10) is exactly the same as of MP with only differences in
the percentage by which each branch is supported. The highest BS percentage occurs only in one case: in
one of the basal clades (BS=100%). Relatively higher BS is where the clade forms the branch of the
studied B.longicaudatus DQ494797 and another one DQ672373, 91% in fact. The supported rate of other
branches is very low.
The BI-phylogram (Figure 11) depicts that almost all branches are highly supported: one of the
basal clades with consisting of Ditylenchus dispaci and Hoplolaimus columbus; in in-group: the clade
comprised of species of the genus Belonolaimus except B.longicaudatus; the branch with Belonolaimus
sp.; and two small branches consisting only out of B.longicaudatus including the studied one are all
supported by 100%. The clade constituted of these two small branches is also well supported, 98% in fact.
The position of out-groups and the studied B.longicaudatus remains the same as in other phylogenetic
Though NJ, MP and BI phylograms (Figures 7-11) are not the same by their topology, they have
some clades which remain unchangeable in all types of trees which will be described below. Since the
relationships in the out-groups are not completely resolved, it is complicated to indicate which species or
which branch is a basal clade and, thus, all three species are considered as basal clades. Another similarity
is found in the clade consisting of only B.longicaudatus species as well as the position of the studied one
is constant as well. Besides, other members of the genus Belonolaimus are also always gathered in one
clade in all 4 trees. The position of other inside clades is rather changeable. Surprisingly, the only one
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B.longicaudatus DQ672384 occurs in different places in different trees. But, nevertheless, MP and ML
trees have exactly the same topology.
In current observation 3 such species as B.euthychilus, B.gracilis and Belonolaimus sp. were chosen
in order to find out their relationship with the species of our choice – B.longicaudatus (DQ494797).
Among 13 isolates 10 were identified as in-group consisting of 6 B. longicaudatus species only and 2
B.gracilis, 1 B.euthychilus, and 1 Belonolaimus sp. “Manteo North Carolina”; while the rest were outgroup
species comprised of Ditylenchus dispaci, Hoplolaimus columbus, and Tylenchorhynchus
annulatus. According to Rau (1963), B.longicaudatus differs from B.gracilis with such morphological
characteristics as having sclerotized plates in the vagina, an elongated rather than spherical metacorpus
and a hemispherical rather than convex-conoid tail shape. Despite the fact that B.euthychilus looks like
B.gracilis, it exhibits sexual dimorphism (males with degenerated stylet and pharynx) and does not
possess a constriction between the labial region and the body.
Since ITS regions are important in analysis of different populations of the same species, a number of
investigations were based on ITS1 and ITS2 parts for studying different populations of the genus
Belonolaimus. Thereby, Cherry et al. (1997) indicated that B. longicaudatus isolates were relatively recent
introduced into the state of California in comparison to Florida and South Carolina. According to
morphological data, the research of Han et al. (2006) showed that females of the isolates from corn
(Scotland County) and citrus (Lake Alfred) fields possess teardrop or kidney-shaped stylet, whereas in
isolates from cotton field (Tifton) is typically oval. The vaginal pieces of isolates from citrus field (Lake
Alfred) were the most prominent and clearly recognized among all isolates, but of those found in corn
field (Scotland County) were weakly developed and not clearly recognized. Based on molecular data it
was possible to conclude that all phylogenetic trees supported that the corn (Columbus, South Carolina),
Madina Rasulova Molecular Systematics of Nematodes Page 12
ermudagrass (Poteet) isolates were clearly different from the bermudagrass (Gainesville), potato
(Hastings), citrus (Lake Alfred), cotton (Tifton) and corn (Scotland County) isolates.
Gozel et al. (2006) studied the D2-D3 and ITS regions of rDNA. The most striking point of her
phylogenetic analysis is that none of the three nominal species (B. longicaudatus, B. euthychilus, and B.
gracilis) are monophyletic. The studies made it possible to identify suites of morphological/morphometric
character states that discriminated between the molecular-derived clades of B.longicaudatus. According to
the tree, relationships between B. euthychilus BePi1 and B. gracilis BgPi2 were unresolved (the presence
or absence of an offset head; the population BgPi2 was identified as B. gracilis, but according to the gene
sequence was much closer to B. euthychilus. However, the character appeared to be intermediate between
the two species, being somewhat less distinctly offset than in B. gracilis). The ratio „stylet length:tail
length‟ has been used like a morphometric character that distinguishes B. longicaudatus from both other
species. Stylets were shorter than tails (ratio < 1.0) in 83-100% of B. longicaudatus specimens from 15
populations (from Florida to New Jersey) and stylets were longer than tails (ratio > 1.0) in all observed
specimens of B. gracilis and B. euthychilus (Rau, 1961; Rau, 1963). On the other hand, five B.
longicaudatus populations (citrus orchards in Polk County, sugarcane in Martin County) had stylets that
were on average longer than tails (Duncan et al., 1999). In fact, the ratios are intermediate between those
reported by Rau (1961) for B. longicaudatus and B. gracilis. Besides, the stylet:tail ratios for B.
euthychilus are very similar to those of B. gracilis. Therefore, it is possible to discriminate B. gracilis and
B. euthychilus from B. longicaudatus. It is possible to conclude that B. longicaudatus populations have a
more recent evolution with a ratio > 1.0 differ in ITS region by no more than two base pairs, while most B.
longicaudatus populations with a ratio < 1.0 have a wide variation in both D2-D3 and ITS nucleotide
sequences (Gozel et al., 2006). Like in our research, comparisons of the MP and ML trees revealed no
significant differences based on the topological features.
