Sergei A. Subbotin

Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences

The evolutionary relationships of 82 species of tylenchid and aphelenchid nematodes were evaluated by use of sequence data of the D2 and D3 expansion fragments of the 28S ribosomal RNA genes. Nine automatic and one culled sequence alignments were analysed using maximum parsimony and Bayesian inference approaches. The molecular data sets showed that the order Tylenchida comprises lineages that largely correspond to two suborders, Hoplolaimina and Criconematina, and other taxonomic divisions as proposed by Siddiqi (2000). Several significant results also derived from our study include: i) the basal position of groups that include entomoparasitic nematodes within tylenchid trees; ii) paraphyly of the superfamily Dolichodoroidea sensu Siddiqi (2000); iii) evidence for a Pratylenchus, Hirschmanniella and Meloidogyne clade; and iv) lack of support for widely held traditional placement of Radopholus within Pratylenchidae and placement of this genus within Hoplolaimidae or Heteroderidae. Congruence and incongruence of molecular phylogeny and traditional classifications and morphological-based hypotheses of phylogeny of tylenchids are discussed.

Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences

RFLP and sequences of ITS-rDNA of 45 populations of cyst-forming nematodes collected from different parts of Iran were analysed and identified as representatives of 21 species. Eight enzymes generated RFLP for all studied populations. Comparison of RFLP profiles and sequences of the ITS regions with published data confirmed the presence of Heterodera avenae, H. filipjevi, H. glycines, H. hordecalis, H. latipons, H. schachtii and H. trifolii in Iran. RFLP patterns and ITS sequences for H. elachista, H. turcomanica, H. mothi and C. cacti were obtained for the first time in this study. Heterodera humuli, H. goettingiana, H. fici, H. elachista, H. turcomanica and Cactodera cacti are recorded for the first time in Iran. These results correspond with morphological and morphometric identification of the populations. Several populations were not identified at the species level and are attributed to Heterodera sp.; some of these may correspond to new species. Twenty-one new sequences from Iranian cyst-forming nematodes and 36 known sequences were used for the phylogenetic analyses. The cyst-forming nematodes formed several clades corresponding to their morphological features. Heterodera mothi and H. elachista clustered with high support with other Cyperi group species and H. turcomanica formed a moderately to highly supported clade with the Humuli group.

Identification of cyst forming nematodes of the genus Heterodera (Nematoda: Heteroderidae) based on the ribosomal DNA-RFLP

Amplified ITS region products of rDNA from 25 valid species and one unidentified species from the genus Heterodera and from Meloidodera alni were digested by 26 restriction enzymes. A combination of seven enzymes clearly separated the agriculturally most important species from each other and from their sibling species. Species specific digestion profiles of ITS regions and a table with approximate sizes of digested fragments for several identification enzymes are given. Heterogeneity of ITS regions was revealed for some cyst forming nematode species. Des fragments amplifies de la region de l’ITS du rDNA de 25 especes valides et d’une espece non identifiee du genre Heterodera et de Meloidodera alni ont ete soumis a une digestion par 26 enzymes de restriction. La combinaison de sept enzymes a permis une separation nette des especes les plus importantes en agriculture, tant les unes par rapport aux autres que par rapport aux especes jumelles. Sont donnes les profils specifiques de digestion des regions de l’ITS et un tableau regroupant les tailles approximatives des fragments digeres pour plusieurs enzymes d’identification. L’heterogeneite des regions de l’ITS a ete revelee chez quelques especes de nematodes a kyste.

A phylogenetic framework for root lesion nematodes of the genus Pratylenchus (Nematoda): Evidence from 18S and D2-D3 expansion segments of 28S ribosomal RNA genes and morphological characters.

The root lesion nematodes of the genus Pratylenchus Filipjev, 1936 are migratory endoparasites of plant roots, considered among the most widespread and important nematode parasites in a variety of crops. We obtained gene sequences from the D2 and D3 expansion segments of 28S rRNA partial and 18S rRNA from 31 populations belonging to 11 valid and two unidentified species of root lesion nematodes and five outgroup taxa. These datasets were analyzed using maximum parsimony and Bayesian inference. The alignments were generated using the secondary structure models for these molecules and analyzed with Bayesian inference under the standard models and the complex model, considering helices under the doublet model and loops and bulges under the general time reversible model. The phylogenetic informativeness of morphological characters is tested by reconstruction of their histories on rRNA based trees using parallel parsimony and Bayesian approaches. Phylogenetic and sequence analyses of the 28S D2-D3 dataset with 145 accessions for 28 species and 18S dataset with 68 accessions for 15 species confirmed among large numbers of geographical diverse isolates that most classical morphospecies are monophyletic. Phylogenetic analyses revealed at least six distinct major clades of examined Pratylenchus species and these clades are generally congruent with those defined by characters derived from lip patterns, numbers of lip annules, and spermatheca shape. Morphological results suggest the need for sophisticated character discovery and analysis for morphology based phylogenetics in nematodes.

