Madina Rasulova Molecular Systematics of Nematodes Page 1
Madina Rasulova Molecular Systematics of Nematodes Page 1
Madina Rasulova Molecular Systematics of Nematodes Page 1
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<strong>Madina</strong> <strong>Rasulova</strong> <strong>Molecular</strong> <strong>Systematics</strong> <strong>of</strong> <strong>Nematodes</strong> <strong>Page</strong> 1
INTRODUCTION<br />
<strong>Nematodes</strong> belonging to the genus <strong>of</strong> Belonolaimus – also known as sting nematodes – are<br />
economically important ectoparasites <strong>of</strong> corn causing severe damage by trimming the lateral roots <strong>of</strong> corn<br />
seedlings even if their number is as low as 1-10 per 100 CC <strong>of</strong> soil. Belonolaimus longicaudatus has a<br />
wide host range including vegetables (e.g., beans, carrot, corn, crucifers, potato), fruits (e.g., citrus,<br />
strawberry), agronomic crops (e.g., cotton, peanut, sorghum, soybean), turfgrasses (e.g., bermudagrass, St.<br />
Augustinegrass, zoysiagrass) and forest crops (pine trees). Currently nine species <strong>of</strong> this genus (Table 1)<br />
are recognized (Fortuner and Luc, 1987).<br />
Table 1. The list <strong>of</strong> species belonging to the genus Belonolaimus.<br />
№ Species <strong>of</strong> genus Belomolaimus Authors<br />
1 Belonolaimus anama (Monteiro and Lordello, 1977) Fortuner and Luc, 1987<br />
2 Belonolaimus euthychilus Rau, 1963<br />
3 Belonolaimus gracilis Steiner, 1949<br />
4 Belonolaimus jara (Monteiro and Lordello, 1977) Fortuner and Luc, 1987<br />
5 Belonolaimus lineatus Roman, 1964<br />
6 Belonolaimus lolii Siviour, 1978<br />
7 Belonolaimus longicaudatus Rau, 1958<br />
8 Belonolaimus maritimus Rau, 1963<br />
9 Belonolaimus nortoni Rau, 1963<br />
Sting nematodes are relatively large worms (between 1.0 – 3.0 mm). B.longicaudatus (Table 2)<br />
possesses such characteristics as long, slender stylet <strong>of</strong> which cone constitutes 70-80% <strong>of</strong> the total stylet<br />
length (Fig. 1, A), oesophageal glands overlapping beginning <strong>of</strong> intestine, female tail cylindroid with a<br />
broadly rounded terminus, lateral fields (Fortuner and Luc, 1987). These worms are widely distributed in<br />
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very sandy soils and are active when soils become warm, while in unfavourable conditions they migrate<br />
deeper into the soil pr<strong>of</strong>ile.<br />
Table 2. The position <strong>of</strong> B.longicaudatus according to the classification.<br />
Phylum Nematoda Potts, 1932<br />
Class Chromadorea Inglis, 1983<br />
Order Rhabditida Chitwood, 1933<br />
Suborder Tylenchina Thorne, 1949<br />
Infraorder Tylenchomorpha De Ley et Blaxter, 2002<br />
Superfamily Tylenchoidea Orley, 1880<br />
Family Belonolaimidae Whitehead, 1959<br />
Genus Belonolaimus Steiner, 1949<br />
Belonolaimus species with a single lateral line occur only in the USA where they are widely spread in<br />
the Southeast and Midwest and occur sporadically in other regions. Belonolaimus species with four lateral<br />
lines are known to occur in Australia, Puerto Rico, Venezuela, and Brazil and are considered by some<br />
authors (Siddiqi, 2000) to constitute a separate genus, Ibipora (Monteiro and Lordello, 1977).<br />
Figure 1. A) The anterior part <strong>of</strong><br />
B.longicaudatus.<br />
Figure 1. B) Life cycle <strong>of</strong><br />
B.longicaudatus.<br />
Figure 1. C) Attack <strong>of</strong> roots by<br />
B.longicaudatus.<br />
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It is common to find both sexes <strong>of</strong> sting nematodes in the soil as they reproduce sexually (Fig. 1, B).<br />
After mating the female lays eggs in pairs in the soil until the food is available. Juveniles hatch out <strong>of</strong> eggs<br />
after about five days and try to find a root <strong>of</strong> a plant in order to survive by feeding on it. Juveniles undergo<br />
three moults before becoming adults. The total life cycle <strong>of</strong> Belonolaimus from egg to reproducing adult<br />
takes about 18-24 days. While feeding the nematodes inject enzymes into root tissues and suck plant<br />
