the its1-5.8s-its2 region of rdna and morphologicalfeatures · s.e. spiridonov et al. the Þrst...

Nematology 2004 Vol 6(4) 547-566

Phylogenetic relationships within the genus Steinernema(Nematoda Rhabditida) as inferred from analyses of sequences of

the ITS1-58S-ITS2 region of rDNA and morphological features

Sergei E SPIRIDONOV 1lowast Alex P REID 2lowastlowast Kasia PODRUCKA 2lowastlowastlowastSergei A SUBBOTIN 1lowastlowastlowastlowast and Maurice MOENS 34

1 Institute of Parasitology of Russian Academy of Sciences Leninskii prospect 33 Moscow 119071 Russia2 CABI-Bioscience Bakeham Lane Egham Surrey TW20 9TY UK

3 Crop Protection Department Agricultural Research Centre Burg Van Gansberghelaan 96 9820 Merelbeke Belgium4 Laboratory for Agrozoology Ghent University Coupure 555 9000 Ghent Belgium

Received 2 March 2004 revised 3 June 2004Accepted for publication 3 June 2004

Summary ndash Eighty four new and four published ITS1-58S-ITS2 sequences of rDNA obtained from different populations of 24 nominalspecies and 28 isolates of 16 putative Steinernema species were analysed using the maximum parsimony method In most of thephylogenetic trees obtained from different ITS alignments and phylogenetic procedures the 84 isolates formed five main highly ormoderately supported clades viz Clade I lsquoaffine-intermediumrsquo Clade II lsquocarpocapsae-scapterisci-tamirsquo Clade III lsquofeltiae-kraussei-oregonensersquo Clade IV lsquobicornutum-ceratophorum-riobraversquo and Clade V lsquoarenarium-glaseri-karii-longicaudumrsquo The ITS rDNAdata were found to be of little utility in resolving relationships between these clades but were useful in studying relationships betweenspecies within certain clades The level of intra-specific variability was different between clades with sequence divergence of 24-28of ITS rDNA for some species Analysis of a combined data matrix of both molecular and morphological features was performed withsix qualitative and three quantitative features

Keywords ndash Entomopathogenic nematodes ITS PCR rDNA sequence analysis Steinernematidae

The genus Steinernema Travassos 1927 is the mostintensively studied group of nematodes associated withinsects Although only about 30 species of the genushave been described to date greater specific diversityis expected (Hominick et al 1997 Kerry amp Hominick2002) Nematodes of this genus are found in virtually allterrestrial habitats supporting vegetation (Elawad et al1997 Waturu et al 1997 Phan et al 2001a b Shahinaet al 2001 Hominick 2002)

Poinar (1993) proposed hypothetical evolutionary linksbetween Steinernema and other rhabditids based on mor-phological data Relationships of the genus Steinernemawith alloionematids and strongyloidids were also sug-gested (Spiridonov amp Belostotskaya 1983) The close-ness of Steinernema with Strongyloides Grassi 1879 and

Corresponding author e-mail s_e_spiridonovramblerru Present address Scottish Agricultural Science Agency 82 Craigs Road Edinburgh EH12 8NJ UK Present address Royal (Dick) School of Veterinary Studies The University of Edinburgh Summerhall Edinburgh EH9 1QH UK Present address Department of Nematology University of California Riverside Riverside CA 92521-0415 USA

some other rhabditid genera was recently proved by analy-ses of 18S rDNA sequences (Blaxter et al 1998)

Several attempts have been made to compare mor-phological and molecular approaches for reconstructingthe phylogeny of the genus Steinernema Liu and Berry(1996) presented phylogenetic relationships derived fromthe analysis of both RAPD fragments and 11 morpholo-gical features for 14 Steinernema species Reid (1994) andReid et al (1997) analysed the restriction fragments ofITS1-58S-ITS2 PCR products generated by 17 restrictionenzymes and proposed a phylogram in which the cladescorrespond to conditional groups of species such as lsquostein-ernematids with long juvenilesrsquo or lsquosteinernematids withshort juvenilesrsquo

copy Koninklijke Brill NV Leiden 2004 547Also available online - wwwbrillnl

SE Spiridonov et al

The first attempt to use sequencing data for Steiner-nema phylogenetic analysis was based on analyses of thepartial 18S gene of rDNA (Liu et al 1997) This re-gion however was found to be too conservative for con-structing a resolved phylogeny of this genus Sequencesof the ITS1 region of rDNA and mitochondrial COII and16S rDNA genes for seven species of Steinernema wereanalysed by Szalanski et al (2000) The authors obtainedtrees which were in good concordance with the morpholo-gical features of the species and considered these genes tobe good candidates for phylogenetic reconstruction of thegroup Analyses of sequences of ITS1-58S-ITS2 regionsobtained from ten steinernematid species revealed sev-eral groups with significant bootstrap supports (Nguyenet al 2001 Nguyen amp Duncan 2002) Comprehensivephylogenetic analyses based on combined morphologicaland molecular (D2-D3 expansion segment of LSU rDNA)data including 21 species of Steinernema were made byStock et al (2001)

All of these studies revealed trends in Steinernema phy-logeny Unfortunately many species and isolates were notincluded so that their position within the genus was leftuncertain In our study the ITS1-58S-ITS2 sequences ofdifferent Steinernema isolates maintained at CABI Bio-science Egham UK and CLO Merelbeke Belgium wereanalysed Several aspects of phylogenetic analyses forthe Steinernematidae were set as the main goals for thepresent work these being i) to study relationships be-tween the main groups of Steinernema using maximumparsimony It was hypothesised that newly obtained se-quences might resolve relationships inside this taxon Inthis analysis we included species belonging to differentgroups (based on gross morphology eg infective juve-nile length) in the genus As alignment ambiguity is acommon event during ITS rDNA analysis of divergent se-quences several alignments were generated under differ-ent combinations of gap opening and gap extension penal-ties and then analysed separately ii) to reveal relation-ships between species and isolates within several maingroups All sequences of isolates belonging to a certaingroup were included in the alignment and analysed usingmaximum parsimony iii) to test congruence of morpholo-gical and molecular phylogeny of Steinernema The mor-phological and morphometrical characters used by Liuand Berry (1996) and Stock et al (2001) were re-analysedand a new matrix proposed The total evidence analy-sis was applied to obtain a tree from the matrix com-bining molecular and morphological data iv) to estimatespecies boundaries in steinernematids based on molecular

data Sequence divergence was estimated firstly betweenisolates which according primarily to their morphologywere identified as belonging to the same species and sec-ondly isolates which on the same basis were identifiedas belonging to different species The presence of autapo-morphies was estimated for related species The speciesdelimitation was finally considered on the basis of a com-bination of molecular (autapomorphies sequence diver-gence) and morphological evidence and v) to reveal eco-logical and biogeographical implications of the contem-porary evolutionary hypothesis for Steinernema We ex-pected that the proposed steinernematid phylogeny mightdemonstrate concordance in features such as habitat pref-erence and geographical distribution patterns

Materials and methods

NEMATODE MATERIAL

The list of studied species and isolates is presented inTable 1 Isolates were obtained either by using Galleriabaiting of soil samples collected during surveys (Beddingamp Akhurst 1975) or were received from other laborato-ries The nematodes were maintained on Galleria cater-pillars and juvenile suspensions were stored at 4C

MORPHOLOGICAL AND ECOLOGICAL STUDIES AND

CROSS-BREEDING TESTS

Juveniles and females were fixed in 6 formalinfor both light microscope and SEM studies For SEMspecimens were processed as described by Kozodoi andSpiridonov (1988) The matrix of morphological featuresfor steinernematids was created on the basis of our ownand published observations (Nguyen amp Smart 1996Artyukhovsky et al 1997 Hominick et al 1997 Stocket al 1998 Pham et al 2000 Nguyen amp Adams2003) Cross-breeding experiments were performed toelucidate the specific status of N and Va isolates ofS feltiae from Merelbeke Belgium Single juvenilesfrom the two populations were injected into Galleriacaterpillars (Akhurst amp Bedding 1978) which were keptin Parafilmreg sealed Petri dishes until the results of theinjection (presence or absence of a new generation)became obvious Information about the habitats of theisolates was obtained during collection of materials forthe present study and from the literature (Hominick ampBriscoe 1990 Gwynn amp Richardson 1996 Miduturi etal 1997 Sturhan 1999)

548 Nematology

Phylogenetic relationships within the genus Steinernema

DNA EXTRACTION PCR AMPLIFICATION CLONING

AND SEQUENCING AT CLO

A few (1-20) juveniles were put in an Eppendorf tubecontaining 8 microl of worm lysis buffer (100 mM KCl20 mM Tris-HCl pH 83 3 mM MgCl2 2 mM DTTand 09 Tween 20) 10 microl double distilled water 2 microlproteinase K (600 microgml) and homogenised (Spiridonovamp Moens 1999) PCR amplification was performed asdescribed by Spiridonov and Moens (1999) Two setsof primers were used in the PCR reactions (1) TW81(5prime-GTTTCCGTAGGTGAACCTGC-3prime) and AB28(5prime-ATATGCTTAAGTTCAGCGGGT-3prime) or (2) 18S(5prime-TTGATTACGTCCCTGCCCTTT-3prime) and 26S(5prime-TTTCACTCGCCGTTACTAAGG-3prime) as described byJoyce et al (1994) and Vrain et al (1992) respectivelyPCR products were purified using QIAquick PCR or GelPurification Kit (Qiagen Ltd Crawley UK)

PCR products were used for direct sequencing us-ing one of the following primers AB28 TW81 18S26S M1 (5prime-ACGAGCCGAGTGATCCACCG-3prime) or M2(5prime-CTTATCGGTGGATCACTCGG-3prime) with the BigDyeTerminator Cycle Sequencing Ready Reaction Kit (Ap-plied Biosystems AH Nieuwerkerk ad Ijissel The Nether-lands) The resulting products were purified using a Cen-triflex Gel Filtration Cartridge (Edge BioSystems IncGaithersburg MD USA)

As initial direct sequencing of several isolates showedambiguous positions with multiple peaks the PCR prod-ucts from these isolates were cloned and re-sequencedThe PCR product was cloned in pGEM-T vector andtransformed into JM109 High Efficiency Competent Cells(Promega Leiden The Netherlands) according to the in-structions of the manufacturer PCR products from cloneswere sequenced as described above Several clones fromeach isolate were sequenced to confirm polymorphismsobserved from the direct sequencing approach


AND SEQUENCING AT CABI

Total genomic DNA was isolated as described by Reidand Hominick (1992) or Joyce et al (1994) Primers 18Sand 26S as described by Vrain et al (1992) were usedfor PCR PCR amplification was performed as describedby Reid et al (1997) All PCR products were cloned andthen sequenced as described above

SEQUENCE ALIGNMENTS AND PHYLOGENETIC

ANALYSIS

Alignments of the ITS1-58S-ITS2 sequences (with nu-cleotides belonging to 18S and 28S rDNA removed) weregenerated using Clustal W (Thompson et al 1994) Sev-eral alignments were generated i) 30 alignments con-taining 48 Steinernema and Caenorhabditis elegans (out-group taxon) sequences with different gap opening andgap extension penalty values for general analysis ii) fivealignments for the analysis of steinernematid groups andiii) alignments with 35 sequences for combined morpho-logical and molecular analysis All studied alignments areavailable from the first author

Maximum parsimony (MP) analyses were conductedwith PAUP 40b8 (Swofford 1998) The heuristic searchprocedure was used with 100 replicates of random taxonaddition Gaps were treated as missing data Bootstrap(BS) analysis with 100 replicates was conducted toassess the degree of support for each branch on thetree (Felsenstein 1985) using simple addition sequenceswith TBR swapping (tree-bisection-reconnection) Theg1 statistic was computed by generating 10 000 randomtrees using the RANDTREES option in PAUP (Hillsamp Huelsenbeck 1992) The partition homogeneity testwas run as implemented in PAUP with 100 replicatesto determine whether the nematode phylogeny inferredfrom molecular data was congruent with morphologicalphylogeny Trees were displayed with TreeView 161(996)

Results

SEQUENCE ANALYSES OF THE ITS REGION FOR

STEINERNEMATIDS

Sequence characteristics

New complete sequences of the ITS1-58S-ITS2 regionof the rDNA cistron were obtained for 81 steinernematidisolates and partial sequences (gt685 bp) were obtainedfor a further three species (S rarum ndash AY 171300Steinernema sp 13 from Pendelton Arkansas ndash AY171277 and Steinernema sp 13 from Nebraska) FourSteinernema sequences deposited in GenBank were alsoused in the analysis (Table 1) The length of full ITS1-58S-ITS2 sequences varied from 712 bp in Steinernemasp 16 to 816 bp in Steinernema sp 7 (E3) from EstoniaThe ITS1 region (243-306 bp) was shorter than ITS2(274-375 bp) except for Steinernema sp 9 from Sri Lanka

Vol 6(4) 2004 549

SE Spiridonov et al

Table 1 Details of Steinernema isolates used in the study

Species Location (strain) GenBank accession number Source of material orreference

