Nematology, 2011, Vol. 13(3), 333-345

Diversity and phylogenetic relationships within the spiralnematodes of Helicotylenchus Steiner, 1945 (Tylenchida:

Hoplolaimidae) as inferred from analysis of the D2-D3expansion segments of 28S rRNA gene sequences

Sergei A. SUBBOTIN 1,2,!, Renato N. INSERRA 3, Mariette MARAIS 4, Peter MULLIN 5,Thomas O. POWERS 5, Philip A. ROBERTS 6, Esther VAN DEN BERG 4,

Gregor W. YEATES 7 and James G. BALDWIN 6

1 Plant Pest Diagnostic Center, California Department of Food and Agriculture, 3294 Meadowview Road,Sacramento, CA 95832-1448, USA

2 Centre of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences,Leninskii Prospect 33, Moscow, 117071, Russia

3 Florida Department of Agriculture and Consumer Services, DPI, Nematology Section, P.O. Box 147100,Gainesville, FL 32614-7100, USA

4 National Collection of Nematodes, Biosystematics Programme, ARC-Plant Protection Research Institute,Private Bag X134, Queenswood, 0121 South Africa

5 Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583, USA6 Department of Nematology, University of California, Riverside, CA 92521, USA

7 P.O. Box 1758, Palmerston North 4440, New Zealand

Received: 2 May 2010; revised: 1 July 2010Accepted for publication: 1 July 2010

Summary – The spiral nematodes of the genus Helicotylenchus are globally distributed and associated with the root system of diversegroups of plants in cultivated and uncultivated areas. Several species are considered serious parasites of crops. The identification ofmany Helicotylenchus species is not always reliable, in part because many species share very similar diagnostic characters and highintraspecific variation. To verify species identification of geographically distant populations of Helicotylenchus, we tested monophylyof some classical morphospecies and studied their phylogenetic relationships; specifically, we conducted sequence and phylogeneticanalysis of 89 sequences of the D2-D3 expansion segments of 28S rRNA gene sequences from 54 Helicotylenchus isolates, includingspecies identified as H. brevis, H. digonicus, H. dihystera, H. labiodiscinus, H. leiocephalus, H. martini, H. multicinctus, H. platyurus,H. pseudorobustus and H. vulgaris, together with three outgroup taxa. Phylogenetic analysis distinguished nine highly or moderatelysupported major clades within Helicotylenchus. Using the molecular approach we were able to confirm congruence with morphological-based identification of samples of H. dihystera and H. multicinctus. However, sequence and phylogenetic analysis using Bayesianinference and maximum parsimony analysis showed that isolates collected in different countries and morphologically identified asH. pseudorobustus, H. digonicus or H. vulgaris were each representative of several different and, sometimes, unrelated lineages.Further detailed comparative morphometrics and morphological studies will help to elucidate if there is some misidentification orif putative species actually comprise a complex of cryptic species. Molecular analysis also revealed that 14 samples were classifiedas representatives of 11 unidentified species. Molecular characterisation of known Helicotylenchus species especially, using samplescollected from type localities, is needed for future reliable identification of species of this genus.

Keywords – Bayesian inference, Helicotylenchus digonicus, Helicotylenchus dihystera, Helicotylenchus multicinctus, Helicotylenchuspseudorobustus, Helicotylenchus vulgaris, maximum parsimony, species delimiting.

! Corresponding author, e-mail: subbotin@ucr.edu

© Koninklijke Brill NV, Leiden, 2011 DOI:10.1163/138855410X520936Also available online - www.brill.nl/nemy 333

S.A. Subbotin et al.

