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Phylogenetic Relationships within the Cyst-Forming Nematodes (Nematoda, Heteroderidae) Based on Analysis of Sequences from the ITS Regions of Ribosomal DNA

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Phylogenetic Relationships within the Cyst-Forming Nematodes (Nematoda, Heteroderidae) Based on Analysis of Sequences from the ITS Regions of Ribosomal DNA
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  PhylogeneticRelationshipswithintheCyst-FormingNematodes(Nematoda,Heteroderidae)BasedonAnalysisof SequencesfromtheITSRegionsof RibosomalDNA Sergei A. Subbotin, *  Andy Vierstraete,   Paul De Ley,   Janet Row e,   Lieven Waeyenberge, § Maurice Moens, §  and Jacques R. Vanfl eteren   * Institute of Parasitology of the Russian Academy of Sciences, Leninskii prospect 33, Moscow 117071, Russia;    Department of  Biology, Ghent University, Ledeganckstraat 35, 9000 Ghent, Belgium;    Entomology and Nematology Department, IACR-Rothamsted,Harpenden, Hertfordshire AL5 2JQ, United Kingdom; and   § Crop Protection Department, Agricultural Research Centre,Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium  Received July 10,2000;revised April5,2001 TheITS1,ITS2,and 5.8S genesequencesof nuclearribosomal DNA from40taxaofthefamilyHeteroderi-dae(includingthegenera Afenestr ata, Cactoder a, H et- er oder a, Globoder a, Punctoder a, Meloidoder a, Cr y- phoder a, and Thecaver miculatus  )weresequencedandanalyzed.TheITS regionsdisplayed high levelsof se-quence divergence within Heteroderinae and com-paredtooutgrouptaxa.Unlikerecentfindingsinrootknotnematodes,ITSsequencepolymorphismdoesnotappear to complicate phylogenetic analysis of cystnematodes. Phylogenetic analyses with maximum-parsimony, minimum-evolution, and maximum-like-lihood methods were performed with a range of computer alignments, including elision and culledalignments.All multiplealignmentsandphylogeneticmethods yielded similar basic structure for phyloge-neticrelationshipsofHeteroderidae.Thecyst-formingnematodes arerepresented by six main clades corre-sponding to morphological characters and host spe-cialization,withcertaincladesassumingdifferentpo-sitions depending on alignment procedure and/ormethod of phylogenetic inference. Hypotheses of monophyly of Punctoderinae and Heteroderinae are,respectively, strongly and moderately supported bythe ITS data across most alignments. Close relation-shipswererevealedbetween theAvenaeandtheSac-chari groupsand between theHumuli group and thespecies  H . sal i xoph i l a   within Heteroderinae. TheGoettingianagroupoccupiesabasalpositionwithinthissubfamily. The validity of the genera  Afenestr ata   and Bider a   wastested and isdiscussed based on moleculardata.WeconcludethatITSsequencedataareappropri-atefor studiesofrelationshipswithinthedifferentspe-cies groups and less so for recovery of more ancientspeciationswithinHeteroderidae.  ©2001Academic Press Key Wor ds:   cyst-forming nematodes; coevolution;Heteroderidae;phylogeny;sequencealignments. INTRODUCTION Cyst-forming nematodes (Heteroderidae) are highlyderived and economically very important plant para-sites. The members of this group are of special scien-tific interest within the order Tylenchida due to someremarkable and efficient parasitic adaptations. Afterroot penetration, initiation of feeding by second-stagejuveniles induce highly specialized nurse cells whichare sustained throughout the life of the parasite. Threemolts transform second-stage juveniles into vermiformmigratory males or sedentary mature females withmost of their swollen body exposed on the root surface.At the end of the life cycle, the female body turns intoa hard-walled protective cyst filled with eggs. Somecyst-forming