S abbasi Sultanate of Oman AY230158 (CABI cl) Elawad et al (1997)S affine Merelbeke Belgium AY171286 (CLO di) SE SpiridonovS affine Wageningen The Netherlands AY171298 (CLO di) SE SpiridonovS affine Aberdeen Scotland UK AY171296 (CLO di) M WilsonS affine Berkshire England UK AY230159 (CABI cl) AP ReidS arenarium Rjazan Russia AY230160 (CABI cl) AP ReidS bicornutum Type locality Vojvodina Yugoslavia AY230163 (CABI cl) R-U EhlersS bicornutum Krasnodar Russia AY171279 (CLO cl) SE SpiridonovS carpocapsae USA (All) AY230164 (CABI cl) AP ReidS carpocapsae Western Europe (isolate 42) AY171283 (CLO cl) MA AnsariS carpocapsae Mozhaisk Moscow region Russia AY171282 (CLO di) SE SpiridonovS ceratophorum Type locality China AY230165 (CABI cl) AP ReidS cubanum Type locality Cuba AY230166 (CABI cl) Z MraacutecekS diaprepesi Florida USA AF122021 Nguyen et al (2001)S diaprepesi Venezuela AY230187 (CABI cl) AP ReidS feltiae Type locality Izhevsk Russia AY171246 (CLO cl) SE SpiridonovS feltiae UK (isolate A1 site 76) AY230169 (CABI cl) AP ReidS feltiae UK (isolate A2 site 107) AY230170 (CABI cl) AP ReidS feltiae Khosrov National Park Armenia AY171256 (CLO di) SE SpiridonovS feltiae Australia (isolate lsquobibionisrsquo) AY171257 (CLO di) R AkhurstS feltiae Merelbeke Belgium (isolate N) AY171254 (CLO cl) SE SpiridonovS feltiae Merelbeke Belgium (isolate Va) AY171274 (CLO cl) SE SpiridonovS feltiae Belovezha Poland AY171266 (CLO cl) SE SpiridonovS feltiae Czech Republic AY171249 (CLO cl) Z MraacutecekS feltiae San Bernardino Switzerland AY171247 (CABI cl) SE SpiridonovS feltiae Tomsk Russia AY171273 (CLO cl) SE SpiridonovS feltiae Artybash Republic Altai Russia (isolate 46) AY171272 (CLO cl) SE SpiridonovS feltiae St Louis USA (isolate 88) AY171275 (CLO cl) K Krasomil-OsterfeldS feltiae Japan (isolate MY9) AY238178 (CABI cl) M YoshidaS glaseri USA (type) AY230171 (CABI di) AP ReidS glaseri Azores Portugal AY171288 (CLO di) C SimoesS intermedium Charleston South Carolina USA (topotype) AY230172 (CABI di) R-U EhlersS intermedium St Louis Missouri USA (isolate 82) AY171290 (CLO di) K Krasomil-OsterfeldS karii Type locality Kibingo Kenya AY230173 (CABI cl) AP ReidS kraussei Type locality Westphalia Germany AY230175 (CABI cl) Z MraacutecekS kraussei Artybash Republic Altai Russia (isolate 35) AY171270 (CLO di) SE SpiridonovS kraussei Artybash Republic Altai Russia (isolate 37) AY171271 (CLO di) SE SpiridonovS kraussei UK (isolate Nash) AY230176 (CLO cl) AP ReidS kraussei Italy AY230174 (CLO cl) AP ReidS kraussei Leignon Belgium AY171250 (CLO cl) SE SpiridonovS kraussei Iceland AY171248 (CLO di) M WilsonS kraussei Moscow region Russia AY171264 (CLO di) SE SpiridonovS kraussei Kirkhill Scotland (isolate K5) AY171253 (CLO di) M WilsonS kraussei S Gottard Switzerland (D) AY171258 (CLO di) SE SpiridonovS kraussei UK (isolate B2 site 216) AY230161 (CABI cl) AP ReidS kraussei S Gottard Switzerland (E) AY171259 (CLO di) SE SpiridonovS kraussei Velikiye Luki Pskov region Russia AY171251 (CLO di) SE SpiridonovS longicaudum Type locality China AY230177 (CABI cl) AP Reid

Place of sequencing CABI CLO cl ndash cloning and sequencing di ndash direct sequencing

550 Nematology


Table 1 (Continued)


S monticolum Type locality Mt Chiri Korea AF122017 Nguyen et al (2001)S neocurtillae Type locality La Crosse Florida USA AF122018 Nguyen et al (2001)S oregonense Type locality Oregon USA AY230180 (CABI cl) AP ReidS pakistanense Type locality Karachi Pakistan AY230181 (CABI cl) Shahina et al (2001)S rarum Type locality Rio Cuarto Argentina AY171300 (CLO cl) ME DoucetS riobrave Type locality Texas USA AY230182 (CABI cl) AP ReidS scapterisci Type locality Uruguay AY230183 (CABI cl) KB NguyenS siamkayai Type locality Lahmsok Thailand AF331917 Stock et al (2001)S tami Type locality Cat Tien Vietnam AY171280 (CLO cl) SE SpiridonovS weiseri UK (isolate D1 site 541 of A Reid) AY230167 (CABI cl) Mraacutecek et al (2003)S weiseri Germany (isolate F of D Sturhan) AY171268 (CLO cl) Mraacutecek et al (2003)S weiseri S Gottard Switzerland AY171269 (CLO di) SE SpiridonovSteinernema sp 1 Missouri USA AY171276 (CLO di) K Krasomil-OsterfeldSteinernema sp 2 Germany (species B) AY171255 (CLO cl) D SturhanSteinernema sp 2 UK (isolate B3 site 249) AY230162 (CABI cl) AP ReidSteinernema sp 3 Binh Chau National Park Vietnam AY171286 (CLO di) SE SpiridonovSteinernema sp 4 Krasnaya Poliana Sotchi Russia AY171291 (CLO di) SE SpiridonovSteinernema sp 5 UK (species E1 of A Reid) AY230168 (CABI cl) R GwynnSteinernema sp 5 Rochefort Belgium (species E1) AY171297 (CLO cl) SE SpiridonovSteinernema sp 5 Polva Estonia (isolate E6) AY171294 (CLO di) SE SpiridonovSteinernema sp 5 Zvenigorod Moscow region Russia AY171295 (CLO di) SE SpiridonovSteinernema sp 5 Artybash Republic Altai Russia (isolate 14) AY171299 (CLO di) SE SpiridonovSteinernema sp 6 Pennsylvania USA (isolate 33) AY171287 (CLO cl) K Krasomil-OsterfeldSteinernema sp 7 Polva Estonia (isolate E3) AY171292 (CLO di) SE SpiridonovSteinernema sp 7 Switzerland (isolate CH 221) AY171293 (CLO di) J GrunderSteinernema sp 8 North Carolina USA (isolate NC 513) AY230179 (CABI cl) R-U EhlersSteinernema sp 8 St Louis Missouri USA (isolate 70) AY171284 (CLO di) K Krasomil-OsterfeldSteinernema sp 9 Sri Lanka (isolate SSL1) AY230184 (CABI cl) Amarasinghe et al (1994)Steinernema sp 10 Tichino Switzerland (isolate CH 213) AY171285 (CLO di) J GrunderSteinernema sp 11 India (isolate 72) AY171281 (CLO di) MA AnsariSteinernema sp 12 Kenya (isolate UH36) AY230186 (CABI cl) AP ReidSteinernema sp 13 Pendelton Arkansas USA AY171277 (CLO cl) K Krasomil-OsterfeldSteinernema sp 13 Nebraska USA (isolate KKO 133) AY171278 (CLO cl) K Krasomil-OsterfeldSteinernema sp 14 Pennsylvania USA (isolate 25) AY171252 (CLO cl) K Krasomil-OsterfeldSteinernema sp 15 St Louis Missouri USA (isolate 73) AY171265 (CLO cl) K Krasomil-OsterfeldSteinernema sp 16 Nebraska USA (isolate KKO 122) AY171260 (CLO di) K Krasomil-OsterfeldSteinernema sp 16 Nebraska USA (isolate KKO 123) AY171261 (CLO di) K Krasomil-OsterfeldSteinernema sp 16 Nebraska USA (isolate KKO 125) AY171262 (CLO di) K Krasomil-OsterfeldSteinernema sp 16 Nebraska USA (isolate KKO 126) AY171267 (CLO di) K Krasomil-OsterfeldSteinernema sp 16 Nebraska USA (isolate KKO 137) AY171263 (CLO di) K Krasomil-Osterfeld

Vol 6(4) 2004 551

SE Spiridonov et al

Fig 1 Fragment of the alignment for ITS1 rDNA of 14 Steinernema feltiae isolates and two ITS-rDNA clones from progeny of cross-breeding Three types of ITS sequences are indicated with letters a ndash without deletions b ndash with 6 bp deletion c ndash with 10 bp deletionThe DdeI restriction site is underlined

Fig 2 Restriction fragments (DdeI) of amplified ITS regions ofsingle infective juveniles from two Belgian isolates of Steiner-nema feltiae Lane 1 ndash 100 bp DNA marker Lanes 2 and 3 ndashtwo individuals of S feltiae isolate Va Lanes 4 and 5 ndash two in-dividuals of S feltiae isolate N Lanes 6 and 7 ndash two individualsfrom the N times Va cross Lane 8 ndash unrestricted PCR fragment ofS feltiae isolate N

(285 bp and 274 bp respectively) The length of the58S gene sequence was constant for most isolates (156bp) except for S tami and Steinernema sp 11 (154 bp)Nucleotide base composition was A ndash 245 (212-287)C ndash 171 (125-217) G ndash 227 (174-269) T ndash 358(299-429)

Intra-specific and inter-specific variation

The intra-specific variability of the S feltiae ITSsequences ranged from 0-16 for European populationsand reached 24 between the British (A2) and the

Armenian isolates Within clones three types of ITS1sequences were distinguished with respect to the presenceof indels in positions 150-170 in the S feltiae sequences(a) without deletions (b) with six nucleotide deletionsand (c) with ten nucleotide deletions (Fig 1) The thirdtype can be distinguished from the first by the absence ofDdeI restriction site (Figs 1 2) Both RFLP and sequencesof the ITS region of S feltiae from Belgium revealedthat individuals contained ITS sequences of the third (Vaclone 4) and first type (Va clone 1) Crosses of the Belgianisolate Va containing the two ITS types with anotherBelgian isolate N having only one ITS type were fertileand the progeny contained both ITS types (Fig 2)

The sequence differences between S kraussei isolatesusually varied between 1-11 bp (up to 1) but reached 21bp (28) between the UK (B2) isolate and the Moscowisolate The sequence divergence of S affine ranged from02-06 (2-5 bp) The difference between sequencesof S carpocapsae strains from Europe and USA was 3bp (04) Sequences of the S bicornutum isolate fromKrasnodar (Russia) differed from the topotype isolate(Yugoslavia) by 17 bp or 22

The maximal inter-specific difference in the ITS rDNAsequences was observed between S pakistanense and Sintermedium (424 305 bp) and the minimal differencewas found between Steinernema sp 5 and Steinernemasp 7 isolate (22-25 18-19 bp) It should be notedhowever that Steinernema sp 5 and Steinernema sp 7have yet to be formally described as new species and thusat present our data cannot be used to create molecularcriteria for species differentiation

552 Nematology


Fig 3 Strict consensus of four maximum parsimony trees for the genus Steinernema obtained after analysis of alignments generatedwith default options of Clustal W Bootstrap numbers (50 and more) are given on appropriate clades

Vol 6(4) 2004 553

SE Spiridonov et al

Table 2 Alignment parameters and tree statistics for 48 ITS rDNA sequences of Steinernema

N GOP GEP Alignment Informative Const Tree Tree CI HI RI RC g1length characters characters length number

D 150 666 921 642 148 3373 4 04429 05571 07395 03468 minus054221 10 05 1496 582 520 2362 5 04484 05116 07466 04397 minus052352 20 05 1514 572 668 2263 16 04693 05307 07509 04608 minus052023 40 05 1332 610 498 2554 36 04677 05323 07460 03894 minus049164 70 05 1146 593 347 2743 2 04642 05358 07466 03843 minus050765 150 05 1069 612 256 3110 9 04493 05507 07338 03610 minus053216 300 05 999 637 205 3493 4 04320 05680 07324 03394 minus052097 10 10 1386 576 543 2226 8 04844 05156 07553 04197 minus053218 20 10 1256 592 453 2369 2 04688 05312 07466 03899 minus051689 40 10 1171 603 254 2769 18 04765 05244 07400 04043 minus0518810 70 10 1094 618 298 2790 1 04635 05365 07470 03770 minus0520411 150 10 1049 623 245 3120 4 04527 05473 07302 03362 minus0556012 300 10 974 639 198 3532 6 04334 05666 07363 03625 minus0515213 10 30 1149 615 342 2482 2 04706 05294 07522 03891 minus0528414 20 30 1128 607 326 2568 2 04633 05367 07459 03802 minus0510515 40 30 1066 612 246 2736 1 04607 05393 07416 03738 minus0513816 70 30 1008 615 215 2922 1 04593 05407 07440 03713 minus0544417 150 30 943 636 157 3266 2 04455 05545 07371 03516 minus0546218 300 30 937 635 165 3581 2 04267 05733 07291 03311 minus0507319 10 666 1006 617 234 2709 32 04634 05366 07486 03741 minus0514620 20 666 984 619 215 2789 6 04562 05438 07438 03646 minus0507521 40 666 962 615 188 2917 4 04572 05428 07431 03663 minus0519422 70 666 939 627 175 3091 2 04418 05582 07395 03493 minus0543523 300 666 919 640 154 3696 6 04138 05862 07250 03176 minus0551924 10 90 963 619 190 2842 6 04546 05454 07450 03654 minus0515625 20 90 940 629 178 2933 4 04605 05395 07486 03668 minus0524126 40 90 936 622 177 3051 2 04535 05465 07466 03609 minus0522027 70 90 927 622 166 3165 6 04413 05587 07414 03490 minus0507628 150 90 912 630 153 3395 2 04309 05691 07331 03349 minus0544729 300 90 916 652 144 3770 4 04181 05819 07207 03181 minus05404