juices out via their stylet (Fig. 1, C) killing the meristematic cells which leads to cessation <strong>of</strong> growing <strong>of</strong><br />
root‟s tip (Grosser et al., 2007). This will lead to abnormal formation <strong>of</strong> roots and consequently result in<br />
dramatically decrease <strong>of</strong> harvest.<br />
Due to their significant damage to different crops, measures based on rotation with alfalfa have been<br />
applied and were successful, while chemical nematicides could only reduce the number <strong>of</strong> sting<br />
nematodes (Rau, 1963). That is the main reason why these nematodes were <strong>of</strong> great interest for many<br />
scientists (Rau, 1961; Rau, 1963; Abu-Gharbieh et al., 1970; Cherry et al., 1997; Koenning et al., 2006;<br />
Han et al., 2006; Grosser et al., 2007; Grosser et al., 2007) and more thoroughly studies, both<br />
morphological and molecular, should be conducted in order to understand their evolutionary origination as<br />
well as establish the most efficient method <strong>of</strong> struggle against these economically important pests.<br />
MATERIAL AND METHODS<br />
A partial nucleotide sequence <strong>of</strong> unknown Belonolaimus genus was chosen for the project work for<br />
further analyzing using different molecular and phylogeny programs. The first step was to find out the<br />
species <strong>of</strong> this genus as well as the type <strong>of</strong> this given sequence in NCBI GenBank by BLAST <strong>of</strong><br />
nucleotides (Appendix, Figure 4).<br />
According to NCBI GenBank, the studied gene was complete sequence <strong>of</strong> ITS1 region as well as<br />
contained other partial parts in both ends. Two ITS regions <strong>of</strong> rDNA, which are located between 18S SSU<br />
and 5.8S for ITS1 and 5.8S and 28S LSU for ITS2, are particularly well-suited for species and population<br />
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level analyses because <strong>of</strong> appreciable nucleotide polymorphism (Campbell et al., 1995; Chilton et al.,<br />
1995; Ferris et al., 1995). 18S and 28S genes <strong>of</strong> rDNA have a characteristic to evolve very slowly and can<br />
be used to compare distant taxa where divergence occurred long ago. In comparison, two ITS regions have<br />
higher evolution rates and consequently have been used for analysis <strong>of</strong> relatively recent evolutionary<br />
events. Consequently, ITS regions are very important in comparison <strong>of</strong> closely related species (Subbotin<br />
and Moens, 2006) and subspecies and play a role <strong>of</strong> a genetic marker in taxonomic studies (Cherry et al.,<br />
1997). Moreover, rDNA sequences which encode for rRNA (only SSU, 5.8S and LSU are present in<br />
mature rRNA after splicing) are present in abundant amount as it is common to find them from hundreds<br />
to thousands <strong>of</strong> tandemly arranged repeats which are separated from each other by intergenic spacer<br />
regions (IGS). Thus, several investigations based on molecular data were carried out in order to improve<br />
the understanding <strong>of</strong> Belonolaimus’ systematics, phylogeny and distribution (Cherry et al., 1997; Gozel et<br />
al., 2006; Han et al., 2006).<br />
For phylogenetic analysis 13 species <strong>of</strong> nematodes were chosen, 9 out <strong>of</strong> which were considered as<br />
in-group to the previously identified from the given sequence Belonolaimus species and they were<br />
selected according to the genus they belong to (all <strong>of</strong> them were members <strong>of</strong> the Belonolaimus genus).<br />
The rest 3 species were chosen from different families or at least different genera and were considered as<br />
out-group.<br />
For analyzing the relationship <strong>of</strong> the chosen species several programs were run which are listed<br />
below:<br />
BLAST (Basic Local Alignment Search Tool) is used for finding regions <strong>of</strong> local similarity<br />
between given sequence and other sequences available in databases. The principle <strong>of</strong> the program is<br />
based on comparison <strong>of</strong> nucleotide or protein sequences to sequence databases and calculates the<br />