nematode species are highly pathogenicon major agricultural crops including cereals, rootcrops, and most legumes. Many of these species aredistributed worldwide, in part because eggs in cystsremain viable under conditions of dispersal that wouldbe fatal to most other organisms (Siddiqi, 1986; Bald-win and Mundo-Ocampo, 1991).The family Heteroderidae contains 18 genera ofwhich 6 belong to cyst-forming nematodes:  Heterodera, and  Afenestrata   in the subfamily Heteroderinae and Globodera, Punctodera, Cactodera,  and  Dolichodera   inthe subfamily Punctoderinae. At present, from 90 tomore than 100 cyst-forming nematode species are rec-ognized by different authors; about two-thirds of thesebelong to the genus  Heterodera   (Siddiqi, 1986; Baldwinand Mundo-Ocampo, 1991; Evans and Rowe, 1998;Wouts and Baldwin, 1998).Studies of phylogenetic relationships among nema-todes are not only essential to taxonomy, but also allow a more complete understanding of the biology of nem-atodes as agricultural pests. Hypotheses of phyloge-netic relationships within Heteroderidae based on tra-ditional morphological characters, scanning electron Molecular Phylogenetics and EvolutionVol. 21, No. 1, October, pp. 1–16, 2001doi:10.1006/mpev.2001.0998, available online at http://www.idealibrary.com on 1055-7903/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved. 1  microscopy surface morphology, cuticle ultrastruc-tures, trophic specialization, cellular host response,and coevolution with hosts have been proposed by sev-eral authors (Krall and Krall, 1970, 1973, 1978; Wouts,1972, 1973, 1985; Stone, 1975, 1979; Ferris, 1979,1985, 1998; Krall, 1990; Baldwin, 1986, 1992; Baldwinand Schouest, 1990). Despite their use of similar char-acters and approaches, some differences were obtainedin the phylogenetic reconstructions proposed by theseauthors. It is evident that homoplastic evolution ofmorphological and biological characters may hamperphylogenetic analyses. The use of molecular data forphylogenetic inference provides an independent ap-proach that may shed new light on the evolution ofsuch characters.Comparative analysis of coding and noncoding re-gions of ribosomal DNA has become a popular tool forconstruction of phylogenetic trees of many organismsincluding nematodes. Recently, phylogenetic analysesbased on the 18S rDNA sequences provided new in-sights into interrelationships within the phylum Nem-atoda and showed that homoplastic morphologicalcharacters may be widespread and that the presenthigher-level classification of the Nematoda needs revi-sion (Aleshin  et al.,  1998; Blaxter  et al.,  1998). Partialsequences of the 18S rDNA gene and the D2–D3 ex-pansion region of the 28S rDNA gene were used toexamine evolutionary relationships among the root-lesion nematodes of the genus  Pratylenchus   and re-lated genera, entomopathogenic nematodes of the gen-era  Steinernema   and  Heterorhabditis,  and bacterial-feeding species of the genus  Acrobeloides   (Al-Banna  et al.,  1997; Liu  et al.,  1997; Duncan  et al.,  1999; De Ley et al.,  1999).The internal transcribed spacers (ITS1 and ITS2)situated between the small nuclear ribosomal subunit(the 18S gene) and the large subunit (the 28S gene) areconsidered appropriate genomic regions, both in termsof ease of isolation and sequencing and in terms ofusefulness in resolving relationships at the specieslevel. The ITS region of rDNA has recently been used tostudy phylogenetic relationships within the genera Heterorhabditis   (Adams  et al.,  1998) and  Meloidogyne  (Hugall  et al.,  1999). The latter study discovered sub-stantial sequence polymorphism within individual rootknot nematodes, and the authors suggested that thismight be due to hybrid srcins of some  Meloidogyne  populations. The pioneering work on Heteroderidae ofFerris and co-authors (Ferris  et al.,  1993, 1994, 1995;Ferris, 1998) demonstrated that nucleotide sequencesof ITS1 and ITS2 are both useful and practical forphylogenetic analysis of the cyst-forming nematodes.They analyzed relationships among a limited numberof species from the genera  Heterodera, Cactodera,  and Globodera   and within the Goettingiana, Avenae, andSchachtii groups of  Heterodera.  