N ndash alignment number GOP ndash gap open penalty GEP ndash gap extension penalty CI ndash consistency index HI ndash homoplasy index RI ndashretention index RC ndash rescaled consistency index g1 ndash reliability value (after Hillis amp Huelsenbeck 1992)

MOLECULAR PHYLOGENY

Phylogenetic relationships between main Steinernemagroups

Forty eight steinernematid sequences and one C ele-gans sequence were aligned with different gap lengthand gap open penalties to study general relationships be-tween Steinernema groups (Table 2) MP analyses of 30alignments generated trees containing five main cladesviz Clade I lsquoaffine-intermediumrsquo group (five sequences)Clade II lsquocarpocapsae-scapterisci-tamirsquo group (four se-quences) Clade III lsquofeltiae-kraussei-oregonensersquo group(14 sequences) Clade IV lsquobicornutum-ceratophorum-riobraversquo group (seven sequences) and Clade V lsquoare-

narium-glaseri-karii-longicaudumrsquogroup (14 sequences)The consensus maximum parsimony tree obtained fromone of the alignments (default options) is presented inFigure 3 Strong support for monophyly of Clades I IIand III was evident in all studied alignments Clade IVwas strongly supported in all alignments except two whichreceived moderate support Monophyly of Clade V wasstrongly supported in trees obtained from 24 alignmentswith moderate or weak support in five trees or one treerespectively (Table 3) The composition of Clades I-V ispresented in detail below

The positions of S monticolum S neocurtillae S ra-rum and Steinernema sp 1 depended on the align-ment (Table 3) Steinernema monticolum was a sister for

554 Nematology


Tabl

e3

Posi

tion

san

dbo

otst

rap

supp

ort

ofse

lect

edcl

ades

and

spec

ies

inth

eM

Ptr

ees

obta

ined

from

the

gene

rala

naly

sis

ofS

tein

erne

ma

sequ

ence

s

Pos

itio

nal

ignm

ent

D1

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

Mon

ophy

lyof

Cla

deI

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

IS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

IVS

MS

SS

SS

MS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

VS

WM

SS

SS

SM

SS

SS

SM

SS

SS

MS

MS

SS

SS

SS

SSt

eine

rnem

asp

p14

15

16+

Sfe

ltia

eM

MM

MM

WW

WM

MM

WW

WW

and

Sw

eise

ricl

ade

Bas

alpo

siti

onof

Stei

nern

ema

spp

14

SM

SM

SW

SW

WS

SW

SS

S15

16

subc

lade

wit

hin

Cla

deII

IS

oreg

onen

seas

asi

ster

toS

krau

ssei

SM

MW

SW

SS

SS

MM

MM

SM

MS

SM

MS

MW

WW

MM

SM

+St

eine

rnem

asp

2S

mon

tico

lum

asa

sist

erto

Cla

deII

IM

WW

MM

MM

WM

SM

WW

MW

MS

MM

WW

SM

Sm

onti

colu

mno

tasi

ster

toC

lade

III

WW

WW

WW

W(p

osit

ione

dne

arot

her

clad

es)

Stei

nern

ema

sp1

asa

sist

erto

MW

WM

MM

WW

WW

MW

WW

WM

MW

MM

MS

SS

mon

tico

lum

+C

lade

III

Stei

nern

ema

sp1

asa

sist

erto

WW

WW

WW

WC

lade

III

wit

hS

mon

tico

lum

outs

ide

this

grou

ping

Bas

alpo

siti

onof

the

Ska

riia

ndS

WW

SS

WW

MW

WW

WW

SM

WW

SM

SM

MW

MM

WS

Stei

nern

ema

spp

912

subc

lade

wit

hin

Cla

deV

Bas

alpo

siti

onof

Sgl

aser

i+

WW

WS

cuba

num

wit

hin

Cla

deV

Spa

kist

anen

seas

asi

ster

toS

riob

rave

SM

WM

WM

MM

WM

SM

SM

MM

WM

WS

MW

WM

WS

MS

MS

+St

eine

rnem

asp

13

Bas

alpo

siti

onof

the

Sbi

corn

utum

SS

SS

SS

SS

SS

SS

SS

SS

SS

S+

Sce

rato

phor

umsu

bcla

dew

ithi

nC

lade

IVB

asal

posi

tion

ofSt

eine

rnem

aab

basi

WW

MS

SS

SS

SS

Sw

ithi

nC

lade

IVS

raru

mas

asi

ster

toC

lade

VW

WW

WW

MW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

Sne

ocur

till

aeas

asi

ster

toS

mon

tico

lum

WW

WW

WW

WW

WW

WM

W+

Stei

nern

ema

sp1

+C

lade

III

Sne

ocur

till

aeas

asi

ster

toC

lade

IIW

WW

WS

neoc

urti

llae

rela

ted

tode

eper

node

sW

WW

WW

WW

WW

WW

ofth

eSt

eine

rnem

aph

ylog

eny

tree

Boo

tstr

apsu

ppor

t(

)fo

rcl

ades

Wlt

70(w

eak)

Mndash

70-9

0(m

oder

ate)

Sgt

90(s

tron

g)

Vol 6(4) 2004 555

SE Spiridonov et al

Fig 4 Strict consensus of four maximum parsimony treesfor Clade I (lsquoaffine-intermediumrsquo) of the genus SteinernemaBootstrap numbers (50 and more) and nucleotide changes aregiven on appropriate clades

Clade III in trees generated from 23 alignments In threecases S monticolum was a sister for the group composedof Clade III species and Steinernema sp 1 In most caseshowever the inverse situation was observed when Stei-nernema sp 1 was a sister for the group formed by theClade III species together with S monticolum The posi-tion of S neocurtillae was uncertain ndash in MP trees ob-tained from 13 alignments S neocurtillae clustered withthe above-mentioned group (Steinernema sp 1 + S mon-ticolum + Clade III) in trees from four alignments it clus-tered with Clade II and in the remaining trees it was ina basal or sub-basal position The bootstrap support forthese positions was always weak The sister taxon statusof S rarum for Clade V was evident in the majority of thealignments but always with low bootstrap support (Ta-ble 3)

Relationships within Steinernema clades

Clade I ndash lsquoaffine-intermediumrsquo The alignment usedfor the analyses comprised 16 sequences along withthose from S rarum and S neocurtillae (outgroups)and contained 825 bp MP analysis revealed four equaltrees of which the strict consensus tree is presented inFigure 4 Bootstrap support for the basal position of twoS intermedium isolates from the USA was very strong

Fig 5 Strict consensus of two maximum parsimony trees forClade II (lsquocarpocapsae-scapterisci-tamirsquo) of the genus Stei-nernema Bootstrap numbers (50 and more) and nucleotidechanges are given on appropriate clades

Relationships between S affine and three putative newspecies (Steinernema spp 4 5 7) were not resolved

Clade II ndash lsquocarpocapsae-scapterisci-tamirsquo For theanalysis of this clade nine sequences were comparedin a 775 bp-alignment S rarum and S monticolumbeing selected as the outgroups MP analysis revealedtwo equal trees the strict consensus being presented inFigure 5 Steinernema scapterisci was always in the basalposition Steinernema carpocapsae was a sister to theclade composed of three isolates originating from tropicalor subtropical regions S siamkayai Steinernema sp 11and S tami

Clade III ndash lsquofeltiae-kraussei-oregonensersquo Sequences of42 isolates were included in the phylogenetic analysis ofthis clade Steinernema monticolum and Steinernema sp 1were chosen as the outgroup for the clade as both speciesusually clustered with this clade in the trees obtained inthe general analyses The length of this alignment was 825bp MP analysis revealed 100 maximum parsimony treesof which the strict consensus is presented in Figure 6 Themonophyly of the clade was clearly supported as well asits subdivision into the three main subclades i) S feltiaeand Steinernema weiseri ii) S kraussei Steinernema sp2 and S oregonense and iii) Steinernema spp 14 15 and16 from the USA

556 Nematology


Fig 6 Strict consensus of 100 maximum parsimony trees for Clade III (lsquofeltiae-kraussei-oregonensersquo) of the genus SteinernemaBootstrap numbers (50 and more) and nucleotide changes are given on appropriate clades

Vol 6(4) 2004 557

SE Spiridonov et al

Fig 7 Strict consensus of four maximum parsimony trees forClade IV (lsquobicornutum-ceratophorum-riobraversquo) of the genusSteinernema Bootstrap numbers (50 and more) and nu-cleotide changes are given on appropriate clades

Clade IV ndash lsquobicornutum-ceratophorum-riobraversquo Se-quences of eight isolates and two outgroups (S rarum andS intermedium) were used for the analysis The lengthof this alignment was 840 bp The single maximum par-simony tree (Fig 7) revealed strong bootstrap support forsister relationships of S bicornutum and S ceratophorumand for S riobrave and Steinernema sp 13

Clade V ndash lsquoarenarium-glaseri-karii-longicaudumrsquoSeventeen sequences were aligned for the analysis of thisclade (alignment length 836 bp outgroups S rarum andS neocurtillae) A single maximum parsimony tree wasobtained (Fig 8) The bootstrap support for monophyly ofthe entire clade was strong The topology of the tree coin-cided with the one presented in the general analyses butdiffered with respect to the relationship between S arena-rium and Steinernema sp 10 (Switzerland) and betweenS diaprepesi and the clade formed by S longicaudum andSteinernema sp 3 (Fig 8) The sequence of the Azoreanpopulation of S glaseri was identical with the topotypeisolate from the USA The lsquotropicalrsquo clade comprising Skarii Steinernema sp 9 (Sri Lanka) and Steinernema sp12 (Kenya) occupied a basal position with strong support

Fig 8 Strict consensus of four maximum parsimony trees forClade V (lsquoarenarium-glaseri-karii-longicaudumrsquo) of the genusSteinernema Bootstrap numbers (50 and more) and nu-cleotide changes are given on appropriate clades

PHYLOGENETIC RELATIONSHIPS BETWEEN SPECIES

AS INFERRED FROM MORPHOLOGICAL AND

COMBINED DATA

Stock et al (2001) included 22 morphological andmorphometrical characters with sequence data from theD2-D3 region of the LSU rDNA and concluded thatthe majority of these were homoplastic We re-evaluatedthe characters of their matrix as follows all highlyhomoplastic qualitative characters were removed and newstates for three qualitative characters were proposednew ranges (borders between states) were given for thethree quantitative features and three new morphologicalcharacters (spermatozoon structure presence of bacterialvesicle and coloration of copulatory apparatus) wereadded

A light microscopy study revealed lateral horn-like pro-jections on the anterior end of infective juveniles of twoisolates of Steinernema sp 13 from USA These featureswere not mentioned in the original descriptions of S ri-obrave (Cabanillas et al 1994) or S abbasi (Elawad etal 1997) although their presence was recently reportedin both species by Nguyen and Adams (2003) A total ofsix qualitative and three quantitative features were used toestimate the relationships between the isolates (Table 4)

558 Nematology


Fig 9 Strict consensus of three trees obtained from maximum parsimony analysis of combined morphological and molecular datasetThe morphological character states are indicated on appropriate branches (state lsquo0rsquo ndash 1 state lsquo1rsquo ndash 2 and state lsquo2rsquo ndash ) and designatedwith numbers corresponding to Table 4

Vol 6(4) 2004 559

SE Spiridonov et al

Table 4 Morphological character states for Steinernemaspecies

SpeciesState 1 2 3 4 5 6 7 8 9

Steinernema abbasi 0 0 0 1 0 1 1 0 0Steinernema affine 2 1 0 0 1 0 1 2 1Steinernema arenarium 0 0 1 1 1 1 0 1 1Steinernema bicornutum 0 0 0 1 0 1 1 2 1Steinernema carpocapsae 0 2 0 1 1 1 1 0 0Steinernema ceratophorum 0 0 0 1 0 1 1 2 1Steinernema cubanum 0 0 1 1 1 1 0 1 1Steinernema feltiae 1 2 0 1 1 1 1 2 1Steinernema glaseri 0 0 1 1 1 1 0 1 1Steinernema intermedium 2 1 0 0 1 0 1 2 1Steinernema karii 0 0 1 1 1 1 0 2 1Steinernema kraussei 1 2 0 1 1 1 1 2 1Steinernema longicaudum 0 0 1 1 1 1 0 1 1Steinernema oregonense 0 1 1 1 1 2 1Steinernema pakistanense 0 0 1 0 1 1 2 0Steinernema rarum 1 0 0 1 1 1 1 0 0Steinernema riobrave 0 0 0 1 0 1 1 2 0Steinernema scapterisci 0 2 0 1 1 1 1 0 0Steinernema tami 0 2 0 1 1 1 1 0 0Steinernema weiseri 1 0 0 1 1 1 1 2 0Steinernema sp 1 1 2 0 1 1 1 1 2 1Steinernema sp 2 1 0 0 1 1 1 1 2 1Steinernema sp 3 0 0 1 1 1 1 0 1 1Steinernema sp 4 2 1 0 0 1 0 1 2 1Steinernema sp 5 2 1 0 0 1 0 1 2 1Steinernema sp 6 0 0 1 1 1 1 0 1 1Steinernema sp 7 2 1 0 0 1 0 1 2 1Steinernema sp 8 0 0 1 1 1 1 0 1 1Steinernema sp 9 0 0 1 1 1 1 0 1 1Steinernema sp 10 0 0 1 1 1 1 0 1 1Steinernema sp 11 0 2 0 1 1 1 1 0 0Steinernema sp 13 0 0 0 1 0 1 1 2 0Steinernema sp 14 1 0 0 1 1 1 1 2 1Steinernema sp 15 1 0 0 1 1 1 1 2 1Steinernema sp 16 1 0 0 1 1 1 1 2 1