statistical significance <strong>of</strong> matches. Two <strong>of</strong> the most important parameters in BLAST are expect value<br />
(represents the rate <strong>of</strong> found hit just by accident, meaning the smaller the E value – the less possibility<br />
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that it was found just by chance, consequently, the more significant) and maximum identity (stands<br />
for the maximum similarities shown in percentages, the higher the percentage – the more identical<br />
sequences). BLAST can be used to infer functional and evolutionary relationships between sequences<br />
as well as help to identify members <strong>of</strong> gene families.<br />
Clustal is a widely used computer program for multiple sequence alignment (Thompson et al., 1997)<br />
existing mainly in two variations: ClustalW and ClustalX. The former has a command line interface,<br />
while the latter has a graphical user interface. Clustal gives a possibility to perform a global-multiple<br />
sequence alignment by the progressive method. The process consists <strong>of</strong> three main steps: (1)<br />
implementation <strong>of</strong> a pairwise alignment; (2) creation <strong>of</strong> a phylogenetic tree (or use a user-defined<br />
tree); (3) usage <strong>of</strong> the phylogenetic tree to carry out a multiple alignment.<br />
GenDoc is a s<strong>of</strong>tware for carrying out editing processes <strong>of</strong> multiple sequence alignment as well as its<br />
visualization and analysis. It is very convenient to use this program manual editing <strong>of</strong> sequence<br />
alignment and prepare it for publication because <strong>of</strong> easy-to-use point as well as click user interface<br />
with extensive keyboard mapping.<br />
Forcon makes it easy to convert alignment files from one format into other, so converting formats<br />
used by all popular s<strong>of</strong>tware packages for sequence alignment and phylogenetic tree inference.<br />
Forcon is able to convert files from CLUSTAL, EMBL, FASTA, GCG/MSF, Hennig86, MEGA,<br />
NBRF/PIR, Parsimony Jackknifer, PAUP/NEXUS, PHYLIP and TREECON to any <strong>of</strong> mentioned<br />
above formats.<br />
PAUP* (Phylogenetic Analysis Using Parsimony *and other methods) is a program for inferring<br />
and interpreting phylogenetic trees. It analyzes molecular sequences data using maximum likelihood,<br />
parsimony and distance methods. An extensive selection <strong>of</strong> analysis options and model choices are<br />
included into PAUP*. Besides, it accommodates DNA, RNA, protein and general data types. The rich<br />
array <strong>of</strong> options for dealing with phylogenetic trees including importing, combining, comparing,<br />
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constraining, rooting and testing hypotheses makes this program very attractive to use (Wilgenbusch<br />
and Sw<strong>of</strong>ford, 2003).<br />
MrBayes is a program for the Bayesian inference <strong>of</strong> phylogeny, which is based on the posterior<br />
probability distribution <strong>of</strong> trees. This probability is the probability <strong>of</strong> a tree conditioned on the<br />
observations, executed using Bayes's theorem. Since it is not possible to calculate the posterior<br />
probability distribution <strong>of</strong> trees analytically, the principle <strong>of</strong> MrBayes is based on usage <strong>of</strong> a<br />
simulation technique called Markov chain Monte Carlo (or MCMC) to approximate the posterior<br />
probabilities <strong>of</strong> trees.<br />
TreeView is a simple, but very useful program for displaying and manipulating phylogenetic trees. It<br />
gives a possibility to view the contents <strong>of</strong> such format tree file as NEXUS, PHYLIP, Hennig86,<br />
Clustal and others. It is very convenient to run several tree files just in one TreeView and compare the<br />
trees obtained from different programs as well as create publication quality trees.<br />
The chosen 13 sequences from BLAST <strong>of</strong> nucleotides were saved as a “fasta” file format and multiple<br />
sequence alignment was performed by the help <strong>of</strong> the program ClustalX v.1.8 which resulted in a new file<br />