In a recent paper, Fer-ris (1998) compared sequences from 15 taxa using par-simony analysis. The positions obtained for  G. vir- giniae, H. carotae,  and  H. bifenestra   in thisphylogenetic tree were in conflict with morphologicaldata. The same author pointed out two problems whichemerged from the use of the rDNA sequences: (1)closely related sibling species, such as members of theSchachtii group, may have rDNA that is too similar topermit sorting on the basis of the ITS sequence dataand (2) other, more phylogenetically distant, taxa mayhave rDNA that is too dissimilar to allow constructionof a plausible alignment of the ITS sequence data,limiting the reliability of subsequent phylogeneticanalysis.In this study we present phylogenetic analyses of 40species and subspecies of the family Heteroderidae.The species cover most of the known taxonomic andmorphological diversity of this nematode group. Ouranalysis is based on 36 new sequences of completeITS1    5.8S    ITS2 regions that we have analyzedwith three phylogenetic procedures: maximum-parsi-mony (MP), minimum-evolution (ME), and maximum-likelihood (ML). The goals of this study were (1) toestimate the phylogenetic relationships between spe-cies, groups of species, and genera of the cyst-formingnematodes, (2) to consider the congruence of the mo-lecular phylogenetic trees with morphological group-ings and coevolution with host plants, and (3) to eval-uate the influence of tree-building methods andalignment procedures on phylogenetic inference forcyst-forming nematodes.Nucleotide insertions or deletions are found com-monly in the ITS regions of the cyst-forming nema-todes and no consensus exists for the alignment of suchsequences. To approach this problem, we generatedseveral alignments: computer alignments with differ-ent gap length and gap opening penalties and an eli-sion alignment combining a range of alignments in asingle matrix and culled alignment exclusion of themost variable fragments. All these alignments werecompared by use of different tree-building methods.For analyses of sensitivity of tree topology we are in-terested in the branching order of clades and theirstatistical support within each analysis. MATERIALS AND METHODS Nematode Samples  Thirty-six new nucleotide sequences of the ITS re-gions of different nematode species and populations ofthe family Heteroderidae were determined during thisstudy. Seven previously determined sequences of spe-cies were included in our phylogenetic analyses. A totalof 28 valid  Heterodera   species, 4  Globodera   species, and1 species of each of the genera  Punctodera, Cactodera, and  Afenestrata   were used in this study (Table 1). Allwere identified by their morphology and morphomet-rics. Two populations of the species  H. avenae   and  H.salixophila,  which revealed polymorphism during re-2  SUBBOTIN ET AL.  striction analysis of the ITS regions in previous studies(Subbotin  et al.,  1999, 2000a), and two populations of H. bifenestra,  which were used by Ferris (1998) as anoutgroup for phylogenetic analysis of the cyst-formingnematodes, were also included. Previously obtainedsequences of  G. rostochiensis, G. pallida, G. tabacum  TABLE 1TheNematodeSpeciesand PopulationsfromtheFamily HeteroderidaeUsed in ThisStudy Classification and