Helicotylenchus Steiner, 1945 is a cosmopolitan genuswith more than 200 species which are commonly calledspiral nematodes because of their coiled habitus mortis(Marais, 2001). These migratory ectoparasitic or semi-endoparasitic nematodes may occur in very high numbersfeeding upon roots of diverse plants and may be abun-dant in soil surrounding host roots (Taylor, 1961; Nor-ton, 1977; Krall, 1978). Species of Helicotylenchus areglobally distributed, spanning many climates, and are as-sociated with the root system of diverse crops of agricul-tural importance. Although data are not available to im-plicate most Helicotylenchus as serious parasites, plantgrowth suppression has been consistently associated withat least five cosmopolitan species: H. digonicus Perryin Perry, Darling & Thorne, 1959, H. dihystera (Cobb,1896) Sher, 1961, H. indicus Siddiqi, 1963, H. multi-cinctus (Cobb, 1893) Golden, 1956 and H. pseudorobus-tus (Steiner, 1914) Golden, 1956. Other species, such asH. cavenessi Sher, 1966, H. erythrinae (Zimmermann,1904) Golden, 1956 and H. microcephalus Sher, 1966,have also been implicated as potentially damaging pests(O’Bannon & Inserra, 1989). The banana spiral nema-tode, H. multicinctus, is endoparasitic and polyphagous,but it is best known for suppressing growth and yield ofbanana in many regions of the world (Krall, 1978; McSor-ley & Parrado, 1986; De Waele & Elsen, 2007). Anotherless known endoparasite is H. variocaudatus Yuen, 1964,which parasitises banana roots in the islands of São Tomeand Príncipe (Vovlas et al., 1995) and also in Rwanda(Van den Berg et al., 2003).

Available dichotomous or polytomous identificationkeys to spiral nematodes (Sher, 1966; Siddiqi, 1972; Boag& Jairajpuri, 1985; Firoza & Maqbool, 1994) are espe-cially helpful in the identification of species that havepeculiar morphological characters. Such species includethose with a posterior gonad less developed than the ante-rior one as in the case of H. multicinctus, which is distin-guished also by a short C-shaped body, a slightly taperingtail, a hemispherical and annulated tail terminus, and nu-merous males (Vovlas et al., 1995). However, the identi-fication of other species is not always reliable, partly be-cause many species share very similar diagnostic charac-ters and species boundaries are not well established. Somefeatures have broad overlapping ranges and intraspecificvariability with characters apparently influenced by en-vironmental conditions, including the host plant (For-tuner, 1979, 1984; Fortuner & Quénéhervé, 1980; For-tuner et al., 1981). Although multivariate analyses canbe useful in reducing the effect of intraspecific variabil-

ity of morphological characters (Fortuner & Maggenti,1991), identification of these nematodes by morphologyalone often remains unresolved or uncertain due to limita-tions of the morphological analysis. Application of non-morphological characters such as DNA sequences canhelp to confirm classical morphology-based identifica-tions and resolve some of the problems experienced in theidentification of Helicotylenchus species.

Application of rRNA gene sequences provides an at-tractive solution for quick and reliable nematode diagnos-tics. Recently, several studies using the ITS-rDNA (Chenet al., 2006), D2-D3 of 28S rRNA (Subbotin et al., 2006,2007; Bae et al., 2009), and 18S rDNA (Holterman et al.,2009) demonstrated the usefulness of this approach foridentification of species of Helicotylenchus. Analysis ofrRNA gene sequences (Subbotin et al., 2007; Bae et al.,2009; Holterman et al., 2009) also provides a basis for re-constructing phylogenetic relationships within this genus.However, such a phylogeny has not been proposed previ-ously based on morphological or molecular datasets.

The major objectives of the present study were to:i) to verify species identification of geographically dis-tant populations of Helicotylenchus by analysing theirfragments of rRNA gene sequences; ii) test monophylyof classical morphospecies and estimate species bound-aries using rRNA gene sequences from large numbersof geographically diverse isolates; and iii) study phy-logenetic relationships within Helicotylenchus using se-quences from the D2-D3 expansion segments of the 28SrRNA gene as inferred from Bayesian inference and max-imum parsimony approaches.

