Madina Rasulova Molecular Systematics of Page 1

INTRODUCTION

Nematodes belonging to the of – 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, ), turfgrasses (e.g., bermudagrass, St.

Augustinegrass, zoysiagrass) and forest crops ( trees). Currently nine 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 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

Madina Rasulova Molecular Systematics of Nematodes Page 2 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 Chitwood, 1933

Suborder Tylenchina Thorne, 1949

Infraorder Tylenchomorpha De Ley et Blaxter, 2002

Superfamily Orley, 1880

Family 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 B.longicaudatus. B.longicaudatus. B.longicaudatus.

Madina Rasulova Molecular Systematics of Nematodes Page 3

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

Madina Rasulova Molecular Systematics of Nematodes Page 4 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

Madina Rasulova Molecular Systematics of Nematodes Page 5

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,

Madina Rasulova Molecular Systematics of Nematodes Page 6

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

Madina Rasulova Molecular Systematics of Nematodes Page 7 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.

Total Variation № Accession Species E value Max identity Length (bp) (bp)

1 DQ494797 Belonolaimus longicaudatus 0.0 100 685 685

Madina Rasulova Molecular Systematics of Nematodes Page 8

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 10 DQ494803 6e-105 94 590 590 Carolina”

11 GQ469496 Ditylenchus dipsaci 6e-61 91 967 946

12 DQ309584 Hoplolaimus columbus 1e-62 89 1269 1248

13 EF030983 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

Madina Rasulova Molecular Systematics of Nematodes Page 9 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

Madina Rasulova Molecular Systematics of Nematodes Page 10 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

Madina Rasulova Molecular Systematics of Nematodes Page 11

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 out- group 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 bermudagrass (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.

Madina Rasulova Molecular Systematics of Nematodes Page 13

Restriction patterns of the Belonolaimus ITS1 region done by Cherry et al. (1997) illustrate that this region differs from one individual 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 Figure 3. B) Nucleotide base pair fragment length patterns of representative Belonolaimus longicaudatus from various digested Belonolaimus isolates. different isolates. KS = Kansas, AR = Arkansas, CA = California, SC = South Carolina, FL = Florida.

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 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.

REFERENCES

Abu-Gharbieh, W. L., and V. G. Perry. (1970) Host differences among Florida population of

Belonolaimus longicaudatus. Journal of Nematology 2:209-216.

Madina Rasulova Molecular Systematics of Nematodes Page 15

Adams, B. J. (1998) Species concepts and the evolutionary paradigm in modern nematology. Journal of

Nematology 30:1-21.

Campbell, A.J.D., R. B. Gasser and N. B. Chilton. (1995) Differences in a ribosomal DNA sequence of

Strengylus species allow identification of single eggs. International Journal for Parasitology,

25:359-365.

Chilton, N. B., R. B. Gasser and I. Beveridge. (1995) Differences in a ribosomal DNA sequence of

morphologically indistinguishable species within the Hypodontus macropi complex

(Nematoda, Strongyloidea). International Journal for Parasitology, 25:647-651.

Cherry T., Szalanski A.L., Todd T.C. and Powers T.O. (1997) The internal transcribed spacer region of

Belonolaimus (Nemata: Belonolaimidae). Journal of Nematology, 29: 23–29.

Duncan, L. W., R. N. Inserra, W. K. Thomas, D. Dunn, I. Mustika, L. M. Frisse, M. L. Mendes, K.

Morris, and D. T. Kaplan. (1999) Molecular and morphological analyses of isolates of

Pratylenchus coffeae and closely related species. Nematropica 29:61-80.

Ferris, V. R., L. I. Miller, J. Faghihi and J. M. Ferris J.M. (1995) Ribosomal DNA comparisons of

Globodera from two continents. Journal of Nematology, 27:273-283.

Fortuner R. and Luc M. (1987) A reappraisal of Tylenchina (Nemata). 6. The family Belonolaimidae

Whitehead, 1960. Revue de Nématologie, 10:183-202.