1 Giant amoeboid cells ndash transformed sperm in female oviductsndash (0) Sperm cells in females forming chains each cell about5-7 microm ndash (1) Sperm cells in females forming chains each cellabout 10-12 microm ndash (2)2 Lateral field of infective juveniles with eight or six equal ridgesndash (0) lateral field of infective juvenile with two central ridgesand less prominent marginal or submarginal lines ndash (1) lateralfield of infective juvenile with three or four pronounced ridgesin the centre with two prominent marginal and less prominentsubmarginal ridges ndash (2)3 Bacterial vesicle well defined with visible walls ndash (0) no clearwalls of bacterial vesicle ndash (1)

The partition homogeneity test indicated substantialcongruence between morphological and molecular data(P = 06) and the two datasets were combined Maxi-mum parsimony analysis generated a single tree (Fig 9)Inclusion of nine morphological and morphometricalcharacters did not influence the relationships between thespecies (data not shown)

Discussion

MOLECULAR AND MORPHOLOGICAL CHARACTERS IN

STEINERNEMATID SPECIES DELIMITATION

Traditionally species delimitation in steinernematidshas been based on morphology and cross-breeding experi-ments (Hominick et al 1997) The availability of molecu-lar data presents a new basis for estimating species bound-aries in this group Adams (1998) considered several par-adigms for species delimitation in nematology and con-cluded that the presence of autapomorphy is the main pre-requisite for the establishment of a new species Stock etal (2001) noted the absence of autapomorphies for fourspecies during the analysis of the LSU domain for Stei-nernema However these were readily found from theITS rDNA analysis of some of these species Our ITSrDNA analysis of a richer set revealed two species (Skarii and S siamkayai) lacking autapomorphies in this do-main It can be expected that reported autapomorphies forsome well established species may disappear as the num-ber of species studied increases In this case the search

4 Spicules and gubernaculum nearly transparent or of slightyellowish coloration ndash (0) spicules and gubernaculum nottransparent well coloured ndash (1)5 Two lateral horns present on both sides of closed mouth ininfective juveniles ndash (0) head end of infective juveniles withoutlateral horns ndash (1)6 Refractive inclusion present in tail tip of some infectivejuveniles in population ndash (0) no refractive inclusion in thejuvenile tail tip ndash (1)7 Infective juvenile excretory pore opening at level of posteriorhalf of pharynx (ie mean value for D gt 55) ndash (0) Excretorypore may be situated on either sides of mid-part or at level ofanterior half of pharynx ndash (1)8 Average infective juvenile body length not exceeding 600 microm ndash(0) more than 1000 microm ndash (1) infective juveniles of intermediatesize ndash 600-1000 microm (2)9 Tail of infective juveniles shorter than 61-62 microm ndash (0) tail ofjuveniles longer than 61-62 microm ndash (1)

560 Nematology


for autapomorphies as an approach to species delimitationwould no longer appear to be a sound procedure

Alternatively sequence divergence might be consideredas an indication for independent evolutionary history insteinernematids In our analysis we revealed different lev-els of intra-specific sequence divergence in Steinernemaclades ranging from 03-06 in Clade I (S intermediumS affine Steinernema sp 5) and up to 24-28 in CladeIII (S feltiae and S kraussei) The difference betweenclosely related species was 24-29 in only two casesand was in excess of 3 for the others

One of the explanations for different levels of intra-specific sequence divergence is the differences in rate ofITS evolution in steinernematids groups It is known thathigher mutation rates occur in the loops than in stems ofrRNA (Dixon amp Hillis 1993) and that differently struc-tured ITS molecules could diverge at different rates Forexample sequence analyses showed that both S feltiaeand S kraussei species with the highest intraspecific di-vergence in our analysis have nucleotide positions withhigh level of substitutions in few loci along the ITS prob-ably in the loops Perhaps these species have a similar sec-ondary RNA structure facilitating the nucleotide changesand thus increasing intra-specific divergence

Hillis and Dixon (1991) suggested that homogenisationmechanisms tend to lower the level of rDNA polymor-phism in a population and the authors hypothesised the ex-istence of lsquoconcerted evolutionrsquo as a result of such mech-anisms These mechanisms however can be disturbedby intracellular factors leading to heterogeneity Hetero-geneity was clearly visualised by the multiple peaks dur-ing direct sequencing of PCR products of both S feltiaeand S bicornutum High levels of intra-specific sequencedivergence were observed in both species If one assumesthat such homogenisation mechanisms would somehowbe less active in these steinernematid species their higherlevel of sequence divergence could then be explainedHowever we did not detect any ITS rDNA heterogene-ity for S kraussei another species with high intraspecificdivergence It is probable that multiple factors may in-fluence the level of intraspecific divergence in steinerne-matids and we acknowledge that the different levels of in-traspecific sequence divergence reported herein may be anartefact caused by the varying number and geographicalspread of isolates within different species

The levels of intra- and inter-specific divergence wereanalysed together with morphological and ecological dataThe results suggested that 27 of the isolates of putativespecies belonged to 15 new species and that only one

isolate Steinernema sp 11 was probably conspecific witha nominal species S siamkayai as a very low level ofsequence divergence (12) was observed between thetwo isolates Study of the RFLP of this isolate revealedits similarity with an undescribed isolate from Sri Lanka(Hussaini et al 2001) which was considered conspecificwith the recently described S asiaticum (Anis et al2002) The proposed differential diagnosis of this speciesis not decisive and additional studies are needed toelucidate its status and its relationships with S siamkayaiand Steinernema sp 11 Our molecular data revealedthat some isolates of species that are currently identifiedon the basis of their morphological similarity as beingconspecific can be separated into several species Forexample our analyses suggest that lsquoS intermediumrsquo-likeisolates from Europe do not belong to S intermedium(USA topotype) but to three as yet undescribed species(Steinernema spp 4 5 7)

PHYLOGENY OF THE GENUS STEINERNEMA

Our comprehensive analyses of the phylogeny basedon the ITS region provides a new insight into the rela-tionships between groups and species of steinernematidsThis study supports the same evolutionary lines previ-ously found by Stock et al (2001) on the basis of thepartially sequenced 28S rDNA gene Five main cladesare observed in both analyses However relationships be-tween the clades are not clear when using ITS sequences(Nguyen et al 2001 present study)

Clearly some of our results differ from previous stud-ies In most of our trees the basal position of Clade I(lsquoaffine-intermediumrsquo-group) was evident whereas theanalysis of the very conservative 58S rDNA by Nguyenet al (2001) lsquotenuously but consistentlyrsquo placed S inter-medium in the basal position for the genus In the phy-lograms of Stock et al (2001) this basal position is oc-cupied by the group S carpocapsae + S scapterisci +S siamkayai and S monticolum

A contradiction between our results and published phy-lograms was also found in the position of S monticolumIn the LSU tree of Stock et al (2001) and in some max-imum parsimony trees for ITS by Nguyen et al (2001)this species clustered with S carpocapsae + S scapterisci+ S siamkayai In our phylogenetic trees S monticolumclustered mainly with Clade II (lsquofeltiae-krausseirsquo group)with weak to strong bootstrap support (Table 3) Basedon its morphometric features such as the infective juve-nile body length of 612-821 microm (Stock et al 1997) thisspecies fits into the group of steinernematids with lsquome-

Vol 6(4) 2004 561

SE Spiridonov et al

dium sizedrsquo juveniles (lsquofeltiae-kraussei-oregonensersquo) butnot into the lsquocarpocapsae-scapterisci-tamirsquo group withshort juveniles

Another difference between the LSU analysis and ourdata is the position of two populations identified asS longicaudum by Stock et al (2001) In the LSU treethe Chinese population of S lsquolongicaudumrsquo clusteredwith S cubanum whereas the American S lsquolongicaudumrsquopopulation was a sister taxon of S karii In our analysesafter excluding several species from the alignment (datanot shown) the topotype Chinese population of S longi-caudum was always related to S karii whereas Steiner-nema sp 8 was a sister group for S cubanum Such aclustering appears to be logical from the biogeographicalpoint of view (Fig 8) It is possible that the two S lsquolongi-caudumrsquo isolates studied by Stock et al (2001) may havebeen mislabelled and that one of the Californian isolatesmay be identical to our Steinernema sp 8 which wascollected from North Carolina and Missouri

MORPHOLOGICAL BASIS FOR STEINERNEMATID

PHYLOGENY

In the analysis by Stock et al (2001) only three char-acters were not homoplastic viz two autapomorphies inS glaseri and S affine (presenceabsence of notch on thespicular lamina and refractive inclusion in tail of infectivejuveniles respectively) and one synapomorphy for S bi-cornutum and S ceratophorum (presence of infective ju-venile lateral projection or horn-like structures) In addi-tion our study showed the highest consistency index (CI= 10) for the following morphological features structureof bacterial vesicles colour of male copulatory apparatusand position of the excretory pore A CI of 067 was ob-served for sperm structure

Spermatozoon morphology is proposed for the firsttime in the reconstruction of steinernematid phylogeny Itwas previously shown that spermatozoa in Steinernemaare diverse (Spiridonov et al 1999) The formationof chains of several steinernematid spermatozoa wasreported by Bovien (1937) and a rich diversity of giantamoeboid cells (transformed sperm) was found betweenSteinernema species Species of Clade I and Clade IIIare characterised by chain-forming spermatozoa gianttransformed sperm only being reported in representativesof Clades II IV and V Features of the sperm may be easilydiscovered during examination of living females and theentire character set of features related to steinernematidspermatozoa is an attractive addition to the analysis

The presence of lateral projections or horn-like struc-tures on the cephalic end of the infective juvenile wassynapomorphic for Clade IV and the character of slightlyyellowish or colourless spicules was synapomorphic forClade I

In our matrix differences in lateral field morphologyof infective juveniles were grouped into states similarto but distinct from those used by Stock et al (2001)The consistency index was still low (04) for this featurethe variability of juvenile lateral field structure in someclades probably being responsible for this Several cladesare characterised by uniform structure of the lateral fieldspecies in Clade I have a lateral field with two ridges inthe centre in Clade II species have four central ridgesand pronounced marginal ridges but weakly developedsubmarginal ridges in Clades IV and V species have eightequal ridges (which appear as nine lines with equal spacesbetween) Different types of lateral field were reportedfor Clade III S kraussei and S feltiae are characterisedby a lateral field with three or four central ridges plusprominent marginal and weakly developed submarginalones whereas some other species of this clade have alateral field with either eight or six (as in S oregonense)equal ridges (Table 4)

The presence of the bacterial vesicle is a stable charac-teristic for steinernematid species (Bird amp Akhurst 1983)In our analysis the CI value for this feature equals 10Representatives of Clade I (lsquoaffine-intermediumrsquo group)have swollen thin-walled vesicles with a large innerspace and fill the body diameter in mature juvenilesSpecies of Clade III (lsquofeltiae-kraussei-oregonensersquo group)are characterised by more compact and thick-walled vesi-cles which usually do not impact upon the general shapeof the intestine Nevertheless the latter type of vesicleis not synapomorphic for this clade as similar thoughsmaller and usually rounded bacterial vesicles are com-mon for the species of Clades II and IV Similar vesicleswere observed in S rarum A true vesicle is probably ab-sent in the species composing Clade V Further research isneeded to validate the use of detailed vesicle morphologyin the phylogenetic analysis of Steinernema

ECOLOGICAL AND GEOGRAPHICAL PATTERNS IN

STEINERNEMA PHYLOGENY

Recently general patterns of natural steinernematid dis-tribution were discussed by Hominick (2002) who re-viewed the available often contradictory informationThe distribution study of Steinernema by Sturhan (1999)which was based on several hundred soil samples col-