“PROJECT_seq.aln” (Appendix, Figure 5). For editing processes GenDoc 2.5 was used and a file<br />
“PROJECT_GenDoc.rtf” was generated in which it was possible to find out the total length <strong>of</strong> sequences<br />
as well as variation. Next, the previously obtained file was converted into Nexus format using the program<br />
ForCon, in fact the “PROJECT.pau” file was generated. This program was several times edited by the<br />
help <strong>of</strong> notepad and additional commands were typed in the end <strong>of</strong> the file (Appendix, Figure 6) for<br />
performing phylogenetic analysis using such programs as PAUP* 4.0 and MrBayes. The program PAUP*<br />
4.0 resulted in such files as “PROJECT_MP.tre” and “PROJECT_MP.txt” for maximum parsimony<br />
method, “PROJECT_NJ.tre” and “PROJECT_NJ.txt” for minimum evolution (neighbor-joining) method,<br />
“PROJECT_ML.tre” and “PROJECT_ML.txt” for maximum likelihood method, and<br />
“PROJECT_distance.txt” for distance analysis. After, the program MrBayes was used in order to generate<br />
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phylogenetic trees based on the posterior probability distribution <strong>of</strong> trees which resulted in files including<br />
“PROJECT_BItree.mcmc”, “PROJECT_BItree.con” and several others. Finally, all obtained phylogenetic<br />
trees were visualized by the help <strong>of</strong> the program TreeView.<br />
RESULTS<br />
The study <strong>of</strong> the given nucleotide sequence <strong>of</strong> Belonolaimus genus by BLAST in NCBI GeneBank<br />
revealed partial sequence <strong>of</strong> 18S ribosomal RNA <strong>of</strong> Belonolaimus longicaudatus GV-2 (accession number<br />
DQ494797) with complete internal transcribed spacer 1 (ITS1) region and partial 5.8S ribosomal RNA<br />
gene the authors <strong>of</strong> which were Han, Jeyaprakash, Weingarther and Dickson (2006).<br />
4 isolates from 3 species <strong>of</strong> the Belonolaimus genus and 6 isolates <strong>of</strong> B.longicaudatus with different<br />
rates <strong>of</strong> similarities in ITS region were studied and used in phylogenetic analysis as in-group (indicated in<br />
pink colour). Such species as Ditylenchus dipsaci (Tylenchina: Anguinidae), Hoplolaimus columbus<br />
(Tylenchina: Hoplolaimidae) and Tylenchorynchus annulatus (Tylenchina: Belonolaimidae) were chosen<br />
as out-group (indicated in blue colour). Despite the fact that all nematodes mentioned above are from the<br />
same suborder Tylenchina, the latter three species belong to other families (except the last species) and<br />
genera, which makes it possible to use them as out-group (Table 3).<br />
Such alignment characteristics as the total length <strong>of</strong> studied sequences as well as variations in length<br />
are given below (Table 3). According to the data, the total length and variation <strong>of</strong> the specimen <strong>of</strong> our<br />
choice is the same, 685 bp in fact.<br />
Table 3. The list <strong>of</strong> species used for the project work.<br />
№ Accession Species E value Max identity<br />
Total<br />
Length<br />
(bp)<br />
Variation<br />
(bp)<br />
1 DQ494797 Belonolaimus longicaudatus 0.0 100 685 685<br />
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2 DQ672373 Belonolaimus longicaudatus 0.0 97 1191 1170<br />
3 DQ672380 Belonolaimus longicaudatus 0.0 95 1182 1161<br />
4 DQ672377 Belonolaimus longicaudatus 0.0 94 1184 1163<br />
5 U89696 Belonolaimus longicaudatus 0.0 90 704 683<br />
6 DQ672384 Belonolaimus longicaudatus 0.0 85 1176 1155<br />
7 DQ672385 Belonolaimus gracilis 6e-105 94 1094 1073<br />
8 DQ672386 Belonolaimus gracilis 0.0 87 1169 1148<br />
9 DQ672382 Belonolaimus euthychilus 6e-105 94 1081 1060<br />
10 DQ494803<br />
Belonolaimus sp. “Manteo North<br />
Carolina”<br />
6e-105 94 590 590<br />
11 GQ469496 Ditylenchus dipsaci 6e-61 91 967 946<br />
12 DQ309584 Hoplolaimus columbus 1e-62 89 1269 1248<br />
13 EF030983 Tylenchorhynchus annulatus 1e-62 87 1198 1177<br />
Distance matrix characteristics are shown below (Figure 2). The total character differences are<br />
illustrated below diagonal, while the mean character differences are given above diagonal. According to<br />