A molecular phylogenetic approach to Longidoridae (Nematoda: Dorylaimida)

The Longidoridae are a group of ectoparasitic nematodes including two subfamilies and six genera with hundreds of species. Sequences of the D2 and D3 expansion region of the large subunit (LSU) rRNA nuclear gene were amplified and used to reconstruct the phylogeny of longidorids. Phylogenetic analyses with maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) were performed with one outgroup taxon and 62 longidorid sequences. Confidence of inferred clades was assessed by non-parametric bootstrapping for MP and Bayesian posterior probability for ML. All analyses placed Paralongidorus species as an inner group within the otherwise monophyletic genus Longidorus. The genus Xiphinema, except for X. americanum-group species, was placed as the sister group of Longidorus with strong support from the ML and BI analyses. The X. americanum-group was strongly supported as an exclusive clade to other genus Xiphinema species. The position of the Xiphidorus clade was not well resolved and the phylogenetic analyses did not support it as a sister group to Longidorus as previously inferred from morphology. Secondary structure models were constructed for the D2/D3 region of LSU rRNA for all studied species. It was found that sequence-based and structural morphometric rRNA phylogenies were incongruent.

Identification of Heterodera avenae group species by morphometrics and rDNA-RFLPs

Summary ‐Canonical discriminant analysis of four morphometric charactersof juvenilesand restriction enzymes analysis of ribosomal DNA sequences were used to distinguish Heterodera arenaria , H. aucklandica , H. avenae, H. elipjevi, H. hordecalis , H. iri, H. latipons, H. litoralis , H. schachtii and an undescribed species from grasslands. The results of unweighted pair group cluster analysis showed that H. avenae populations formed three groups and H. elipjevi two groups at the 80% level of similarity. Intraspecie c polymorphism was revealed by rDNA-RFLP studies and two types of ITS regions within H. avenae populations can be distinguished. The pattern of restriction bands obtained with BsuRI, PstI and TaqI clearly distinguished populations of H. elipjevifrom other species of the H. avenae group. Further enzymes and their combinations distinguished the other species. There are no enzymes which differentiate European populations of H. avenae from H. arenaria. Morphometrics, restriction endonuclease cleavage maps of ITS regions and a dendrogram of putative phylogenetic relations of several cyst-forming nematode species are given. Resume ‐ Identie cation des espe ces du groupe Heterodera avenae par la morphometrie et les rDNA-RFLP ‐ L’ analyse canonique discriminante sur quatre caracte res morphome triques des juve niles et l’ analyse des enzymes de restriction de l’ ADN ribosomal ont e te utilise es pour identie er Heterodera arenaria , H. aucklandica , H. avenae, H. elipjevi, H. hordecalis , H. iri, H. latipons, H. litoralis , H. schachtii et une nouvelle espe ce originaire de prairies.Lesre sultats de l’ analyse UPGMAont montre que les populations d’H. avenae formaient trois groupes et celles d’H. elipjevi deux groupes a un niveau de similarite de 80%. Le polymorphisme intraspe cie que a e te re ve le par des e tudes de rDNA-RFLP et deux types de re gions de l’ ITS peuvent e tre mis en e vidence dans les populations d’H. avenae. Les mode les de bandes de restriction obtenus avec BsuRI, PstI et TaqI ont identie e clairement les populations d’H. elipjevi des autres espe ces du groupe H. avenae. D’ autres enzymes et leurs combinaisons ont identie e les autres espe ces. Aucun enzyme n’ a diffe rencie les populations europe ennes d’H. avenae de H. arenaria. Les caracte res morphome triques, les cartes de clivage des re gions des ITS par l’ endonucle ase de restriction et un dendogramme des relations phyloge ne tiques suppose es sont donne s.

