Madina Rasulova Molecular Systematics of Nematodes Page 1

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Madina Rasulova Molecular Systematics of Nematodes Page 1

Madina Rasulova Molecular Systematics of Nematodes Page 1


INTRODUCTION

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

B.longicaudatus.

Figure 1. B) Life cycle of

B.longicaudatus.

Figure 1. C) Attack of roots by

B.longicaudatus.

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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

out-group.

For analyzing the relationship of the chosen species several programs were run which are listed

below:

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

above formats.

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.

RESULTS

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

Total

Length

(bp)

Variation

(bp)

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

10 DQ494803

Belonolaimus sp. “Manteo North

Carolina”

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

trees.

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.

DISCUSSION

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),

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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.,

1997).

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.

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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

this genus.

CONCLUSION

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.

ACKNOWLEDGEMENTS

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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APPENDIX

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

consensus tree.

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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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