562 Nematology


lected throughout Germany revealed that some specieshave habitat preferences (eg S affine is a species char-acteristic of grasslands and arable soil) The habitat pref-erences of steinernematids are presumably largely deter-mined by the distribution of their host insects (Peters1996) and other factors including soil type and temper-ature As insects correspond in their distribution to plantassociations habitat preference of steinernematids mayalso be expected Some cases of congruence between eco-logical features and the genetic relationships of stein-ernematids became obvious during mapping of our dataSpecies of Clade I are divided into several groups eachwith characteristic habitat preferences (Fig 4) S affine isa species of open landscapes viz grassland or cultivatedsoil whereas S intermedium (USA) and Steinernema sp5 (Europe) appear to be inhabitants of woodlands (Spiri-donov amp Moens 1999) This result contradicts that ofKramer et al (2000) who reported that S intermedium isa common inhabitant of grasslands in Swiss lowlands Theidentification of this species however was based on mor-phological characters only and the conspecific status ofthis Swiss isolate with the North American S intermediumwas not evident Sequence analysis of one of these Swissisolates (CH 221) revealed its similarity to Steinernemasp 7 from Estonia This latter isolate was only found ingrasslands of Southern Estonia whereas Steinernema sp5 (isolate E6) was common under the canopy of deciduousand mixed forests in the same area

Habitat preference patterns were also observed inClade III (Fig 6) All of our S feltiae isolates originatedfrom grasslands or cultivated soil though Sturhan (1999)also reported it from woodland Steinernema kraussei iso-lates in our collection were mainly obtained from wood-lands However S kraussei was also reported from alpinegrasslands in Switzerland (Steiner 1996) and Scotland(Gwynn amp Richardson 1996) and from alpine meadowsin Bulgaria (Shishinova et al 2000) The Iceland iso-late of S kraussei was obtained from a swampy treelessarea in a volcanic valley The distribution of S kraussei inwoodland habitats is almost a rule in lowland parts of Eu-rope although this species can also be commonly foundoutside woodlands at high altitudes and latitudes

An attempt was recently made to analyse the geograph-ical distribution of steinernematids (Hominick 2002)Any such estimation based on morphological identifica-tion only might be risky because of the difficulties in ac-curately identifying Steinernema species Kerry and Ho-minick (2002) indicated that accurate identification is a

key factor in understanding of geographical distributionand biodiversity of steinernematids

Several geographical patterns of steinernematids canbe observed from the data presented here Some speciesare found throughout the temperate belt of the northernhemisphere Hence S feltiae from the USA (isolate 88) isidentical in its ITS sequence to the topotype isolate fromEurope However this must be interpreted with the caveatthat the distribution could be the result of introductionevents

Steinernematids quite often form groups of closelyrelated isolates with a specific geographical area ofdistribution Separate clades for North American andEurasian species are obvious in Clades I III and IVwith some level of independence of subtropical andtropical species from those inhabiting temperate regionsin Clades II and V

Conclusions

The analysed set of sequence data from the ITS rDNAregion of steinernematids provided new information con-cerning the composition of five main clades in the genusSteinernema and the phylogenetic relationships insidethese main steinernematid evolutionary lineages ITSrDNA data were confirmed to be of little value for resolv-ing relationships between clades something previouslysuggested by Nguyen et al (2001) The level of intra-specific variability differs between clades reaching 25in S feltiae These different levels of variability should betaken into account when delimitating new steinernematidspecies The morphology of steinernematids adds littledata for the reconstruction of their phylogeny but can sup-port decisions about species independence when the ob-served differences in ITS rDNA nucleotides are close tothe intra-specific level (eg Steinernema weiseri and Stei-nernema sp 2 vs S feltiae and S kraussei respectively)Although original data about habitat preferences of stein-ernematids are sometimes contradictory ndash and this phe-nomenon can be latitude- or altitude-dependent ndash the datasuggest some preferences for specific ecosystems betweenspecies of Clades I and III Biogeographical patterns areapparent in the phylogeny of some clades

Acknowledgements

The authors are grateful to all colleagues who presentedtheir steinernematid isolates for molecular and morpho-

Vol 6(4) 2004 563

SE Spiridonov et al

logical examination We would like to thank two anony-mous reviewers for their helpful comments This workwas made possible through the support of the Russianco-authors by the Agricultural Research Centre in Merel-beke Belgium The work in Russia was supported byRFBR grant 02-04-48389

References

ADAMS BJ (1998) Species concept and the evolutionaryparadigm in modern nematology Journal of Nematology 301-21

AKHURST RJ amp BEDDING RA (1978) A simple cross-breeding technique to facilitate species determination in thegenus Neoaplectana Nematologica 24 328-330

AMARASINGHE LD HOMINICK WM BRISCOE BRamp REID AP (1994) Occurrence and distribution of ento-mopathogenic nematodes (Rhabditida Heterorhabditidae andSteinernematidae) in Sri Lanka Journal of Helminthology 6877-86

ANIS M SHAHINA F REID AP amp ROWE J (2002)Steinernema asiaticum sp n (Rhabditida Steinernematidae)from Pakistan International Journal of Nematology 12 220-231

ARTYUKHOVSKY AK KOZODOI EM REID AP ampSPIRIDONOV SE (1997) Redescription of Steinernemaarenarium (Artyukhovsky 1967) topotypes from CentralRussia and a proposal for S anomalae (Kozodoi 1984) asa junior synonym Russian Journal of Nematology 5 31-37

BEDDING RA amp AKHURST RJ (1975) A simple techniquefor the detection of insect parasitic rhabditid nematodes insoil Nematologica 21 109-110

BIRD AF amp AKHURST RJ (1983) Bacterial vesicle in thenematodes of the genus Steinernema (Rhabditida Steinerne-matidae) International Journal of Parasitology 13 599-606

BLAXTER ML DE LEY P GAREY JR LIU LXSCHELDEMANN P VIERSTRAETE A VANFLETTERENJR MACKEY LY DORRIS M FRISSE LM VIDAJT amp THOMAS WK (1998) A molecular evolutionaryframework for the phylum Nematoda Nature 392 71-75

BOVIEN P (1937) Some types of association between ne-matodes and insects Videnskabelige Meddelelser fra DanskNaturhistorisk Forening Kobenhavn 101 1-114

CABANILLAS HE POINAR JR GO amp RAULSTON JR(1994) Steinernema riobravis n sp (Rhabditida Steinerne-matidae) from Texas Fundamental and Applied Nematology17 123-131

DIXON MT amp HILLIS DM (1993) Ribosomal RNA sec-ondary structure compensatory mutations and implicationsfor phylogenetic analysis Molecular Biology and Evolution10 256-267

ELAWAD S AHMAD W amp REID AP (1997) Steinernemaabbasi sp n (Nematoda Steinernematidae) from the Sul-

tanate of Oman Fundamental and Applied Nematology 20433-442

FELSENSTEIN J (1985) Confidence limits on phylogenies anapproach using the bootstrap Evolution 39 783-791

GWYNN RL amp RICHARDSON PN (1996) Incidence ofentomopathogenic nematodes in soil samples collected fromScotland England and Wales Fundamental and AppliedNematology 19 427-431

HILLIS DM amp DIXON MT (1991) Ribosomal DNA mole-cular evolution and phylogenetic inference The QuarterlyReview of Biology 66 411-428

HILLIS DM amp HUELSENBECK JP (1992) Signal noiseand reliability in molecular phylogenetic analyses Journal ofHeredity 83 189-195

HOMINICK WM (2002) Biogeography In Gaugler R (Ed)Entomopathogenic nematology Wallingford UK CABI Pub-lishing pp 115-144

HOMINICK WM amp BRISCOE BR (1990) Occurrence ofentomopathogenic nematodes (Rhabditida Steinernematidaeand Heterorhabditidae) in British soils Parasitology 100295-302

HOMINICK WM BRISCOE BR DEL PINO FGJIAN HENG HUNT DJ KOZODOY E MRAacuteCEK ZNGUYEN KB REID AP SPIRIDONOV SE STOCKP STURHAN D WATURU C amp YOSHIDA M (1997)Biosystematics of entomopathogenic nematodes current sta-tus protocols and definitions Journal of Helminthology 71271-298

HUSSAINI SS ANSARI MA AHMAD W amp SUBBOTINSA (2001) Identification of some Indian populations ofSteinernema species (Nematoda) by RFLP analysis of the ITSregion of rDNA International Journal of Nematology 11 73-76

JOYCE SA REID A DRIVER F amp CURRAN J (1994)Application of polymerase chain reaction (PCR) methods tothe identification of entomopathogenic nematodes In Bur-nell AM Ehlers R-U amp Masson J-P (Eds) COST 812Biotechnology Genetics of entomopathogenic nematodes-bacterium complexes Proceedings of symposium and work-shop St Patrickrsquos College Maynooth County Kildare Ire-land Luxembourg European Commission DGXII pp 178-187

KERRY BR amp HOMINICK WM (2002) Biological controlIn Lee DL (Ed) The biology of nematodes London amp NewYork Taylor amp Francis pp 483-510

KOZODOI EM amp SPIRIDONOV SE (1988) Cuticular ridgeson lateral fields of larvae of Neoaplectana Folia Parasitolog-ica 35 359-362

KRAMER I HIRSCHY O amp GRUNDER JM (2000) Sur-vey of baited insect parasitic nematodes from Swiss lowlandIn Griffin CT Burnell AM Downes MJ amp Mulder R(Eds) COST 819 Developments in entomopathogenic ne-matodebacterial research Luxembourg European Commis-sion DGXII pp 172-176

564 Nematology


LIU J amp BERRY RE (1996) Phylogenetic analysis of thegenus Steinernema by morphological characters and ran-domly amplified polymorphic DNA fragments Fundamentaland Applied Nematology 19 463-469

LIU J BERRY RE amp MOLDENKE AF (1997) Phyloge-netic relationships of entomopathogenic nematodes (Hete-rorhabditidae and Steinernematidae) inferred from partial 18SrRNA gene sequences Journal of Invertebrate Pathology 69246-252

MIDUTURI J WAEYENBERGE L amp MOENS M (1997)Natural distribution of entomopathogenic nematodes (Hete-rorhabditidae and Steinernematidae) in Belgian soils RussianJournal of Nematology 5 155-165

MRAacuteCEK Z STURHAN D amp REID A (2003) Steinernemaweiseri n sp (Rhabditida Steinernematidae) a new ento-mopathogenic nematode from Europe Systematic Parasito-logy 56 37-47

NGUYEN KB amp ADAMS BJ (2003) SEM and systematicstudies on Steinernema abbasi Elawad et al 1997 andSteinernema riobrave Cabanillas et al 1994 Zootaxa 1791-10

NGUYEN KB amp DUNCAN LW (2002) Steinernema di-aprepesi n sp (Rhabditida Steinernematidae) a parasite ofthe citrus root weevil Diaprepes abbreviatus (L) (ColeopteraCurculionidae) Journal of Nematology 34 159-170

NGUYEN KB amp SMART GC JR (1996) Identification of en-tomopathogenic nematodes in the Steinernematidae and He-terorhabditidae (Nemata Rhabditida) Journal of Nematology28 286-300

NGUYEN KB MARUNIAK J amp ADAMS BJ (2001)Diagnostic and phylogenetic utility of the rDNA internaltranscribed spacer sequences of Steinernema Journal ofNematology 33 73-82

PAGE RDM (1996) TREEVIEW an application to viewphylogenetic trees on personal computer CABIOS 12 357-358

PETERS A (1996) The natural host range of Steinernema andHeterorhabditis spp and their impact on insect populationBiocontrol Science and Technology 6 389-402

PHAM VL NGUYEN BK REID AP amp SPIRIDONOVSE (2000) Steinernema tami sp n (Rhabditida Steinerne-matidae) from Cat Tien forest Vietnam Russian Journal ofNematology 8 33-43

PHAN KL NGUYEN NC amp MOENS M (2001a) Steiner-nema sangi sp n (Rhabditida Steinernematidae) from Viet-nam Russian Journal of Nematology 9 1-17

PHAN KL NGUYEN NC amp MOENS M (2001b) Steiner-nema loci sp n and Steinernema thanhi sp n (RhabditidaSteinernematidae) from Vietnam Nematology 3 503-514

POINAR JR GO (1993) Origins and phylogenetic relation-ships of the entomophilic rhabditids Heterorhabditis and Stei-nernema Fundamental and Applied Nematology 16 333-338

REID AP (1994) Molecular taxonomy of SteinernemaIn Burnell AM Ehlers R-U amp Masson J-P (Eds)COST 812 Biotechnology Genetics of entomopathogenicnematodes-bacterium complexes Proceedings of symposiumand workshop St Patrickrsquos College Maynooth County Kil-dare Ireland Luxembourg European Commission DG XIIpp 49-58

REID AP amp HOMINICK WM (1992) Restriction fragmentlength polymorphisms within the ribosomal DNA repeatunit of British entomopathogenic nematodes (RhabditidaSteinernematidae) Parasitology 105 317-323

REID AP HOMINICK WM amp BRISCOE BR (1997)Molecular taxonomy and phylogeny of entomopathogenicnematode species (Rhabditida Steinernematidae) by RFLPanalysis of the ITS region of the ribosomal DNA repeat unitSystematic Parasitology 37 187-193

SHAHINA F ANIS M REID AP ROWE J amp MAQBOOLMA (2001) Steinernema pakistanense sp n (RhabditidaSteinernematidae) from Pakistan International Journal ofNematology 11 124-133

SHISHINOVA M BUDUROVA L amp GRADINAROV D(2000) Entomopathogenic nematodes from Steinernematidaeand Heterorhabditidae (Nematoda Rhabditida) in BulgariaIOBC Bulletin 23 75-78

SPIRIDONOV SE amp BELOSTOTSKAYA FN (1983) [Newdata about morphology and biology of the nematodes ofthe genus Neoaplectana and their position in the system ofrhabditids] Parasitologyia 17 119-125