the table, Belonolaimus longicaudatus <strong>of</strong> our choice (DQ494797) has differences only in 13 nucleotides<br />
comparing with Belonolaimus longicaudatus DQ672373. The maximum differences in nucleotide<br />
sequence are with Hoplolaimus columbus DQ309584, 251 nucleotides in fact. If consider the mean<br />
character differences, the lowest percentage is 1,906% between Belonolaimus longicaudatus DQ494797<br />
and Belonolaimus longicaudatus DQ672373, whereas the highest percentage is 40,426% between<br />
Belonolaimus longicaudatus DQ494797 and Ditylenchus dispaci GQ469496. Indeed the species which<br />
have the maximum differences in nucleotides and the highest percentage <strong>of</strong> dissimilarity are out-group<br />
species as they differ from the specimen <strong>of</strong> our choice a lot. On the other hand, Belonolaimus<br />
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longicaudatus DQ672373 is very similar to the one we are studying in the current work (DQ494797) and<br />
that is the reason why they both occur in the same clade in all phylogenetic trees (discussed below).<br />
Figure 2. Distance matrix.<br />
Trees obtained from 4 phylogenetic methods <strong>of</strong> analysis have a bit different topology except MP and<br />
ML cases. According to NJ-phylogram (Figure 7, 8) the basal clade (since it is unresolved situation)<br />
consists <strong>of</strong> 2 branches, one is EF030983, while the other one divides into GQ469496 and DQ309584. The<br />
latter basal clade consists <strong>of</strong> only out-group species and is highly supported, 100% in fact. The in-group<br />
species are divided into 2 clades: one <strong>of</strong> which is highly supported (100%) and composed <strong>of</strong> DQ672385,<br />
DQ672382 and DQ494803. Despite the fact that the second clade is supported only by 77%, it includes all<br />
B.longicaudatus and only one B.gracilis (DQ672386) with BS=98%. The position <strong>of</strong> studied sequence<br />
sample is in one branch with DQ672373. This clade and its sister clade as well as the branch forming<br />
these two clades are all supported by 100%.<br />
In MP-cladogram (Figure 7, 9) the basal clades are similar as in NJ-phylogram and one <strong>of</strong> them is<br />
supported by 100%. Then it forms one clade with only one DQ672386 which in its turn divides into two<br />
clades: the first one consisting <strong>of</strong> only B.longicaudatus species (BS=88%) and the second one comprised<br />
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<strong>of</strong> other species <strong>of</strong> the same genus (BS=100%) as well as only one B.longicaudatus DQ672384. If<br />
considering the first sister clade, it is very well supported: it has one branch supported by 98% which<br />
divides into two clades both <strong>of</strong> which are supported by 100%. The position <strong>of</strong> the studied B.longicaudatus<br />
is exactly the same as in NJ-phylogram.<br />
The phylogenetic tree <strong>of</strong> ML (Figure 7, 10) is exactly the same as <strong>of</strong> MP with only differences in<br />
the percentage by which each branch is supported. The highest BS percentage occurs only in one case: in<br />
one <strong>of</strong> the basal clades (BS=100%). Relatively higher BS is where the clade forms the branch <strong>of</strong> the<br />
studied B.longicaudatus DQ494797 and another one DQ672373, 91% in fact. The supported rate <strong>of</strong> other<br />
branches is very low.<br />
The BI-phylogram (Figure 11) depicts that almost all branches are highly supported: one <strong>of</strong> the<br />
basal clades with consisting <strong>of</strong> Ditylenchus dispaci and Hoplolaimus columbus; in in-group: the clade<br />
comprised <strong>of</strong> species <strong>of</strong> the genus Belonolaimus except B.longicaudatus; the branch with Belonolaimus<br />
sp.; and two small branches consisting only out <strong>of</strong> B.longicaudatus including the studied one are all<br />
supported by 100%. The clade constituted <strong>of</strong> these two small branches is also well supported, 98% in fact.<br />
The position <strong>of</strong> out-groups and the studied B.longicaudatus remains the same as in other phylogenetic<br />