speciesGenBankAccession No. Host plants (family) Location SourceHeteroderinae* Heterodera   (28/62)** a  H. arenaria   AF274396  Ammophila arenaria   (Poaceae) Linconshire, England J . Rowe, UK H. aucklandica   AF274398  Microlaena stipoides   (Poaceae) One Tree Hill, Auckland,New ZealandW. Wouts, New Zealand H. avenae   AF274395 Cereals (Poaceae) Argentan, France R. Rivoal, France H. avenae   AF274397 Cereals (Poaceae) India J . Rowe, UK H. bifenestra   AF274384 Grasses (Poaceae) Sweden J . Rowe, UK H. bifenestra   AF274385 Grasses (Poaceae) Luxembourg province, Belgium S. A. Subbotin, Russia H. cajani   AF274389  Cajanus cajan   (Fabaceae) India J . Rowe, UK H. carotae   AF274413  Daucus   sp. (Umbelliferae) Cre´ances, France M. Bossis, France H. ciceri   AF274393  Cicer   sp. (Fabaceae) Syria N. Vovlas, Italy H. cruciferae   AF274411  Brassica   sp. (Cruciferae) Brielle, The Netherlands B. Schoemaker,the Netherlands H. cynodontis   AF274386  Cynodon dactylon   (Poaceae) Pakistan F. Shahina, Pakistan H. cyperi   AF274388  Cyperus   sp. (Cyperaceae) Spain M. Romero, Spain H. fici   AF274409  Ficus carica   (Moraceae) Sukhumi, Georgia S. A. Subbotin, Russia H. filipjevi   AF274399 Cereals (Poaceae) Saratov, Russia E. Osipova, Russia H. glycines   AF274390  Glycine max   (Fabaceae) Arkansas, USA R. Robbins, USA H. goettingiana   AF274414  Pisum   sp. (Fabaceae) Germany J . Rowe, UK H. hordecalis   AF274401 Grasses (Poaceae) Montrose, Scotland, UK S. A. Subbotin, Russia H. humuli   AF274408  Humulus lupulus   (Cannabaceae) Tsivilsk, Chuvashija, Russia Yu. Danilova, Russia H. iri   AF274400 Grasses (Poaceae) Forfar, Scotland, UK S. A. Subbotin, Russia H. latipons   AF274402 Cereals (Poaceae) Breda, Syria U. Scholz, Germany H. litoralis   AF274410  Sarcocornia quinqueflora  (Chenopodiaceae)Glen Innes, Auckland,New ZealandW. Wouts, New Zealand H. medicaginis   AF274391  Medicago sativa   (Fabaceae) Stavropol region, Russia S. A. Subbotin, Russia H. oryzicola   AF274387  Oryza sativa   (Poaceae) Kerala, India J . Rowe, UK H. riparia   AF274407  Urtica dioica   (Urticaceae) Germany D. Sturhan, Germany H. sacchari   AF274403  Saccharum officinale   (Poaceae) Ivory Coast J . Rowe, UK H. salixophila   AF274405  Salix album   (Salicaceae) Kherson, the Ukraine S. A. Subbotin, Russia H. salixophila   AF274406  Salix   sp. (Salicaceae) Nieuwpoort, Belgium S. A. Subbotin, Russia H. schachtii   AF274394  Beta vulgaris   (Chenopodiaceae) Germany D. Sturhan, Germany H. sorghi   AF274404  Sorghum   sp. (Poaceae) New Delhi, India J . Rowe, UK H. trifolii   AF274392  Trifolium   sp. (Fabaceae) UK J . Rowe, UK H. urticae   AF274412  Urtica   sp. (Urticaceae) Diksmuide, Belgium S. A. Subbotin, Russia Afenestrata   (1/5) a  A. orientalis Miscanthus purpurecens   (Poaceae) Primorskii territory, Russia Eroshenko  et al. (unpublished)Punctoderinae Globodera   (4/8) a  G. artemisiae   AF274415  Artemisia   sp. (Asteraceae) China D. Peng, China G. pallida Solanum tuberosum   (Solanaceae) Risby, UK Subbotin  et al.  (2000b) G. rostochiensis Solanum tuberosum   (Solanaceae) Moscow region, Russia Subbotin  et al.  (2000b) G. tabacum G. t. solanacearum Solanum dulcamara   (Solanaceae) Connecticut, USA Subbotin  et al.  (2000b) G. t. tabacum Solanum dulcamara   (Solanaceae) Virginia, USA Subbotin  et al.  (2000b) G. t. virginiae Solanum dulcamara   (Solanaceae) Virginia, USA Subbotin  et al.  (2000b) Cactodera   (1/9) a  C. estonica   AF274417  Polygonum   sp. (Polygonaceae) The Netherlands G. Karssen, The Netherlands Punctodera   (1/3) a  P. punctata   AF274416 Grasses (Poaceae) Luxembourg province, Belgium S. A. Subbotin, RussiaAtaloderinae Thecavermiculatus   (1/4) b  T. crassicrustata Mertensia maritima   (Poaceae) Kamchatka, Russia Eroshenko  et al.  (unpublished)Cryphoderinae Cryphodera   (1/6) c  C. brinkmani   AF274418  Pinus thunbergii   (Pinaceae) Yokokowa, Saitama-Ken, J apan G. Karssen, The NetherlandsMeloidoderinae Meloidodera   (1/9) b  M. alni   AF274419  Alnus   sp. (Betulaceae) Luxembourg province, Belgium S. A. Subbotin, Russia * Classification of the family Heteroderidae according to Wouts (1985).** The number of species sampled/total number of valid species in genera according to  a  Wouts and Baldwin (1998),  b  Evans and Rowe(1998),  c  Karssen and Van Aelst (1999). 3 PHYLOGENY OF CYST-FORMING NEMATODES  solanacearum, G. t. virginiae,  and  G. t. tabacum   (Sub-botin  et al.,  2000b) were added to the analysis. Thenon-cyst-forming sedentary nematode species  The- cavermiculatus crassicrustata   was used as an ingrouptaxon to examine its position with respect to cyst-forming nematode genera. Sequences of  A. orientalis  and  T. crassicrustata   were obtained from an unpub-lished study (A. S. Eroshenko  et al.,  unpublished). Thenon-cyst-forming sedentary nematode species  Meloid- odera alni   and  Cryphodera brinkmani   were sequencedand designated outgroup taxa (Table 1). DNA Extraction, PCR, Cloning, and Sequencing  For each population, one to four cysts or femaleswere transferred into 10   l of double-distilled water inan Eppendorf tube and crushed with a microhomog-enizer. Eight microliters of nematode lysis buffer (125mM KCl, 25 mM Tris–Cl, pH 8.3, 3.75 mM MgCl 2 , 2.5mM dithiothreitol, 1.125%Tween 20, 0.025%gelatine)and 2   l of proteinase K (600   g/ml) were added. Thetubes were incubated at 65°C (1 h) and 95°C (10 min)consecutively. After centrifugation (1 min; 16,000 g  ) 10  l of the resulting DNA suspension was added to thePCR mixture containing 10   l of 10   Taq   incubationbuffer, 20   l of 5  Q solution, 200   M each dNTP ( Taq  PCR Core Kit, Qiagen, Germany), 1.5   M each primer(synthesized by Eurogentec, Belgium), 0.8 U of  Taq  Polymerase ( Taq   PCR Core Kit, Qiagen), and double-distilled water to a final volume of 100   l. The forwardprimer TW81 (5  -GTTTCCGTAGGTGAACCTGC-3  )and the reverse primer AB28 (5  -ATATGCTTAAGT-TCAGCGGGT-3  ) were used in the PCR. The DNAamplification profile, carried out in a GeneE DNA ther-mal cycler (New Brunswick Scientific, Wezembeek-Oppem, Belgium), consisted of 4 min at 94°C; 35 cyclesof 1 min at 94°C, 1.5 min at 55°C, and 2 min at 72°C;and 10 min at 72°C. After DNA amplification, 5   l ofthe product was run on a 1%agarose gel. The remain-der was stored at   20°C and then used for sequencing.Amplified products were treated with shrimp alka-line phosphatase (1 U/  l; Amersham E70092Y) andexonuclease I (10 U/  l; Amersham, E70073Z) for 15min at 37°C, followed by 15 min at 80°C to kill theenzymes. Sequences of  H. avenae, H. urticae, H. oryzi- cola, H. sorghi, H. sacchari, H. cruciferae, P. punctata,G. artemisiae,  and  M. alni   showed one or several am-biguous positions. Therefore, for these species, the nu-cleotide position was scored according to the IUB con-vention. DNA fragments were sequenced in bothdirections with TF1 (5  -GTAGGTGAACCTGCTGC-TGG-3  ), 28R1 (5  -TGATATGCTTAANTTCAGCG-GGT-3  ), and two additional internal forward and re-verse primers MITSF (5  -ATGAAGAACGCAGC-3  )and MITSR (5  -AATGACCCTGAACC-3  ) with BigDyeTerminator Cycle Sequencing Ready Reaction Kit (PEApplied Biosystems, UK) according to the manufactur-er’s instructions. The following program was used forall sequencing reactions: 94°C for 30 s, 50°C for 15 s,and 60°C for 4 min. The resulting products were pre-cipitated by addition of 50   l of 95%ethanol and 2   l of3 M sodium acetate, pH 4.6, to each cycle sequencingreaction tube (20   l). The pellet was rinsed