SPIRIDONOV SE amp MOENS M (1999) Two previouslyunreported species of steinernematids from woodlands inBelgium Russian Journal of Nematology 7 39-42

SPIRIDONOV SE HOMINICK WM amp BRISCOE BR(1999) Morphology of amoeboid cells in the uterus ofSteinernema species (Rhabditida Steinernematidae) RussianJournal of Nematology 7 49-56

STEINER WA (1996) Distribution of entomopathogenic ne-matodes in the Swiss Alps Revue Suisse de Zoologie 103439-452

STOCK SP CHOO HY amp KAYA HK (1997) Steiner-nema monticolum sp n (Rhabditida Steinernematidae) anentomopathogenic nematode from Korea with a key to otherspecies Nematologica 43 15-29

STOCK SP SOMSOOK V amp REID AP (1998) Steiner-nema siamkayai n sp (Rhabditida Steinernematidae) an en-tomopathogenic nematode from Thailand Systematic Parasi-tology 41 105-113

STOCK SP CAMPBELL JF amp NADLER SA (2001)Phylogeny of Steinernema Travassos 1927 (CephalobinaSteinernematidae) inferred from ribosomal DNA sequencesand morphological characters Journal of Parasitology 87877-889

STURHAN D (1999) Prevalence and habitat specificity of en-tomopathogenic nematodes in Germany In Gwynn RLSmith PH Griffin C Ehlers R-U Boemare N amp Mas-

Vol 6(4) 2004 565

SE Spiridonov et al

son J-P (Eds) COST 819 Entomopathogenic nematodesapplication and persistence of entomopathogenic nematodesLuxembourg European Commission DG XII pp 123-132

SWOFFORD DL (1998) PAUP Phylogenetic analysis usingparsimony Version 4 Sunderland MA USA Sinauer

SZALANSKI AP TAYLOR DB amp MULLIN PG (2000)Assessing nuclear and mitochondrial DNA sequence vari-ation within Steinernema (Rhabditida Steinernematidae)Journal of Nematology 32 229-233

THOMPSON JD HIGGINS DG amp GIBSON TJ (1994)Clustal W improving the sensitivity of progressive multiplesequence alignment though sequence weighting position gap

penalties and weight matrix choice Nucleic Acids Research22 4673-4680

VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RJ (1992) Intraspecific rDNA restriction frag-ment length polymorphism in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-573

WATURU CN HUNT DJ amp REID AP (1997) Steiner-nema karii sp n (Nematoda Steinernematidae) a new ento-mopathogenic nematode from Kenya International Journalof Nematology 7 68-75

566 Nematology

SE Spiridonov et al

The first attempt to use sequencing data for Steiner-nema phylogenetic analysis was based on analyses of thepartial 18S gene of rDNA (Liu et al 1997) This re-gion however was found to be too conservative for con-structing a resolved phylogeny of this genus Sequencesof the ITS1 region of rDNA and mitochondrial COII and16S rDNA genes for seven species of Steinernema wereanalysed by Szalanski et al (2000) The authors obtainedtrees which were in good concordance with the morpholo-gical features of the species and considered these genes tobe good candidates for phylogenetic reconstruction of thegroup Analyses of sequences of ITS1-58S-ITS2 regionsobtained from ten steinernematid species revealed sev-eral groups with significant bootstrap supports (Nguyenet al 2001 Nguyen amp Duncan 2002) Comprehensivephylogenetic analyses based on combined morphologicaland molecular (D2-D3 expansion segment of LSU rDNA)data including 21 species of Steinernema were made byStock et al (2001)

All of these studies revealed trends in Steinernema phy-logeny Unfortunately many species and isolates were notincluded so that their position within the genus was leftuncertain In our study the ITS1-58S-ITS2 sequences ofdifferent Steinernema isolates maintained at CABI Bio-science Egham UK and CLO Merelbeke Belgium wereanalysed Several aspects of phylogenetic analyses forthe Steinernematidae were set as the main goals for thepresent work these being i) to study relationships be-tween the main groups of Steinernema using maximumparsimony It was hypothesised that newly obtained se-quences might resolve relationships inside this taxon Inthis analysis we included species belonging to differentgroups (based on gross morphology eg infective juve-nile length) in the genus As alignment ambiguity is acommon event during ITS rDNA analysis of divergent se-quences several alignments were generated under differ-ent combinations of gap opening and gap extension penal-ties and then analysed separately ii) to reveal relation-ships between species and isolates within several maingroups All sequences of isolates belonging to a certaingroup were included in the alignment and analysed usingmaximum parsimony iii) to test congruence of morpholo-gical and molecular phylogeny of Steinernema The mor-phological and morphometrical characters used by Liuand Berry (1996) and Stock et al (2001) were re-analysedand a new matrix proposed The total evidence analy-sis was applied to obtain a tree from the matrix com-bining molecular and morphological data iv) to estimatespecies boundaries in steinernematids based on molecular

data Sequence divergence was estimated firstly betweenisolates which according primarily to their morphologywere identified as belonging to the same species and sec-ondly isolates which on the same basis were identifiedas belonging to different species The presence of autapo-morphies was estimated for related species The speciesdelimitation was finally considered on the basis of a com-bination of molecular (autapomorphies sequence diver-gence) and morphological evidence and v) to reveal eco-logical and biogeographical implications of the contem-porary evolutionary hypothesis for Steinernema We ex-pected that the proposed steinernematid phylogeny mightdemonstrate concordance in features such as habitat pref-erence and geographical distribution patterns

Materials and methods

NEMATODE MATERIAL

The list of studied species and isolates is presented inTable 1 Isolates were obtained either by using Galleriabaiting of soil samples collected during surveys (Beddingamp Akhurst 1975) or were received from other laborato-ries The nematodes were maintained on Galleria cater-pillars and juvenile suspensions were stored at 4C

MORPHOLOGICAL AND ECOLOGICAL STUDIES AND

CROSS-BREEDING TESTS

Juveniles and females were fixed in 6 formalinfor both light microscope and SEM studies For SEMspecimens were processed as described by Kozodoi andSpiridonov (1988) The matrix of morphological featuresfor steinernematids was created on the basis of our ownand published observations (Nguyen amp Smart 1996Artyukhovsky et al 1997 Hominick et al 1997 Stocket al 1998 Pham et al 2000 Nguyen amp Adams2003) Cross-breeding experiments were performed toelucidate the specific status of N and Va isolates ofS feltiae from Merelbeke Belgium Single juvenilesfrom the two populations were injected into Galleriacaterpillars (Akhurst amp Bedding 1978) which were keptin Parafilmreg sealed Petri dishes until the results of theinjection (presence or absence of a new generation)became obvious Information about the habitats of theisolates was obtained during collection of materials forthe present study and from the literature (Hominick ampBriscoe 1990 Gwynn amp Richardson 1996 Miduturi etal 1997 Sturhan 1999)

548 Nematology











ANALYSIS


Maximum parsimony (MP) analyses were conductedwith PAUP 40b8 (Swofford 1998) The heuristic searchprocedure was used with 100 replicates of random taxonaddition Gaps were treated as missing data Bootstrap(BS) analysis with 100 replicates was conducted toassess the degree of support for each branch on thetree (Felsenstein 1985) using simple addition sequenceswith TBR swapping (tree-bisection-reconnection) Theg1 statistic was computed by generating 10 000 randomtrees using the RANDTREES option in PAUP (Hillsamp Huelsenbeck 1992) The partition homogeneity testwas run as implemented in PAUP with 100 replicatesto determine whether the nematode phylogeny inferredfrom molecular data was congruent with morphologicalphylogeny Trees were displayed with TreeView 161(Page 1996)

Results


STEINERNEMATIDS



Vol 6(4) 2004 549

SE Spiridonov et al





550 Nematology


Table 1 (Continued)



Vol 6(4) 2004 551

SE Spiridonov et al









552 Nematology



Vol 6(4) 2004 553

SE Spiridonov et al





MOLECULAR PHYLOGENY





554 Nematology


Tabl

e3

Posi

tion

san

dbo

otst

rap

supp

ort

ofse

lect

edcl

ades

and

spec

ies

inth

eM

Ptr

ees

obta

ined

from

the

gene

rala

naly

sis

ofS

tein

erne

ma

sequ

ence

s

Pos

itio

nal

ignm

ent

D1

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

Mon

ophy

lyof

Cla

deI

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

IS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

IVS

MS

SS

SS

MS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

VS

WM

SS

SS

SM

SS

SS

SM

SS

SS

MS

MS

SS

SS

SS

SSt

eine

rnem

asp

p14

15

16+

Sfe

ltia

eM

MM

MM

WW

WM

MM

WW

WW

and

Sw

eise

ricl

ade

Bas

alpo

siti

onof

Stei

nern

ema

spp

14

SM

SM

SW

SW

WS

SW

SS

S15

16

subc

lade

wit

hin

Cla

deII

IS

oreg

onen

seas

asi

ster

toS

krau

ssei

SM

MW

SW

SS

SS

MM

MM

SM

MS

SM

MS

MW

WW

MM

SM

+St

eine

rnem

asp

2S

mon

tico

lum

asa

sist

erto

Cla

deII

IM

WW

MM

MM

WM

SM

WW

MW

MS

MM

WW

SM

Sm

onti

colu

mno

tasi

ster

toC

lade

III

WW

WW

WW

W(p

osit

ione

dne

arot

her

clad

es)

Stei

nern

ema

sp1

asa

sist

erto

MW

WM

MM

WW

WW

MW

WW

WM

MW

MM

MS

SS

mon

tico

lum

+C

lade

III

Stei

nern

ema

sp1

asa

sist

erto

WW

WW

WW

WC

lade

III

wit

hS

mon

tico

lum

outs

ide

this

grou

ping

Bas

alpo

siti

onof

the

Ska

riia

ndS

WW

SS

WW

MW

WW

WW

SM

WW

SM

SM

MW

MM

WS

Stei

nern

ema

spp

912

subc

lade

wit

hin

Cla

deV

Bas

alpo

siti

onof

Sgl

aser

i+

WW

WS

cuba

num

wit

hin

Cla

deV

Spa

kist

anen

seas

asi

ster

toS

riob

rave

SM

WM

WM

MM

WM

SM

SM

MM

WM

WS

MW

WM

WS

MS

MS

+St

eine

rnem

asp

13

Bas

alpo

siti

onof

the

Sbi

corn

utum

SS

SS

SS

SS

SS

SS

SS

SS

SS

S+

Sce

rato

phor

umsu

bcla

dew

ithi

nC

lade

IVB

asal

posi

tion

ofSt

eine

rnem

aab

basi

WW

MS

SS

SS

SS

Sw

ithi

nC

lade

IVS

raru

mas

asi

ster

toC

lade

VW

WW

WW

MW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

Sne

ocur

till

aeas

asi

ster

toS

mon

tico

lum

WW

WW

WW

WW

WW

WM

W+

Stei

nern

ema

sp1

+C

lade

III

Sne

ocur

till

aeas

asi

ster

toC

lade

IIW

WW

WS

neoc

urti

llae

rela

ted

tode

eper

node

sW

WW

WW

WW

WW

WW

ofth

eSt

eine

rnem

aph

ylog

eny

tree

Boo

tstr

apsu

ppor

t(

)fo

rcl

ades

Wlt

70(w

eak)

Mndash

70-9

0(m

oder

ate)

Sgt

90(s

tron

g)

Vol 6(4) 2004 555

SE Spiridonov et al









556 Nematology



Vol 6(4) 2004 557

SE Spiridonov et al







COMBINED DATA



558 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology











ANALYSIS


Maximum parsimony (MP) analyses were conductedwith PAUP 40b8 (Swofford 1998) The heuristic searchprocedure was used with 100 replicates of random taxonaddition Gaps were treated as missing data Bootstrap(BS) analysis with 100 replicates was conducted toassess the degree of support for each branch on thetree (Felsenstein 1985) using simple addition sequenceswith TBR swapping (tree-bisection-reconnection) Theg1 statistic was computed by generating 10 000 randomtrees using the RANDTREES option in PAUP (Hillsamp Huelsenbeck 1992) The partition homogeneity testwas run as implemented in PAUP with 100 replicatesto determine whether the nematode phylogeny inferredfrom molecular data was congruent with morphologicalphylogeny Trees were displayed with TreeView 161(Page 1996)

Results


STEINERNEMATIDS



Vol 6(4) 2004 549

SE Spiridonov et al





550 Nematology


Table 1 (Continued)