Materials and methods

NEMATODE POPULATIONS, SPECIES IDENTIFICATION

AND DELIMITING

Nematode populations used in this study were obtainedfrom soil samples collected from geographically diverselocations (Table 1). The nematodes were extracted fromsamples using the Baermann funnel, centrifugal flotationor elutriation techniques (Hooper, 1986). Specimens werekilled by gentle heat, fixed by 4% formalin, TAF orFPG and mounted in anhydrous glycerin or immobilisedby gently heating and then mounting in water agarfor examination (Netscher & Seinhorst, 1969; Esser,1986). All morphological identifications of specimens,except for the South African ones, were done by usingidentification keys and descriptions provided by Siddiqi

334 Nematology

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Vol. 13(3), 2011 335

S.A. Subbotin et al.Ta

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336 Nematology

Diversity and phylogeny of HelicotylenchusTa

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Vol. 13(3), 2011 337

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(1972), Krall (1978), Anderson and Eveleigh (1982),Boag and Jairajpuri (1985) and Firoza and Maqbool(1994). The South African materials were identified usingthe relevant species descriptions without the use of any ofpublished diagnostic keys.

For some populations, species were delimited anddefined based on an integrated approach that consid-ered morphological evaluation combined with molecular-based phylogenetic inference (tree based methods) andsequence analyses (genetic distance methods) (Sites &Marshall, 2004).

DNA EXTRACTION, PCR, CLONING AND

SEQUENCING

Nematode DNA was extracted from several individualsusing proteinase K. Detailed protocols for DNA extractionand PCR were as described by Tanha Maafi et al. (2003).The forward D2A (5"-ACAAGTACCGTGAGGGAAAGTTG-3") and reverse D3B (5"-TCGGAAGGAACCAGCTACTA-3") primers were used for amplification and se-quencing of the fragment of D2-D3 regions of the 28SrRNA gene (Subbotin et al., 2006). PCR products werepurified using QIAquick (Qiagen, Valencia, CA, USA)gel extraction kits and then cloned using pGEM-T Vec-tor System II kit (Promega, Madison, WI, USA). One ortwo clones were sequenced from each sample. The result-ing products were purified and run on a DNA sequencer atthe University of California, Riverside, Genomics Center.The newly obtained sequences have been submitted to theGenBank database under accession numbers indicated inTable 1.

SEQUENCE AND PHYLOGENETIC ANALYSES

The newly obtained sequences were aligned usingClustalX (Thompson et al., 1997) with default parametersand with sequences published for Helicotylenchus inGenBank (De Ley et al., 2005; Subbotin et al., 2007;Bae et al., 2009) and with Rotylenchus magnus Zancada,1985, Hoplolaimus galeatus (Cobb, 1913) Thorne, 1935and H. seinhorsti Luc, 1958 used as outgroup taxa(Subbotin et al., 2007; Bae et al., 2008; Vovlas etal., 2008). Sequence and phylogenetic analysis of thedataset was performed with Bayesian inference (BI) usingMrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001) andmaximum parsimony (MP) using PAUP* 4b10 (Swofford,2003). BI analysis under the GTR + I + G modelwas initiated with a random starting tree and was runwith four chains for 1.0 # 106 generations. The Markov

chains were sampled at intervals of 100 generations.Two runs were performed for each analysis. The log-likelihood values of the sample points stabilised afterapproximately 103 generations. The topologies were usedto generate a 50% majority rule consensus tree. Posteriorprobabilities (PP) are given on appropriate clades. ForMP we used a heuristic search setting with ten replicatesof random taxon addition (max. tree number = 1000),tree bisection-reconnection branch swapping to seek themost parsimonious trees. Gaps were treated as missingdata. To obtain an estimate of support for each node,a bootstrap analysis (BS) with 100 replicates (max treenumber = 100) was done. Sequence differences betweensamples were calculated with PAUP* 4b10 as an absolutedistance matrix and the percentage was adjusted formissing data.