trees.<br />
Though NJ, MP and BI phylograms (Figures 7-11) are not the same by their topology, they have<br />
some clades which remain unchangeable in all types <strong>of</strong> trees which will be described below. Since the<br />
relationships in the out-groups are not completely resolved, it is complicated to indicate which species or<br />
which branch is a basal clade and, thus, all three species are considered as basal clades. Another similarity<br />
is found in the clade consisting <strong>of</strong> only B.longicaudatus species as well as the position <strong>of</strong> the studied one<br />
is constant as well. Besides, other members <strong>of</strong> the genus Belonolaimus are also always gathered in one<br />
clade in all 4 trees. The position <strong>of</strong> other inside clades is rather changeable. Surprisingly, the only one<br />
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B.longicaudatus DQ672384 occurs in different places in different trees. But, nevertheless, MP and ML<br />
trees have exactly the same topology.<br />
DISCUSSION<br />
In current observation 3 such species as B.euthychilus, B.gracilis and Belonolaimus sp. were chosen<br />
in order to find out their relationship with the species <strong>of</strong> our choice – B.longicaudatus (DQ494797).<br />
Among 13 isolates 10 were identified as in-group consisting <strong>of</strong> 6 B. longicaudatus species only and 2<br />
B.gracilis, 1 B.euthychilus, and 1 Belonolaimus sp. “Manteo North Carolina”; while the rest were outgroup<br />
species comprised <strong>of</strong> Ditylenchus dispaci, Hoplolaimus columbus, and Tylenchorhynchus<br />
annulatus. According to Rau (1963), B.longicaudatus differs from B.gracilis with such morphological<br />
characteristics as having sclerotized plates in the vagina, an elongated rather than spherical metacorpus<br />
and a hemispherical rather than convex-conoid tail shape. Despite the fact that B.euthychilus looks like<br />
B.gracilis, it exhibits sexual dimorphism (males with degenerated stylet and pharynx) and does not<br />
possess a constriction between the labial region and the body.<br />
Since ITS regions are important in analysis <strong>of</strong> different populations <strong>of</strong> the same species, a number <strong>of</strong><br />
investigations were based on ITS1 and ITS2 parts for studying different populations <strong>of</strong> the genus<br />
Belonolaimus. Thereby, Cherry et al. (1997) indicated that B. longicaudatus isolates were relatively recent<br />
introduced into the state <strong>of</strong> California in comparison to Florida and South Carolina. According to<br />
morphological data, the research <strong>of</strong> Han et al. (2006) showed that females <strong>of</strong> the isolates from corn<br />
(Scotland County) and citrus (Lake Alfred) fields possess teardrop or kidney-shaped stylet, whereas in<br />
isolates from cotton field (Tifton) is typically oval. The vaginal pieces <strong>of</strong> isolates from citrus field (Lake<br />
Alfred) were the most prominent and clearly recognized among all isolates, but <strong>of</strong> those found in corn<br />
field (Scotland County) were weakly developed and not clearly recognized. Based on molecular data it<br />
was possible to conclude that all phylogenetic trees supported that the corn (Columbus, South Carolina),<br />
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ermudagrass (Poteet) isolates were clearly different from the bermudagrass (Gainesville), potato<br />
(Hastings), citrus (Lake Alfred), cotton (Tifton) and corn (Scotland County) isolates.<br />
Gozel et al. (2006) studied the D2-D3 and ITS regions <strong>of</strong> rDNA. The most striking point <strong>of</strong> her<br />
phylogenetic analysis is that none <strong>of</strong> the three nominal species (B. longicaudatus, B. euthychilus, and B.<br />
gracilis) are monophyletic. The studies made it possible to identify suites <strong>of</strong> morphological/morphometric<br />
character states that discriminated between the molecular-derived clades <strong>of</strong> B.longicaudatus. According to<br />
the tree, relationships between B. euthychilus BePi1 and B. gracilis BgPi2 were unresolved (the presence<br />