Vol 6(4) 2004 551

SE Spiridonov et al









552 Nematology



Vol 6(4) 2004 553

SE Spiridonov et al





MOLECULAR PHYLOGENY





554 Nematology


Tabl

e3

Posi

tion

san

dbo

otst

rap

supp

ort

ofse

lect

edcl

ades

and

spec

ies

inth

eM

Ptr

ees

obta

ined

from

the

gene

rala

naly

sis

ofS

tein

erne

ma

sequ

ence

s

Pos

itio

nal

ignm

ent

D1

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

Mon

ophy

lyof

Cla

deI

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

IS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

IVS

MS

SS

SS

MS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

VS

WM

SS

SS

SM

SS

SS

SM

SS

SS

MS

MS

SS

SS

SS

SSt

eine

rnem

asp

p14

15

16+

Sfe

ltia

eM

MM

MM

WW

WM

MM

WW

WW

and

Sw

eise

ricl

ade

Bas

alpo

siti

onof

Stei

nern

ema

spp

14

SM

SM

SW

SW

WS

SW

SS

S15

16

subc

lade

wit

hin

Cla

deII

IS

oreg

onen

seas

asi

ster

toS

krau

ssei

SM

MW

SW

SS

SS

MM

MM

SM

MS

SM

MS

MW

WW

MM

SM

+St

eine

rnem

asp

2S

mon

tico

lum

asa

sist

erto

Cla

deII

IM

WW

MM

MM

WM

SM

WW

MW

MS

MM

WW

SM

Sm

onti

colu

mno

tasi

ster

toC

lade

III

WW

WW

WW

W(p

osit

ione

dne

arot

her

clad

es)

Stei

nern

ema

sp1

asa

sist

erto

MW

WM

MM

WW

WW

MW

WW

WM

MW

MM

MS

SS

mon

tico

lum

+C

lade

III

Stei

nern

ema

sp1

asa

sist

erto

WW

WW

WW

WC

lade

III

wit

hS

mon

tico

lum

outs

ide

this

grou

ping

Bas

alpo

siti

onof

the

Ska

riia

ndS

WW

SS

WW

MW

WW

WW

SM

WW

SM

SM

MW

MM

WS

Stei

nern

ema

spp

912

subc

lade

wit

hin

Cla

deV

Bas

alpo

siti

onof

Sgl

aser

i+

WW

WS

cuba

num

wit

hin

Cla

deV

Spa

kist

anen

seas

asi

ster

toS

riob

rave

SM

WM

WM

MM

WM

SM

SM

MM

WM

WS

MW

WM

WS

MS

MS

+St

eine

rnem

asp

13

Bas

alpo

siti

onof

the

Sbi

corn

utum

SS

SS

SS

SS

SS

SS

SS

SS

SS

S+

Sce

rato

phor

umsu

bcla

dew

ithi

nC

lade

IVB

asal

posi

tion

ofSt

eine

rnem

aab

basi

WW

MS

SS

SS

SS

Sw

ithi

nC

lade

IVS

raru

mas

asi

ster

toC

lade

VW

WW

WW

MW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

Sne

ocur

till

aeas

asi

ster

toS

mon

tico

lum

WW

WW

WW

WW

WW

WM

W+

Stei

nern

ema

sp1

+C

lade

III

Sne

ocur

till

aeas

asi

ster

toC

lade

IIW

WW

WS

neoc

urti

llae

rela

ted

tode

eper

node

sW

WW

WW

WW

WW

WW

ofth

eSt

eine

rnem

aph

ylog

eny

tree

Boo

tstr

apsu

ppor

t(

)fo

rcl

ades

Wlt

70(w

eak)

Mndash

70-9

0(m

oder

ate)

Sgt

90(s

tron

g)

Vol 6(4) 2004 555

SE Spiridonov et al









556 Nematology



Vol 6(4) 2004 557

SE Spiridonov et al







COMBINED DATA



558 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology

SE Spiridonov et al





550 Nematology


Table 1 (Continued)



Vol 6(4) 2004 551

SE Spiridonov et al









552 Nematology



Vol 6(4) 2004 553

SE Spiridonov et al





MOLECULAR PHYLOGENY





554 Nematology


Tabl

e3

Posi

tion

san

dbo

otst

rap

supp

ort

ofse

lect

edcl

ades

and

spec

ies

inth

eM

Ptr

ees

obta

ined

from

the

gene

rala

naly

sis

ofS

tein

erne

ma

sequ

ence

s

Pos

itio

nal

ignm

ent

D1

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

Mon

ophy

lyof

Cla

deI

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

IS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

IVS

MS

SS

SS

MS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

VS

WM

SS

SS

SM

SS

SS

SM

SS

SS

MS

MS

SS

SS

SS

SSt

eine

rnem

asp

p14

15

16+

Sfe

ltia

eM

MM

MM

WW

WM

MM

WW

WW

and

Sw

eise

ricl

ade

Bas

alpo

siti

onof

Stei

nern

ema

spp

14

SM

SM

SW

SW

WS

SW

SS

S15

16

subc

lade

wit

hin

Cla

deII

IS

oreg

onen

seas

asi

ster

toS

krau

ssei

SM

MW

SW

SS

SS

MM

MM

SM

MS

SM

MS

MW

WW

MM

SM

+St

eine

rnem

asp

2S

mon

tico

lum

asa

sist

erto

Cla

deII

IM

WW

MM

MM

WM

SM

WW

MW

MS

MM

WW

SM

Sm

onti

colu

mno

tasi

ster

toC

lade

III

WW

WW

WW

W(p

osit

ione

dne

arot

her

clad

es)

Stei

nern

ema

sp1

asa

sist

erto

MW

WM

MM

WW

WW

MW

WW

WM

MW

MM

MS

SS

mon

tico

lum

+C

lade

III

Stei

nern

ema

sp1

asa

sist

erto

WW

WW

WW

WC

lade

III

wit

hS

mon

tico

lum

outs

ide

this

grou

ping

Bas

alpo

siti

onof

the

Ska

riia

ndS

WW

SS

WW

MW

WW

WW

SM

WW

SM

SM

MW

MM

WS

Stei

nern

ema

spp

912

subc

lade

wit

hin

Cla

deV

Bas

alpo

siti

onof

Sgl

aser

i+

WW

WS

cuba

num

wit

hin

Cla

deV

Spa

kist

anen

seas

asi

ster

toS

riob

rave

SM

WM

WM

MM

WM

SM

SM

MM

WM

WS

MW

WM

WS

MS

MS

+St

eine

rnem

asp

13

Bas

alpo

siti

onof

the

Sbi

corn

utum

SS

SS

SS

SS

SS

SS

SS

SS

SS

S+

Sce

rato

phor

umsu

bcla

dew

ithi

nC

lade

IVB

asal

posi

tion

ofSt

eine

rnem

aab

basi

WW

MS

SS

SS

SS

Sw

ithi

nC

lade

IVS

raru

mas

asi

ster

toC

lade

VW

WW

WW

MW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

Sne

ocur

till

aeas

asi

ster

toS

mon

tico

lum

WW

WW

WW

WW

WW

WM

W+

Stei

nern

ema

sp1

+C

lade

III

Sne

ocur

till

aeas

asi

ster

toC

lade

IIW

WW

WS

neoc

urti

llae

rela

ted

tode

eper

node

sW

WW

WW

WW

WW

WW

ofth

eSt

eine

rnem

aph

ylog

eny

tree

Boo

tstr

apsu

ppor

t(

)fo

rcl

ades

Wlt

70(w

eak)

Mndash

70-9

0(m

oder

ate)

Sgt

90(s

tron

g)

Vol 6(4) 2004 555

SE Spiridonov et al









556 Nematology



Vol 6(4) 2004 557

SE Spiridonov et al







COMBINED DATA



558 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology


Table 1 (Continued)



Vol 6(4) 2004 551

SE Spiridonov et al









552 Nematology



Vol 6(4) 2004 553

SE Spiridonov et al





MOLECULAR PHYLOGENY





554 Nematology


Tabl

e3

Posi

tion

san

dbo

otst

rap

supp

ort

ofse

lect

edcl

ades

and

spec

ies

inth

eM

Ptr

ees

obta

ined

from

the

gene

rala

naly

sis

ofS

tein

erne

ma

sequ

ence

s

Pos

itio

nal

ignm

ent

D1

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

Mon

ophy

lyof

Cla

deI

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

IS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

IVS

MS

SS

SS

MS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

VS

WM

SS

SS

SM

SS

SS

SM

SS

SS

MS

MS

SS

SS

SS

SSt

eine

rnem

asp

p14

15

16+

Sfe

ltia

eM

MM

MM

WW

WM

MM

WW

WW

and

Sw

eise

ricl

ade

Bas

alpo

siti

onof

Stei

nern

ema

spp

14

SM

SM

SW

SW

WS

SW

SS

S15

16

subc

lade

wit

hin

Cla

deII

IS

oreg

onen

seas

asi

ster

toS

krau

ssei

SM

MW

SW

SS

SS

MM

MM

SM

MS

SM

MS

MW

WW

MM

SM

+St

eine

rnem

asp

2S

mon

tico

lum

asa

sist

erto

Cla

deII

IM

WW

MM

MM

WM

SM

WW

MW

MS

MM

WW

SM

Sm

onti

colu

mno

tasi

ster

toC

lade

III

WW

WW

WW

W(p

osit

ione

dne

arot

her

clad

es)

Stei

nern

ema

sp1

asa

sist

erto

MW

WM

MM

WW

WW

MW

WW

WM

MW

MM

MS

SS

mon

tico

lum

+C

lade

III

Stei

nern

ema

sp1

asa

sist

erto

WW

WW

WW

WC

lade

III

wit

hS

mon

tico

lum

outs

ide

this

grou

ping

Bas

alpo

siti

onof

the

Ska

riia

ndS

WW

SS

WW

MW

WW

WW

SM

WW

SM

SM

MW

MM

WS

Stei

nern

ema

spp

912

subc

lade

wit

hin

Cla

deV

Bas

alpo

siti

onof

Sgl

aser

i+

WW

WS

cuba

num

wit

hin

Cla

deV

Spa

kist

anen

seas

asi

ster

toS

riob

rave

SM

WM

WM

MM

WM

SM

SM

MM

WM

WS

MW

WM

WS

MS

MS

+St

eine

rnem

asp

13

Bas

alpo

siti

onof

the

Sbi

corn

utum

SS

SS

SS

SS

SS

SS

SS

SS

SS

S+

Sce

rato

phor

umsu

bcla

dew

ithi

nC

lade

IVB

asal

posi

tion

ofSt

eine

rnem

aab

basi

WW

MS

SS

SS

SS

Sw

ithi

nC

lade

IVS

raru

mas

asi

ster

toC

lade

VW

WW

WW

MW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

Sne

ocur

till

aeas

asi

ster

toS

mon

tico

lum

WW

WW

WW

WW

WW

WM

W+

Stei

nern

ema

sp1

+C

lade

III

Sne

ocur

till

aeas

asi

ster

toC

lade

IIW

WW

WS

neoc

urti

llae

rela

ted

tode

eper

node

sW

WW

WW

WW

WW

WW

ofth

eSt

eine

rnem

aph

ylog

eny

tree

Boo

tstr

apsu

ppor

t(

)fo

rcl

ades

Wlt

70(w

eak)

Mndash

70-9

0(m

oder

ate)

Sgt

90(s

tron

g)

Vol 6(4) 2004 555

SE Spiridonov et al









556 Nematology



Vol 6(4) 2004 557

SE Spiridonov et al







COMBINED DATA



558 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology

SE Spiridonov et al









552 Nematology



Vol 6(4) 2004 553

SE Spiridonov et al





MOLECULAR PHYLOGENY





554 Nematology


Tabl

e3

Posi

tion

san

dbo

otst

rap

supp

ort

ofse

lect

edcl

ades

and

spec

ies

inth

eM

Ptr

ees

obta

ined

from

the

gene

rala

naly

sis

ofS

tein

erne

ma

sequ

ence

s

Pos

itio

nal

ignm

ent

D1

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

Mon

ophy

lyof

Cla

deI

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

IS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

IVS

MS

SS

SS

MS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

VS

WM

SS

SS

SM

SS

SS

SM

SS

SS

MS

MS

SS

SS

SS

SSt

eine

rnem

asp

p14

15

16+

Sfe

ltia

eM

MM

MM

WW

WM

MM

WW

WW

and

Sw

eise

ricl

ade

Bas

alpo

siti

onof

Stei

nern

ema

spp

14

SM

SM

SW

SW

WS

SW

SS

S15

16

subc

lade

wit

hin

Cla

deII

IS

oreg

onen

seas

asi

ster

toS

krau

ssei

SM

MW

SW

SS

SS

MM

MM

SM

MS

SM

MS

MW

WW

MM

SM

+St

eine

rnem

asp

2S

mon

tico

lum

asa

sist

erto

Cla

deII

IM

WW

MM

MM

WM

SM

WW

MW

MS

MM

WW

SM

Sm

onti

colu

mno

tasi

ster

toC

lade

III

WW

WW

WW

W(p

osit

ione

dne

arot

her

clad

es)