Results

SPECIES IDENTIFICATION AND DELIMITING

Eighty-six sequences from 54 Helicotylenchus isolateswere included in the analysis. Sixty-eight sequences werenewly obtained in the present study. Using traditionalmorphological taxonomic characters and molecular crite-ria (apomorphies and DNA distances), we distinguishedthe following species within the samples: Helicotylen-chus brevis (Whitehead, 1958) Fortuner, 1960, H. digo-nicus, H. dihystera, H. labiodiscinus Sher, 1966, H. leio-cephalus Sher, 1966, H. martini Sher, 1966, H. multicinc-tus, H. platyurus Perry in Perry, Darling & Thorne, 1959,H. pseudorobustus and H. vulgaris Yuen, 1964. Severalsamples, which were identified morphologically as repre-sentative of the same species, showed differences in mole-cular characteristics, and were thus classified as differ-ent species types: H. pseudorobustus type ‘A’, ‘B’, ‘C’and ‘D’, H. vulgaris type ‘A’ and ‘B’ and H. digoni-cus type ‘A’ and ‘B’. Fourteen samples were classified asrepresentatives of 11 unidentified species. More detailedmorphological and molecular analysis is required to fur-ther evaluate and identify these samples. Sequence andphylogenetic analysis confirmed that each analysed sam-ple used in the present study contained representatives ofa single species only. One exception, collected in KawaiiIsland, included a mixture of specimens with H. dihysteraand Helicotylenchus spIV.

338 Nematology

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SEQUENCE ANALYSIS

Amplification of D2-D3 of the 28S rRNA gene usingPCR produced a single fragment of ca 680 bp for the sam-ples studied. The sequence alignment for Helicotylenchusand outgroup taxa included 89 sequences and was 596 bpin length. Sequence diversity within all studied taxa in-cluding outgroup taxa reached 20.7% (118 nucleotides(nt)) and for Helicotylenchus it reached 19.9% (115 nt).Minimal interspecific sequence variation was observedfor taxa belonging to clades I, III, V and IX (Figs 1, 2).Intraspecific sequence diversity varied for H. pseudoro-bustus type A from 0.2-0.5% (1-3 nt), H. pseudorobus-tus type B from 0-0.5% (0-3 nt), H. labiodiscinus from0.5-1.5% (3-9 nt), H. multicinctus from 0.5-1.0% (3-6 nt),H. vulgaris type A from 0.3-0.9% (2-5 nt), and H. dihys-tera from 0-2.3% (0-13 nt). Heterogeneity was observedfor many taxa among sequenced clones originated fromthe same PCR product. The largest difference was foundbetween two sequenced clones for a H. martini sample,which reached 6% (35 nt).

PHYLOGENETIC ANALYSIS

Phylogenetic relationships within Helicotylenchus asinferred from Bayesian inference and maximum parsi-mony are given in Figures 1 and 2, respectively. Topolo-gies of BI and MP trees were congruent, except for po-sitions of some weakly supported clades. Nine highlyor moderately supported major clades were distinguishedwithin Helicotylenchus. Clade I (PP = 100%, BS = 74%)and included 11 putative taxa as follows: H. pseudoro-bustus type A, B, C and D, H. leiocephalus, H. digoni-cus type B, H. platyurus, Helicotylenchus spI-5, spI-8,spI-9 and spI-10. Clade II (PP = 100%, BS = 97%)consisted of 22 sequences obtained from 14 samplesidentified here as H. dihystera. Clade III (PP = 72%,BS < 50%) included five sequences of H. multicinc-tus and sequences from two unidentified Helicotylen-chus taxa (Helicotylenchus spIII-1 and spIII-2). Clade IV(PP = 100%, BS = 100%) included only one unidentifiedHelicotylenchus sample (Helicotylenchus spIV). Clade V(PP = 100%, BS = 100%) contained four sequences fromsamples identified as H. vulgaris type B and H. digonicustype C. The highly supported clades VI and VIII each in-cluded only a single taxon, H. labiodiscinus and H. mar-tini, respectively. Clade VII (PP = 100%; BS = 97%)consisted of H. brevis and an unidentified Helicotylenchussample (Helicotylenchus spVII). Clade IX (PP = 100%;BS = 96%) included H. vulgaris type A, H. digonicus

type A, and three unidentified Helicotylenchus samples(Helicotylenchus spIX-1, spIX-3, spIX-4).