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Restriction patterns of the Belonolaimus ITS1 region done by Cherry et al. (1997) illustrate that this
region differs from one individual nematode to another. For example, Hinc II digestion of ITS1 from the
Arkansas population (Figure 3, A) displayed three fragments between 300 and 400 bp, while in Kansas
population there are two bands in one case and one band in two cases (Figure 3, B). The populations from
other areas displayed only one fragment in the same range. Despite of very similar morphologies the
studied isolates gave a unique restriction profile. This may be explained by evolutionary divergence that
has occurred in allopatry, since Belonolaimus populations live in sandy soils that are often geographically
isolated. ITS1 heterogeneity within individuals has been observed in Meloidogyne (Zijlstra et al., 1995)
because of mitotically parthenogenetic polyploidy, but the structural nature of this heterogeneity in
Belonolaimus is unclear since Belonolaimus is a taxon of diploid, amphimictic species (Cherry et al.,
According to Gozel et al. (2006), the phylogeny inferred from the DNA sequences of the sting
nematode populations indeed support the likelihood that B. longicaudatus and B. euthychilus are species
complexes (Adams, 1998). Robbins and Hirschmann (1974) propose that populations of B. longicaudatus
Figure 3. A) Hinc II digestion patterns of
representative Belonolaimus longicaudatus from
different isolates. KS = Kansas, AR = Arkansas, CA =
California, SC = South Carolina, FL = Florida.
Figure 3. B) Nucleotide base pair fragment length patterns of
various digested Belonolaimus isolates.
Madina Rasulova Molecular Systematics of Nematodes Page 14
outside of Florida are reproductively isolated. Besides, several studies report that B. longicaudatus
populations differ within each other showing various host range and wide morphometric variations (Abu-
Gharbieh and Perry, 1970; Robbins and Hirschmann, 1974; Duncan et al., 1996). All of the above
mentioned prove, that indeed B. longicaudatus populations are very complex and require additional
studies of reproductive compatibility, behavior and morphology of specific genotypes (Gozel et al., 2006)
in order to be able to distinguish it from other species as well as have the complete picture of phylogeny of
Polymorphism within populations of the same species makes it an ideal tool for application in many
aspects such as phylogeny in order to distinguish individuals among populations (in this case
B.longicaudatus). These genetic differences, on one hand serve as convenient diagnostic markers; on the
other hand proves that the genus Belonolaimus is far more complex than currently recognized.
Many thanks to Professor Sergei A. Subbotin for providing us with necessary skills, so we are able to
work with a number of phylogeny programs ourselves without any assistance. I believe that this acquired
knowledge would be valuable in our further research activities as well as future career.
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Current Protocols in Bioinformatics, Wiley and Sons, New York. Pages 6.4.1-6.4.28.
Zijlstra, C., A. E. M. Lever, B.J. Uenk, and C. H. Van Silfhout. (1995) Differences between ITS regions
of isolates of root-knot nematodes Meloidogyne hapla and M. chitwoodi. Phytopathology
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Figure 4. Results of BLAST search in NCBI GeneBank.
Figure 5. Sequence multiple alignment by ClustalX.
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Figure 6. The notepad with all commands for phylogeny programs (PAUP* and MrBayes).
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Figure 7. NJ, MP and ML trees with the supported rate (BS).
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Figure 8. Phylogenetic tree NJ. Neighbor Joining: number of bootstrap replicates = 10000. Bootstrap 50% majority-rule
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Figure 9. Phylogenetic tree MP. Maximum Parsimony: number of bootstrap replicates = 1000. Search = heuristic.
Among 1311 characters: 579 – constant characters; 323 – variable parsimony-uninformative characters; 409 –
parsimony-informative characters. Gaps are treated as “missing”. Bootstrap 50% majority-rule consensus tree.
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Figure 10. Phylogenetic tree ML. Maximum Likelihood: number of bootstrap replicates = 100. GTR+G+I model.
Number of substitution types = 6. Assumed nucleotide frequencies: A=0.19270 C=0.22540 G=0.28520 T=0.29670.
Bootstrap 50% majority-rule consensus tree.
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Figure 11. Phylogenetic tree MrB. Bayesian inference: 1000000 replicates; Markov Chains.
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