with 250   lof 70%ethanol, dried in a Speedvac concentrator, re-dissolved in loading buffer, and run on 48-cm 4%acryl-amide sequencing gels with a Perkin–Elmer ABI Prism377 DNA sequencer.Initial sequences of  H. carotae, H. humuli, H.schachtii, H. ciceri, H. trifolii,  and  Cr. brinkmani  showed many ambiguous nucleotide positions for bothdirect forward- and reverse-primed sequencing. PCRproducts of these species were therefore cloned andresequenced. Low-yield PCR products of  H. bifenestra  and  A. orientalis   were also cloned and resequenced.Amplified products were excised from 1%TBE bufferedagarose gels with the QIAquick Gel Extraction Kit(Qiagen), cloned into the pGEM-T vector, and trans-formed into J M109 High Efficiency Competent Cells(Promega Corp., Madison, WI). Several clones of eachpopulation were isolated by blue/white selection, sub-mitted to PCR, and then cycle-sequenced. Only onesequence was used for analyses, because sequencevariants from each species with polymorphism alwayscluster per species and do not affect tree topology withregard to interspecific relationships (data not shown). Sequence Alignments  DNA sequences were edited with Chromas 1.45(©1996–1998 Conor McCarthy), aligned with ClustalW 1.7 (Thompson  et al.,  1994) and slightly modifiedmanually with the DCSE v 3.4 for X-Windows (Linux;De Rijk and De Wachter, 1993) or GeneDOC 2.5.0(Nicholas and Nicholas, 1997). Only sequences of ITS1,the 5.8S gene, and ITS2 were used for further analyses.The boundaries of ITS1 and ITS2 were determined bycomparison of the aligned sequence with previouslypublished sequences of  Globodera   (Bulman and Mar-shall, 1997; Blok  et al.,  1998).Several methods were used for generating align-ments. Nine computer alignments were created, withgap open penalty parameters of 5, 15, and 30 and gaplength penalty parameters of 3, 6.66, and 10. All ofthese alignments were subjected to minor manual ed-iting followed by independent phylogenetic analysis(Table 2) and then combined into a single matrix forelision analysis (cf. Wheeler  et al.,  1995; Liston  et al., 1999). A single culled alignment was also obtained, asfollows. Two ingroup alignments for Heteroderinaeand Punctoderinae, comprising, respectively, 1154 and922 positions, resulted from aligning to each other andto  T. crassicrustata,  with Clustal Wset to gap open/gaplength penalty of 15/6.66 (default). Next, the two align-ments were combined and aligned with outgroup spe-cies, resulting in a final alignment comprising 1185positions. Subsequent phylogenetic analysis of the ITSregions was applied to an 853-character data set, afterexclusion of most ambiguous positions. The original4  SUBBOTIN ET AL.  sequences from this study are available in the Gen-Bank database. Multiple alignments are available athttp://allserv.rug.ac.be/  avierstr/maintained by AndyVierstraete (Department of Biology, Ghent Univer-sity). Sequence and Phylogenetic Analyses  All alignments were analyzed with maximum-parsi-mony, maximum-likelihood, and neighbor-joining (NJ )algorithms to resolve the interspecific and intergenericphylogenetic relationships within Heteroderidae.  M.alni   and  Cr. brinkmani   were considered representa-tives of primitive genera in this family (Krall andKrall, 1978; Wouts 1985; Baldwin, 1992)and were usedas outgroup taxa for phylogenetic analyses.Equally weighted MP analyses were performed withPAUP* 4.0b4a (Swofford, 1998). Heuristic search set-tings were 100 replicates of random taxon addition,tree bisection–reconnection branch swapping, multipletrees retained, no steepest descent, and acceleratedtransformation. Gaps were treated as missing data.Bootstrap (BS) analysis with 1000 replicates was per-formed, to assess the support for each