Discussion

INTEGRATED APPROACH FOR HELICOTYLENCHUSSYSTEMATICS

Identification of Helicotylenchus species is often not aneasy task because of high intra- and interspecific variabil-ity and a large number of poorly described species (For-tuner, 1979, 1984; Fortuner & Quénéhervé, 1980). Vari-ous authors have published dichotomous keys for Helico-tylenchus, but none of these keys is reliable for speciesdiagnostics (Fortuner & Wong, 1984). To try to overcomethe inherent flaws of dichotomous keys a number of com-pendia have been published. However, compendia, likekeys, rapidly become outdated (Boag & Jairajpuri, 1985;Firoza & Maqbool, 1994; Vovlas et al., 1995). The useof such diagnostic keys and compendia can consequentlylead to unresolved or uncertain identification of Helicoty-lenchus species.

Phylogenetic and DNA sequence analyses of nema-tode samples provide additional criteria for identifyingand delimiting species within Helicotylenchus. Our find-ings show that there was congruence between the resultsof the molecular and morphological analyses of H. di-hystera and H. multicinctus. However, the morphologicalidentification of a large number of the spiral nematodesstudied seems not to be reliable. For example, the prelim-inary results comparing morphological identification fromdifferent nematology laboratories failed to delimit speciesboundaries and conflicted with results based on a molecu-lar approach. Common species collected and morpholog-ically identified from different countries as H. pseudoro-bustus, H. vulgaris or H. digonicus were assessed as be-ing different and often not closely related taxa when theywere subjected to molecular analysis. In this study we pro-visionally distinguished such samples by a letter code: H.pseudorobustus type A, B, C and D, H. vulgaris type Aand B and H. digonicus type A and B. Comparative de-tailed morphometrics and morphological studies can helpto elucidate if there is some misidentification or if each ofthese putative species is actually comprised of a complexof cryptic species. Identification of these samples will bepossible after careful molecular and morphological char-acterisation of type representatives of these species, in-cluding new material collected from the type localities.Similarly, several samples each were identified as repre-sentatives of H. leiocephalus, H. platyurus, H. labiodisci-

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nus, H. brevis and H. martini, although none of these werefrom the type locality, thereby underscoring the need forfurther work to confirm these identifications.

In several cases, molecular approaches failed to delimitboundaries of recognised species. For example: i) twosequence clones from the D2-D3 rRNA PCR productobtained from a single sample and identified as H.platyurus did not cluster together; and ii) two sequences ofH. martini showed a high level of nucleotide differencesbeyond the level of intraspecific variation common forHelicotylenchus species.

These observations, coupled with the indistinct natureof species boundaries, emphasise the importance of usingan integrated approach to delimiting species and cautionagainst reliance on any single dataset or method for thispurpose. Particularly for groups such as Helicotylenchus,these considerations also challenge defining species con-cepts and how to operationally address such concepts.

PHYLOGENY AND TAXONOMY OF HELICOTYLENCHUS

Fortuner (1991) suggested that Helicotylenchus mostlikely originated from ancestral forms close to Pararoty-lenchus and he also noted that it was not known whetherHelicotylenchus and the other Hoplolaiminae are mono-phyletic. In phylogenetic analyses using 18S rRNA genesequences (Holterman et al., 2009; van Megen et al.,2009), Helicotylenchus was supported as monophyleticand its representatives formed a single clade but withbootstrap support varying from strong to weak. In theD2-D3 regions of 28S trees reconstructed under the GTRmodel of DNA evolution, the genus Helicotylenchus wasparaphyletic (Subbotin et al., 2006; Vovlas et al., 2008;Bae et al., 2009) and composed of two distinct lineages.However, application of the secondary structure model forthe same dataset (Subbotin et al., 2006) led to a tree withlower resolution of relationships among the main cladesand suggested that the paraphyly was the result of an arte-fact of the conventional models used. Based on these re-sults, we conclude that the presently available moleculardata do not provide convincing evidence in support of aparaphyletic origin of this genus.