Molecular and morphological characterisation of the Heterodera avenae species complex (Tylenchida: Heteroderidae)

Species of the Heterodera avenae complex, including populations of H. arenaria, H. aucklandica, H. australis, H. avenae, H. filipjevi, H. mani, H. pratensis and H. ustinovi, obtained from different regions of the world were analysed with PCR-RFLP and sequencing of the ITS-rDNA, RAPD and light microscopy. Phylogenetic relationships between species and populations of the H. avenae complex as inferred from analyses of 70 sequences of the ITS region and of 237 RAPD markers revealed that the cereal cyst nematode H. avenae is a paraphyletic taxon. The taxonomic status of the Australian cereal cyst nematode H. australis based on sequences of the ITS-rDNA and RAPD data is confirmed. Morphometrical and ITS-rDNA sequence analyses revealed that the Chinese cereal cyst nematode is different from other H. avenae populations infecting cereals and is related to H. pratensis. Bidera riparia Kazachenko, 1993 is transferred to the genus Heterodera as H. riparia (Kazachenko, 1993) comb. n. As a consequence, H. riparia Subbotin, Sturhan, Waeyenberge & Moens, 1997 becomes a junior secondary homonym and is renamed as H. ripae nom. nov. Morphological, morphometrical characters and RFLP profiles for identification of the nine species presently placed in the H. avenae species complex are given.

Evolution of the gall-forming plant parasitic nematodes (Tylenchida: Anguinidae) and their relationships with hosts as inferred from Internal Transcribed Spacer sequences of nuclear ribosomal DNA.

Phylogenetic relationships among gall-forming plant parasitic nematodes of the subfamily Anguininae are reconstructed by maximum parsimony and maximum likelihood analyses. Sequences of the ITS of rDNA from 53 populations and species of gall-forming nematodes and five populations of the Ditylenchus dipsaci species complex were analysed. The phylogenetic trees strongly support monophyly of the genus Anguina and show nonmonophyly for the genera Mesoanguina and Heteroanguina. Morphological and biological characters are generally congruent with the anguinid groups identified in the rDNA phylogeny. Analyses of evolution of different gall types among anguinids reveal that there are apparent evolutionary trends in gall evolution: from abnormal swelling and growth of infested plant organs toward small localised galls, and from infestation of vegetative toward generative organs. Our study demonstrates that the main anguinid groups are generally associated with host plants belonging to the same or related systematic groups. The comparison of the ITS phylogenies of anguinids parasitising Poaceae and their host grasses shows a high level of cospeciation events.

A rapid method for the identification of the soybean cyst nematode Heterodera glycines using duplex PCR

A method for rapid identification of juveniles and cysts of the soybean cyst nematode based on PCR with species specific primers is described. The PCR assay was tested on 53 populations originating from China, Russia, USA and Brazil. A single cyst or second stage juvenile of Heterodera glycines alone or in a mixture with other soil inhabiting nematodes was detectable.

Heterorhabditis baujardi sp. n. (Rhabditida: Heterorhabditidae) from Vietnam and morphometric data for H. indica populations

Summary ‐ A survey of entomopathogenic nematodes in Vietnam yielded several Heterorhabditis isolates. The majority belonged to H. indica; their morphometrics are given in this paper. Three isolates collected in forests in Backan, Ninhbinh and Kontum provinces, Vietnam are here described as a new species Heterorhabditis baujardi sp. n. The new species is distinguished from the other ten valid Heterorhabditis species by a combination of morphological, morphometrical, and DNA characters. Heterorhabditis baujardi sp. n. is morphometrically similar to H. indica, but can be separated from this species by the shape of the gubernaculum and the number of normal pairs of genital papillae. The gubernaculum of H. baujardi sp. n. with the proximal end ventrally curved resembles that of H. bacteriophora. Heterorhabditis baujardi sp. n. can be separated from this latter species by a shorter body length of infective juveniles and longer spicules, longer gubernaculum, and a higher spicule length to anal body diameter ratio of males. The canonical discriminant analysis of morphometrical characters of both infective juveniles and males failed to discriminate Heterorhabditis baujardi sp. n. from H. indica. However, the new species was easily distinguished from H. downesi, H. marelatus , H. megidis and H. bacteriophora . Heterorhabditis baujardi sp. n. was slightly separated from H. bacteriophora by variables of the infective juveniles, but was clearly distinguished by variables of the males. Cross-breeding tests using isolates of the new species and H.indicadid not yield fertileprogeny. Analysis of the ITS1 sequence of rDNA of H. baujardi sp. n. revealed substantial differences with other known ITS1 Heterorhabditis sequences. Phylogenetic relationships between Heterorhabditis species and the usefulness of morphological and molecular characters for identie cation of species from this group are discussed.