or absence <strong>of</strong> an <strong>of</strong>fset head; the population BgPi2 was identified as B. gracilis, but according to the gene<br />
sequence was much closer to B. euthychilus. However, the character appeared to be intermediate between<br />
the two species, being somewhat less distinctly <strong>of</strong>fset than in B. gracilis). The ratio „stylet length:tail<br />
length‟ has been used like a morphometric character that distinguishes B. longicaudatus from both other<br />
species. Stylets were shorter than tails (ratio < 1.0) in 83-100% <strong>of</strong> B. longicaudatus specimens from 15<br />
populations (from Florida to New Jersey) and stylets were longer than tails (ratio > 1.0) in all observed<br />
specimens <strong>of</strong> B. gracilis and B. euthychilus (Rau, 1961; Rau, 1963). On the other hand, five B.<br />
longicaudatus populations (citrus orchards in Polk County, sugarcane in Martin County) had stylets that<br />
were on average longer than tails (Duncan et al., 1999). In fact, the ratios are intermediate between those<br />
reported by Rau (1961) for B. longicaudatus and B. gracilis. Besides, the stylet:tail ratios for B.<br />
euthychilus are very similar to those <strong>of</strong> B. gracilis. Therefore, it is possible to discriminate B. gracilis and<br />
B. euthychilus from B. longicaudatus. It is possible to conclude that B. longicaudatus populations have a<br />
more recent evolution with a ratio > 1.0 differ in ITS region by no more than two base pairs, while most B.<br />
longicaudatus populations with a ratio < 1.0 have a wide variation in both D2-D3 and ITS nucleotide<br />
sequences (Gozel et al., 2006). Like in our research, comparisons <strong>of</strong> the MP and ML trees revealed no<br />
significant differences based on the topological features.<br />
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Restriction patterns <strong>of</strong> the Belonolaimus ITS1 region done by Cherry et al. (1997) illustrate that this<br />
region differs from one individual nematode to another. For example, Hinc II digestion <strong>of</strong> ITS1 from the<br />
Arkansas population (Figure 3, A) displayed three fragments between 300 and 400 bp, while in Kansas<br />
population there are two bands in one case and one band in two cases (Figure 3, B). The populations from<br />
other areas displayed only one fragment in the same range. Despite <strong>of</strong> very similar morphologies the<br />
studied isolates gave a unique restriction pr<strong>of</strong>ile. This may be explained by evolutionary divergence that<br />
has occurred in allopatry, since Belonolaimus populations live in sandy soils that are <strong>of</strong>ten geographically<br />
isolated. ITS1 heterogeneity within individuals has been observed in Meloidogyne (Zijlstra et al., 1995)<br />
because <strong>of</strong> mitotically parthenogenetic polyploidy, but the structural nature <strong>of</strong> this heterogeneity in<br />
Belonolaimus is unclear since Belonolaimus is a taxon <strong>of</strong> diploid, amphimictic species (Cherry et al.,<br />
1997).<br />
According to Gozel et al. (2006), the phylogeny inferred from the DNA sequences <strong>of</strong> the sting<br />
nematode populations indeed support the likelihood that B. longicaudatus and B. euthychilus are species<br />
complexes (Adams, 1998). Robbins and Hirschmann (1974) propose that populations <strong>of</strong> B. longicaudatus<br />
Figure 3. A) Hinc II digestion patterns <strong>of</strong><br />
representative Belonolaimus longicaudatus from<br />
different isolates. KS = Kansas, AR = Arkansas, CA =<br />
California, SC = South Carolina, FL = Florida.<br />
Figure 3. B) Nucleotide base pair fragment length patterns <strong>of</strong><br />
various digested Belonolaimus isolates.<br />
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outside <strong>of</strong> Florida are reproductively isolated. Besides, several studies report that B. longicaudatus<br />
populations differ within each other showing various host range and wide morphometric variations (Abu-<br />
Gharbieh and Perry, 1970; Robbins and Hirschmann, 1974; Duncan et al., 1996). All <strong>of</strong> the above<br />
mentioned prove, that indeed B. longicaudatus populations are very complex and require additional<br />