Whitehead (1958) proposed the genus RotylenchoidesWhitehead, 1958 with R. brevis Whitehead, 1958 as thetype species. Rotylenchoides only differed from Helico-

tylenchus in a single characteristic that is in the regres-sion of the posterior genital branch. Rotylenchoides wasmade a junior synonym of Helicotylenchus by Fortuner(1984). Fortuner (1984) did not consider this characteras sufficient justification for establishing a genus, becauseof observation of a transformation series of regression ofthis organ documented throughout additional species ofthe genus Helicotylenchus or presence of the so called in-termediate forms. Siddiqi (1986, 2000) rejected the syn-onymy but Fortuner’s opinion was widely supported andthe synonymy of Rotylenchoides accepted (Ebsary, 1991;Vovlas et al., 1995; Marais, 1998, 2001; Van den Berg etal., 2003). The results of our phylogenetic analysis showthat H. brevis clusters within Helicotylenchus and thussupports the synonymisation of Rotylenchoides with Heli-cotylenchus.

SPECIES COMPLEXES WITHIN HELICOTYLENCHUS

Both H. dihystera and H. pseudorobustus have a world-wide distribution and have been reported from many dif-ferent host plants. Helicotylenchus dihystera is the typespecies of the genus, whereas H. pseudorobustus is con-sidered, after H. dihystera and H. multicinctus, to be themost frequently reported species of Helicotylenchus in theworld literature (Fortuner et al., 1984). In our tree, H. di-hystera was represented by 14 populations which werecollected from different plants in subtropical and tropicalregions and formed clade II. Fortuner et al. (1981) madeH. rotundicauda Sher, 1966 a junior synonym of H. dihys-tera on the grounds that the species shares the same rangeof variation as H. dihystera. Furthermore, as originally de-fined, the two species only differ in tail shape and shape ofthe stylet knobs and he did not consider those sufficient toaccept them as distinct species. The synonymy was ac-cepted by some taxonomists (Boag & Jairajpuri, 1985;Ebsary, 1991; Marais, 2001) but rejected by Siddiqi (Sid-diqi, 1986, 2000). The results of our phylogenetic analy-sis show that one of the samples identified by morpho-logical characters as H. rotundicauda clusters within H.dihystera and thus supports the synonymy of H. rotundi-cauda with H. dihystera. Samples from Burkina Faso,West Africa were identified morphologically as H. dihys-tera (Sawadogo et al., 2009) and clustered in Clade III,which includes H. multicinctus. These samples also clus-

Fig. 1. Phylogenetic relationships within Helicotylenchus populations and species: Bayesian 50% majority rule consensus tree fromtwo runs as inferred from analysis of D2-D3 of 28 rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilitiesequal or more than 70% are given for appropriate clades.

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tered with samples from Florida, USA, one of which wasidentified as H. microlobus by Bae et al. (2009). Sher(1966) synonymised H. microlobus with H. pseudorobus-tus because he could not morphologically distinguish thetopotypes of H. microlobus from those of H. pseudoro-bustus. The opinion of Sher was supported by a numberof authors. These Clade III relationships show no obviousinterpretive pattern of association based on morphologyor geographical distribution, and require further analysisat the morphological and molecular levels.