Gozel U., Adams B.J., Nguyen K.B. Inserra P.N. Giblin-Davis R.M. and Duncan L.W. (2006) A

phylogeny of Belonolaimus populations in Florida inferred from DNA sequences.

Nematropica, 36: 155–171.

Grosser J.D., Chandler J.L. and Duncan L.W. (2007) Production of mandarin plus pummelo somatic

hybrid citrus rootstocks with potential for improved tolerance/resistance to sting nematode.

Scientia Horticulturae, 113: 33–36.

Madina Rasulova Molecular Systematics of Nematodes Page 16

Han H-R., Jeyaprakash A., Weingarther D.P. and Dickson D.W. (2006) Morphological and molecular

biological characterization of Belonolaimus longicaudatus. Nematropica, 36: 37–52.

Koenning S.R., Overstreet C., Noling J.W., Donald P.A., Becker J.O. and Fortnum B. (1999) A survey of

crop losses in response to phytoparasitic nematodes in the United States for 1994. Journal of

Nematology, 31: 587–618.

Koenning S.R., Bowman D.T., Morris R.H. (2006) Quantifying potential tolerance of selected cotton

cultivars to Belonolaimus longicaudatus. Journal of Nematology, 38: 187–191.

Monteiro A.R. and Lordello L.G.E. (1977) Dois novos nematoides encontrados associados á cana de

acuar. Revista de Agricultura. Piracicaba, Brazil 52, 5–11.

Rau G. J. (1961) Amended descriptions of Belonolaimus gracilis Steiner, 1949, and B. longicaudatus Rau,

1958 (Nematoda: ). Proceedings of the Helminthological Society of Washington

28:198-200.

Rau G.J. (1963) Three new species of Belonolaimus (Nematoda: Tylenchida) with additional data on B.

longicaudatus and B. gracilis. Proceedings of the Helminthological Society of Washington, 30:

119–128.

Robbins, R. T., and H. Hirschmann. (1974) Variation among populations of Belonolaimus longicaudatus.

Journal of Nematology 6:87-94.

Siddiqi M.R. (2000) Tylenchida parasites of plants and insects. Wallingford, UK, CABI Publishing, 833.

Subbotin S.A. and Moens M. (2006) Molecular and Phylogeny. In Book: Plant Nematology

edited by Perry R.N. & Moens M. Plant Nematology. UK: CABI, P. 33–58.

Thompson, J.D., Gibson, T.J., Plewiniak, F. and Higgins, D.G. (1997) The ClustalX windows interface:

flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic

Acids Research, 24: 4876–4882.

Madina Rasulova Molecular Systematics of Nematodes Page 17

Wilgenbusch, J. C. and D. L. Swofford. (2003) Inferring Evolutionary Trees with PAUP*. In A. D.

Baxevanis, D. B. Davison, R. D. M. Page, G. A. Petsko, L. D. Stein, and G. D. Stormo (eds.)

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

85:1231-1237.

Madina Rasulova Molecular Systematics of Nematodes Page 18

APPENDIX

Figure 4. Results of BLAST search in NCBI GeneBank.

Figure 5. Sequence multiple alignment by ClustalX.

Madina Rasulova Molecular Systematics of Nematodes Page 19

Figure 6. The notepad with all commands for phylogeny programs (PAUP* and MrBayes).

Madina Rasulova Molecular Systematics of Nematodes Page 20

Figure 7. NJ, MP and ML trees with the supported rate (BS).

Madina Rasulova Molecular Systematics of Nematodes Page 21

Figure 8. Phylogenetic tree NJ. Neighbor Joining: number of bootstrap replicates = 10000. Bootstrap 50% majority-rule

consensus tree.

Madina Rasulova Molecular Systematics of Nematodes Page 22

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.

Madina Rasulova Molecular Systematics of Nematodes Page 23

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.

Madina Rasulova Molecular Systematics of Nematodes Page 24

Figure 11. Phylogenetic tree MrB. Bayesian inference: 1000000 replicates; Markov Chains.

Madina Rasulova Molecular Systematics of Nematodes Page 25