branch on thecorresponding tree(s) obtained by heuristic search withsimple addition sequences (Felsenstein, 1985). For eli-sion analysis, mean and standard deviation of boot-strap results were calculated from the bootstrap sup-port values obtained from individual alignments.Consistency index (CI) (Kluge and Farris, 1969), reten-tion index (RI), rescaled consistency index (RC)(Farris,1989), and the  g  1 statistic (Hillis and Huelsenbeck,1992) were computed to estimate the amount of phylo-genetic signal available for parsimony analysis. The  g  1statistic, a measure of skewness of tree-length distri-bution, was computed by generation of 10,000 randomtrees with the RANDTREES option in PAUP. Pairwisedivergences between taxa were computed as absolutedistance values and as percentage mean distance val-ues based on whole alignment, with adjustment formissing data.For ML analysis, the appropriate substitution modelof DNA evolution that best fitted the data set wasdetermined by the likelihood ratio test (LRT) and theAkaike Information Criterion (AIC) with ModelTest3.04 (Posada and Crandall, 1998). Alternative topolo-gies were tested by the ML method of Kishino andHasegawa (1989) as implemented in PAUP*.Minimum-evolution analysis was performed by ap-plication of the selected ML substitution model to theNJ algorithm. Bootstrap analysis with 1000 replicateswas conducted to assess the degree of support for eachclade on the trees. The orders of corresponding planthosts were mapped onto the strict consensus trees ofMP, ME, and ML of the elision alignment with Mac-Clade 3.07 (Maddison and Maddison, 1993). Treeswere displayed with TreeView 1.6.1 (Page, 1996). RESULTS Analysis of DNA Sequence  The ITS region sequence length was shorter in spe-cies of Punctoderinae than in those of Heteroderinaeand ranged from 898 bp ( C. estonica, G. rostochiensis  )to 910 bp ( G. artemisiae  ) and from 931 bp ( H. oryzicola  )to 1021 bp ( H. cyperi  ). The lengths of the nine align-ments varied from 1124 to 1298 positions, and theelision alignment comprised 10,734 positions (Table 2).Sequence divergence for ingroup alignment rangedfrom 0.0 to 31.4% and from 0.3 to 14.7% within Het-eroderinae and Punctoderinae, respectively, as ob-served by pairwise comparisons of the whole DNAalignment with adjustment for missing data.A broad range of G    C content was observed inentire sequences of different species of Heteroderidae.A relatively higher value of G   C content was found inthe ITS1 region than in the 5.8S gene or the ITS2region. Although ITS2 was shorter than ITS1, se-quence divergence between genera, and within  Het- erodera   and  Globodera,  was higher in this region than TABLE 2Alignment Parametersand TreeStatisticsfor 43ITS Region Sequencesof SpeciesfromtheFamily Heteroderidae No.computeralignmentGap openpenaltyGaplengthpenaltyAlignedlengthInformativecharactersConstantcharactersTreelengthTreenumberCI(w/o uninf)HI(w/o unif) RI RC  g  1Elision 10734 6130 3239 27050 6 0.4854 0.5146 0.7409 0.3845   0.5955Culled 853 464 300 2003 2 0.4616 0.5384 0.7307 0.3597   0.58411 5 3 1298 623 460 2425 12 0.5185 0.4815 0.7644 0.4366   0.56242 5 6.66 1228 653 397 2638 6 0.5068 0.4932 0.7553 0.4134   0.62573 5 10 1192 652 384 2773 12 0.4807 0.5193 0.7438 0.3860   0.53104 15 3 1215 665 378 2875 6 0.4895 0.5105 0.7482 0.3953   0.62205 15 6.66 1192 687 356 2967 6 0.4906 0.4906 0.7425 0.3892   0.60136 15 10 1159 692 337 3183 6 0.4728 0.5272 0.7315 0.3663   0.61827 30 3 1170 713 312 3279 6 0.4856 0.5144 0.7435 0.3841   0.67658 30 6.66 1156 719 312 3317 12 0.4795 0.5205 0.7193 0.3641   0.67789 30 10 1124 726 305 3498 12 0.4751 0.5249 0.7443 0.3681   0.5629 5 PHYLOGENY OF CYST-FORMING NEMATODES
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