Stei

nern

ema

sp1

asa

sist

erto

MW

WM

MM

WW

WW

MW

WW

WM

MW

MM

MS

SS

mon

tico

lum

+C

lade

III

Stei

nern

ema

sp1

asa

sist

erto

WW

WW

WW

WC

lade

III

wit

hS

mon

tico

lum

outs

ide

this

grou

ping

Bas

alpo

siti

onof

the

Ska

riia

ndS

WW

SS

WW

MW

WW

WW

SM

WW

SM

SM

MW

MM

WS

Stei

nern

ema

spp

912

subc

lade

wit

hin

Cla

deV

Bas

alpo

siti

onof

Sgl

aser

i+

WW

WS

cuba

num

wit

hin

Cla

deV

Spa

kist

anen

seas

asi

ster

toS

riob

rave

SM

WM

WM

MM

WM

SM

SM

MM

WM

WS

MW

WM

WS

MS

MS

+St

eine

rnem

asp

13

Bas

alpo

siti

onof

the

Sbi

corn

utum

SS

SS

SS

SS

SS

SS

SS

SS

SS

S+

Sce

rato

phor

umsu

bcla

dew

ithi

nC

lade

IVB

asal

posi

tion

ofSt

eine

rnem

aab

basi

WW

MS

SS

SS

SS

Sw

ithi

nC

lade

IVS

raru

mas

asi

ster

toC

lade

VW

WW

WW

MW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

Sne

ocur

till

aeas

asi

ster

toS

mon

tico

lum

WW

WW

WW

WW

WW

WM

W+

Stei

nern

ema

sp1

+C

lade

III

Sne

ocur

till

aeas

asi

ster

toC

lade

IIW

WW

WS

neoc

urti

llae

rela

ted

tode

eper

node

sW

WW

WW

WW

WW

WW

ofth

eSt

eine

rnem

aph

ylog

eny

tree

Boo

tstr

apsu

ppor

t(

)fo

rcl

ades

Wlt

70(w

eak)

Mndash

70-9

0(m

oder

ate)

Sgt

90(s

tron

g)

Vol 6(4) 2004 555

SE Spiridonov et al









556 Nematology



Vol 6(4) 2004 557

SE Spiridonov et al







COMBINED DATA



558 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology



Vol 6(4) 2004 553

SE Spiridonov et al





MOLECULAR PHYLOGENY





554 Nematology


Tabl

e3

Posi

tion

san

dbo

otst

rap

supp

ort

ofse

lect

edcl

ades

and

spec

ies

inth

eM

Ptr

ees

obta

ined

from

the

gene

rala

naly

sis

ofS

tein

erne

ma

sequ

ence

s

Pos

itio

nal

ignm

ent

D1

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

Mon

ophy

lyof

Cla

deI

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

IS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

IVS

MS

SS

SS

MS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

VS

WM

SS

SS

SM

SS

SS

SM

SS

SS

MS

MS

SS

SS

SS

SSt

eine

rnem

asp

p14

15

16+

Sfe

ltia

eM

MM

MM

WW

WM

MM

WW

WW

and

Sw

eise

ricl

ade

Bas

alpo

siti

onof

Stei

nern

ema

spp

14

SM

SM

SW

SW

WS

SW

SS

S15

16

subc

lade

wit

hin

Cla

deII

IS

oreg

onen

seas

asi

ster

toS

krau

ssei

SM

MW

SW

SS

SS

MM

MM

SM

MS

SM

MS

MW

WW

MM

SM

+St

eine

rnem

asp

2S

mon

tico

lum

asa

sist

erto

Cla

deII

IM

WW

MM

MM

WM

SM

WW

MW

MS

MM

WW

SM

Sm

onti

colu

mno

tasi

ster

toC

lade

III

WW

WW

WW

W(p

osit

ione

dne

arot

her

clad

es)

Stei

nern

ema

sp1

asa

sist

erto

MW

WM

MM

WW

WW

MW

WW

WM

MW

MM

MS

SS

mon

tico

lum

+C

lade

III

Stei

nern

ema

sp1

asa

sist

erto

WW

WW

WW

WC

lade

III

wit

hS

mon

tico

lum

outs

ide

this

grou

ping

Bas

alpo

siti

onof

the

Ska

riia

ndS

WW

SS

WW

MW

WW

WW

SM

WW

SM

SM

MW

MM

WS

Stei

nern

ema

spp

912

subc

lade

wit

hin

Cla

deV

Bas

alpo

siti

onof

Sgl

aser

i+

WW

WS

cuba

num

wit

hin

Cla

deV

Spa

kist

anen

seas

asi

ster

toS

riob

rave

SM

WM

WM

MM

WM

SM

SM

MM

WM

WS

MW

WM

WS

MS

MS

+St

eine

rnem

asp

13

Bas

alpo

siti

onof

the

Sbi

corn

utum

SS

SS

SS

SS

SS

SS

SS

SS

SS

S+

Sce

rato

phor

umsu

bcla

dew

ithi

nC

lade

IVB

asal

posi

tion

ofSt

eine

rnem

aab

basi

WW

MS

SS

SS

SS

Sw

ithi

nC

lade

IVS

raru

mas

asi

ster

toC

lade

VW

WW

WW

MW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

Sne

ocur

till

aeas

asi

ster

toS

mon

tico

lum

WW

WW

WW

WW

WW

WM

W+

Stei

nern

ema

sp1

+C

lade

III

Sne

ocur

till

aeas

asi

ster

toC

lade

IIW

WW

WS

neoc

urti

llae

rela

ted

tode

eper

node

sW

WW

WW

WW

WW

WW

ofth

eSt

eine

rnem

aph

ylog

eny

tree

Boo

tstr

apsu

ppor

t(

)fo

rcl

ades

Wlt

70(w

eak)

Mndash

70-9

0(m

oder

ate)

Sgt

90(s

tron

g)

Vol 6(4) 2004 555

SE Spiridonov et al









556 Nematology



Vol 6(4) 2004 557

SE Spiridonov et al







COMBINED DATA



558 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology

SE Spiridonov et al





MOLECULAR PHYLOGENY





554 Nematology


Tabl

e3

Posi

tion

san

dbo

otst

rap

supp

ort

ofse

lect

edcl

ades

and

spec

ies

inth

eM

Ptr

ees

obta

ined

from

the

gene

rala

naly

sis

ofS

tein

erne

ma

sequ

ence

s

Pos

itio

nal

ignm

ent

D1

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

Mon

ophy

lyof

Cla

deI

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

IS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

IVS

MS

SS

SS

MS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

VS

WM

SS

SS

SM

SS

SS

SM

SS

SS

MS

MS

SS

SS

SS

SSt

eine

rnem

asp

p14

15

16+

Sfe

ltia

eM

MM

MM

WW

WM

MM

WW

WW

and

Sw

eise

ricl

ade

Bas

alpo

siti

onof

Stei

nern

ema

spp

14

SM

SM

SW

SW

WS

SW

SS

S15

16

subc

lade

wit

hin

Cla

deII

IS

oreg

onen

seas

asi

ster

toS

krau

ssei

SM

MW

SW

SS

SS

MM

MM

SM

MS

SM

MS

MW

WW

MM

SM

+St

eine

rnem

asp

2S

mon

tico

lum

asa

sist

erto

Cla

deII

IM

WW

MM

MM

WM

SM

WW

MW

MS

MM

WW

SM

Sm

onti

colu

mno

tasi

ster

toC

lade

III

WW

WW

WW

W(p

osit

ione

dne

arot

her

clad

es)

Stei

nern

ema

sp1

asa

sist

erto

MW

WM

MM

WW

WW

MW

WW

WM

MW

MM

MS

SS

mon

tico

lum

+C

lade

III

Stei

nern

ema

sp1

asa

sist

erto

WW

WW

WW

WC

lade

III

wit

hS

mon

tico

lum

outs

ide

this

grou

ping

Bas

alpo

siti

onof

the

Ska

riia

ndS

WW

SS

WW

MW

WW

WW

SM

WW

SM

SM

MW

MM

WS

Stei

nern

ema

spp

912

subc

lade

wit

hin

Cla

deV

Bas

alpo

siti

onof

Sgl

aser

i+

WW

WS

cuba

num

wit

hin

Cla

deV

Spa

kist

anen

seas

asi

ster

toS

riob

rave

SM

WM

WM

MM

WM

SM

SM

MM

WM

WS

MW

WM

WS

MS

MS

+St

eine

rnem

asp

13

Bas

alpo

siti

onof

the

Sbi

corn

utum

SS

SS

SS

SS

SS

SS

SS

SS

SS

S+

Sce

rato

phor

umsu

bcla

dew

ithi

nC

lade

IVB

asal

posi

tion

ofSt

eine

rnem

aab

basi

WW

MS

SS

SS

SS

Sw

ithi

nC

lade

IVS

raru

mas

asi

ster

toC

lade

VW

WW

WW

MW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

Sne

ocur

till

aeas

asi

ster

toS

mon

tico

lum

WW

WW

WW

WW

WW

WM

W+

Stei

nern

ema

sp1

+C

lade

III

Sne

ocur

till

aeas

asi

ster

toC

lade

IIW

WW

WS

neoc

urti

llae

rela

ted

tode

eper

node

sW

WW

WW

WW

WW

WW

ofth

eSt

eine

rnem

aph

ylog

eny

tree

Boo

tstr

apsu

ppor

t(

)fo

rcl

ades

Wlt

70(w

eak)

Mndash

70-9

0(m

oder

ate)

Sgt

90(s

tron

g)

Vol 6(4) 2004 555

SE Spiridonov et al









556 Nematology



Vol 6(4) 2004 557

SE Spiridonov et al







COMBINED DATA



558 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology


Tabl

e3

Posi

tion

san

dbo

otst

rap

supp

ort

ofse

lect

edcl

ades

and

spec

ies

inth

eM

Ptr

ees

obta

ined

from

the

gene

rala

naly

sis

ofS

tein

erne

ma

sequ

ence

s

Pos

itio

nal

ignm

ent

D1

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

Mon

ophy

lyof

Cla

deI

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

Mon

ophy

lyof

Cla

deII

IS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

IVS

MS

SS

SS

MS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SM

onop

hyly

ofC

lade

VS

WM

SS

SS

SM

SS

SS

SM

SS

SS

MS

MS

SS

SS

SS

SSt

eine

rnem

asp

p14

15

16+

Sfe

ltia

eM

MM

MM

WW

WM

MM

WW

WW

and

Sw

eise

ricl

ade

Bas

alpo

siti

onof

Stei

nern

ema

spp

14

SM

SM

SW

SW

WS

SW

SS

S15

16

subc

lade

wit

hin

Cla

deII

IS

oreg

onen

seas

asi

ster

toS

krau

ssei

SM

MW

SW

SS

SS

MM

MM

SM

MS

SM

MS

MW

WW

MM

SM

+St

eine

rnem

asp

2S

mon

tico

lum

asa

sist

erto

Cla

deII

IM

WW

MM

MM

WM

SM

WW

MW

MS

MM

WW

SM

Sm

onti

colu

mno

tasi

ster

toC

lade

III

WW

WW

WW

W(p

osit

ione

dne

arot

her

clad

es)

Stei

nern

ema

sp1

asa

sist

erto

MW

WM

MM

WW

WW

MW

WW

WM

MW

MM

MS

SS

mon

tico

lum

+C

lade

III

Stei

nern

ema

sp1

asa

sist

erto

WW

WW

WW

WC

lade

III

wit

hS

mon

tico

lum

outs

ide

this

grou

ping

Bas

alpo

siti

onof

the

Ska

riia

ndS

WW

SS

WW

MW

WW

WW

SM

WW

SM

SM

MW

MM

WS

Stei

nern

ema

spp

912

subc

lade

wit

hin

Cla

deV

Bas

alpo

siti

onof

Sgl

aser

i+

WW

WS

cuba

num

wit

hin

Cla

deV

Spa

kist

anen

seas

asi

ster

toS

riob

rave

SM

WM

WM

MM

WM

SM

SM

MM

WM

WS

MW

WM

WS

MS

MS

+St

eine

rnem

asp

13

Bas

alpo

siti

onof

the

Sbi

corn

utum

SS

SS

SS

SS

SS

SS

SS

SS

SS

S+

Sce

rato

phor

umsu

bcla

dew

ithi

nC

lade

IVB

asal

posi

tion

ofSt

eine

rnem

aab

basi

WW

MS

SS

SS

SS

Sw

ithi

nC

lade

IVS

raru

mas

asi

ster

toC

lade

VW

WW

WW

MW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

WW

Sne

ocur

till

aeas

asi

ster

toS

mon

tico

lum

WW

WW

WW

WW

WW

WM

W+

Stei

nern

ema

sp1

+C

lade

III

Sne

ocur

till

aeas

asi

ster

toC

lade

IIW

WW

WS

neoc

urti

llae

rela

ted

tode

eper

node

sW

WW

WW

WW

WW

WW

ofth

eSt

eine

rnem

aph

ylog

eny

tree

Boo

tstr

apsu

ppor

t(

)fo

rcl

ades

Wlt

70(w

eak)

Mndash

70-9

0(m

oder

ate)

Sgt

90(s

tron

g)

Vol 6(4) 2004 555

SE Spiridonov et al









556 Nematology



Vol 6(4) 2004 557

SE Spiridonov et al







COMBINED DATA



558 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology

SE Spiridonov et al









556 Nematology



Vol 6(4) 2004 557

SE Spiridonov et al







COMBINED DATA



558 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology



Vol 6(4) 2004 557

SE Spiridonov et al







COMBINED DATA



558 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology

SE Spiridonov et al







COMBINED DATA



558 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology



Vol 6(4) 2004 559

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology

SE Spiridonov et al






Discussion





560 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology












Vol 6(4) 2004 561

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology

SE Spiridonov et al




PHYLOGENY









562 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology








Conclusions


Acknowledgements


Vol 6(4) 2004 563

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology

SE Spiridonov et al


References


























564 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology





























Vol 6(4) 2004 565

SE Spiridonov et al








566 Nematology

SE Spiridonov et al








566 Nematology

the its1-5.8s-its2 region of rdna and morphologicalfeatures · s.e. spiridonov et al. the Þrst...

Documents