studies <strong>of</strong> reproductive compatibility, behavior and morphology <strong>of</strong> specific genotypes (Gozel et al., 2006)<br />
in order to be able to distinguish it from other species as well as have the complete picture <strong>of</strong> phylogeny <strong>of</strong><br />
this genus.<br />
CONCLUSION<br />
Polymorphism within populations <strong>of</strong> the same species makes it an ideal tool for application in many<br />
aspects such as phylogeny in order to distinguish individuals among populations (in this case<br />
B.longicaudatus). These genetic differences, on one hand serve as convenient diagnostic markers; on the<br />
other hand proves that the genus Belonolaimus is far more complex than currently recognized.<br />
ACKNOWLEDGEMENTS<br />
Many thanks to Pr<strong>of</strong>essor Sergei A. Subbotin for providing us with necessary skills, so we are able to<br />
work with a number <strong>of</strong> phylogeny programs ourselves without any assistance. I believe that this acquired<br />
knowledge would be valuable in our further research activities as well as future career.<br />
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Morris, and D. T. Kaplan. (1999) <strong>Molecular</strong> and morphological analyses <strong>of</strong> isolates <strong>of</strong><br />
Pratylenchus c<strong>of</strong>feae and closely related species. Nematropica 29:61-80.<br />
Ferris, V. R., L. I. Miller, J. Faghihi and J. M. Ferris J.M. (1995) Ribosomal DNA comparisons <strong>of</strong><br />
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Fortuner R. and Luc M. (1987) A reappraisal <strong>of</strong> Tylenchina (Nemata). 6. The family Belonolaimidae<br />
Whitehead, 1960. Revue de Nématologie, 10:183-202.<br />
Gozel U., Adams B.J., Nguyen K.B. Inserra P.N. Giblin-Davis R.M. and Duncan L.W. (2006) A<br />
phylogeny <strong>of</strong> Belonolaimus populations in Florida inferred from DNA sequences.<br />
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Grosser J.D., Chandler J.L. and Duncan L.W. (2007) Production <strong>of</strong> mandarin plus pummelo somatic<br />
hybrid citrus rootstocks with potential for improved tolerance/resistance to sting nematode.<br />
Scientia Horticulturae, 113: 33–36.<br />
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Han H-R., Jeyaprakash A., Weingarther D.P. and Dickson D.W. (2006) Morphological and molecular<br />
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Wilgenbusch, J. C. and D. L. Sw<strong>of</strong>ford. (2003) Inferring Evolutionary Trees with PAUP*. In A. D.<br />
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APPENDIX<br />
Figure 4. Results <strong>of</strong> BLAST search in NCBI GeneBank.<br />
Figure 5. Sequence multiple alignment by ClustalX.<br />
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Figure 6. The notepad with all commands for phylogeny programs (PAUP* and MrBayes).<br />
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Figure 7. NJ, MP and ML trees with the supported rate (BS).<br />
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Figure 8. Phylogenetic tree NJ. Neighbor Joining: number <strong>of</strong> bootstrap replicates = 10000. Bootstrap 50% majority-rule<br />
consensus tree.<br />
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Figure 9. Phylogenetic tree MP. Maximum Parsimony: number <strong>of</strong> bootstrap replicates = 1000. Search = heuristic.<br />
Among 1311 characters: 579 – constant characters; 323 – variable parsimony-uninformative characters; 409 –<br />
parsimony-informative characters. Gaps are treated as “missing”. Bootstrap 50% majority-rule consensus tree.<br />
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Figure 10. Phylogenetic tree ML. Maximum Likelihood: number <strong>of</strong> bootstrap replicates = 100. GTR+G+I model.<br />
Number <strong>of</strong> substitution types = 6. Assumed nucleotide frequencies: A=0.19270 C=0.22540 G=0.28520 T=0.29670.<br />
Bootstrap 50% majority-rule consensus tree.<br />
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Figure 11. Phylogenetic tree MrB. Bayesian inference: 1000000 replicates; Markov Chains.<br />
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