Since Sher (1966) redescribed H. pseudorobustus fromtopotypes, many populations have been described fromdifferent countries. These populations show a high degreeof variability in several taxonomic characters, a fact thatoften confounds differentiation of this species from sim-ilar species (Fortuner et al., 1984). Fortuner et al. (1984)noted that this may be interpreted as a high degree ofintraspecific variability or it may be seen as evidence ofseveral species under the name of H. pseudorobustus. Us-ing multivariate analyses of characters for 28 populationsidentified as H. pseudorobustus, Fortuner et al. (1984) re-vealed some morphological differences among the popu-lations of H. pseudorobustus, mostly between samplesfrom North America and Western Europe. The differenceswere most apparent in the pattern of the junction of theinner lines of lateral field on the tail, as well as the posi-tion of the phasmids and the dorsal gland opening. Theyconcluded that multivariate analyses are a valuable iden-tification tool that can overcome the problem of intraspe-cific variability. They also noticed that a few samples orig-inally proposed as H. pseudorobustus were, in fact, moresimilar to H. dihystera or could represent another, uniden-tified species. Against this background it is not surpris-ing that in our study we were not able to identify un-ambiguously some samples as H. pseudorobustus and in-stead we proposed four possible candidates named hereas H. pseudorobustus type A, B, C and D. Most likely, thetype B found in Europe and having a wider distributionrepresents the true H. pseudorobustus. Future molecularanalysis of H. pseudorobustus samples collected from thetype locality in Switzerland could give a reliable sequencesignature for this species and will provide a basis to clarifyidentification of our samples.

The grouping of H. pseudorobustus type A and species,morphologically identified as H. labiatus, from NewZealand (clade I (7)), despite the consistent differences inlip region shape and the lateral fields on their tails, clearlyraises questions about their distinctness. Yeates and Wouts(1992) found only four Helicotylenchus species across the159 managed soils they sampled, with H. pseudorobus-tus being recorded from 52% of the sites and H. labiatusfrom 35% of the sites and with no males being recognised.However, Wouts and Yeates (1994) found eight Helicoty-lenchus species from native vegetation and undisturbedsoils but did not report either H. pseudorobustus or H.labiatus. Thus, these two nominal species were consid-ered to be apparently introduced to New Zealand, withthe probability of multiple introductions. They each havewide distribution within New Zealand and their variabilityin both morphological and molecular criteria may reflectthe global pool of populations from which introductionswere derived.

Clade VII consists of a single species, H. martini. Thisspecies was described from Zimbabwe and had since onlybeen reported from Africa (Ali et al., 1973; Marais, 1998).This species has a unique set of characteristics that placeit apart from all the other Helicotylenchus species. Adultsdo not have lip annuli and internal fasciculi are describedas present. Another interesting feature for females of thisspecies is the relatively long tail ranging from 17 to 49 µm(Van den Berg, 1978; Marais, 1998).

The results of the present study suggest that observedgenetic diversity of Helicotylenchus is significantly higherthan has been shown by morphological observations.Integration of morphological and morphometric studieswith molecular analyses may clarify the identification ofspecies within this complex genus. Molecular character-isation of Helicotylenchus species using analysis of theD2-D3 expansion segments of 28S rRNA gene sequencesand sequences of more variable genes, such as ITS-rRNAgene and coxI of mtDNA, can become an important stepin verification of identified samples and diagnostics of thespiral nematodes.

Fig. 2. Phylogenetic relationships within Helicotylenchus populations and species: Strict consensus of 1000 maximum parsimony treesas inferred from analysis of D2-D3 of 28 rRNA gene sequence alignment. (Tree length = 744; CI (excl. uninformative characters) =0.5045; HI (excl. uninformative characters) = 0.4955; RI = 0.8518; RC = 0.4763). Bootstrap values equal or more than 70% are givenfor appropriate clades. Numbers of apomorphies for a clade representing the same species are given in parentheses.

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Acknowledgements

The authors thank Dr R. Fortuner for valuable com-ments for improving of the manuscript draft. The first andlast authors acknowledge support of the US National Sci-ence Foundation PEET grant DEB-0731516.

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