A molecular phylogenetic approach to Longidoridae ...baldwin/sergei/reprints/... · Nematology ,...

Nematology 2005 Vol 7(1) 111-124

A molecular phylogenetic approach to Longidoridae(Nematoda Dorylaimida)

Yu HE 1 Sergei A SUBBOTIN 2 Tatiana V RUBTSOVA 2 Franco LAMBERTI 3daggerDerek JF BROWN 4 and Maurice MOENS 15lowast

1 Agricultural Research Centre Burg Van Gansberghelaan 96 9820 Merelbeke Belgium2 Institute of Parasitology RAS Leninskii Prospekt 33 Moscow 177071 Russia

3 Istituto per la Protezione delle Piante Sezione di Bari CNR Bari Italy4 Central Laboratory of General Ecology Gagarin Street 2 1113 Sofia Bulgaria

5 Laboratory for Agrozoology Ghent University Coupure 555 9000 Ghent Belgium

Received 6 July 2004 revised 7 December 2004Accepted for publication 14 December 2004

Summary ndash The Longidoridae are a group of ectoparasitic nematodes including two subfamilies and six genera with hundreds ofspecies Sequences of the D2 and D3 expansion region of the large subunit (LSU) rRNA nuclear gene were amplified and used toreconstruct the phylogeny of longidorids Phylogenetic analyses with maximum parsimony (MP) maximum likelihood (ML) andBayesian inference (BI) were performed with one outgroup taxon and 62 longidorid sequences Confidence of inferred clades wasassessed by non-parametric bootstrapping for MP and Bayesian posterior probability for ML All analyses placed Paralongidorusspecies as an inner group within the otherwise monophyletic genus Longidorus The genus Xiphinema except for X americanum-groupspecies was placed as the sister group of Longidorus with strong support from the ML and BI analyses The X americanum-group wasstrongly supported as an exclusive clade to other genus Xiphinema species The position of the Xiphidorus clade was not well resolvedand the phylogenetic analyses did not support it as a sister group to Longidorus as previously inferred from morphology Secondarystructure models were constructed for the D2D3 region of LSU rRNA for all studied species It was found that sequence-based andstructural morphometric rRNA phylogenies were incongruent

Keywords ndash amphid structure evolution large subunit Longidorus Paralongidorus phylogeny rRNA secondary structureXiphidorus Xiphinema Xiphinema americanum-group

The family Longidoridae Thorne 1935 belongs to theDorylaimida Pearse 1942 and is subdivided into two sub-families Longidorinae Thorne 1935 and Xiphinemati-nae Dalmasso 1969 Within the Longidorinae the ge-nera Longidorus Micoletzky 1922 (107 valid species)Paralongidorus Siddiqi Hooper amp Khan 1963 (42 validspecies) Longidoroides Khan Chawla amp Saha 1978(19 valid species) Xiphidorus Monteiro 1976 (eightvalid species) Paraxiphidorus Coomans amp Chaves 1995(three valid species) and Australodorus Coomans Ol-mos Casella amp Chaves 2004 (monotypic) are classi-fied into two tribes Xiphidorini Coomans 1985 with thegenera Australodorus Xiphidorus and Paraxiphidorusand Longidorini Coomans 1985 for the remaining threegenera (Coomans 1985) One genus Xiphinema Cobb

dagger Franco Lamberti died in 2004 Corresponding author e-mail mmoensclofgovbe

1913 is classified in the Xiphinematinae with 296 nom-inal taxa corresponding to 234 valid taxa 49 junior syn-onyms and 13 species inquirendae (Coomans et al 2001)All species live ectoparasitically Some can vector plantviruses (nepoviruses) and are classified as quarantinepests (Taylor amp Brown 1997)

Within the longidorids the Xiphinema americanum-group attracts special attention According to Loof andLuc (1990) the common morphological characters for thisgroup are spiral or C-shaped small body two well de-veloped genital branches no uterine differentiation shortconical to broadly convex-conoid tail and vulva posi-tioned at 40-60 of the body length from the head Asmore populations of X americanum were sampled and in-vestigated from different geographical localities taxono-

copy Koninklijke Brill NV Leiden 2005 111Also available online - wwwbrillnl

Y He et al

mists encountered many morphological varieties amongpopulations studied Lima (1965) and Tarjan (1969) sug-gested that X americanum is a complex of several speciesSince then 51 nominal species have been placed in thisgroup (Lamberti et al 2000) The traditional identifica-tion of these species by morphology and morphometricsis very difficult because many of the characters used over-lap Moreover questions concerning the true phylogenybehind such a complicated group and the position of thespecies within this group have been raised (Lamberti etal 2002) So far phylogenetic analyses of Longidoridaeare few The phylogeny based on selected morphologicalcharacters of the genus Xiphinema was analysed in greatdetail by Coomans et al (2001) Rubtsova et al (2001)used a molecular approach to investigate the phylogeneticposition of a few species from the genera Longidorus andParalongidorus Recently the phylogeny of longidoridswas inferred from ITS1 analyses (Ye et al 2004) and 18SrDNA sequences (Oliveira et al 2004)

Molecular systematic approaches are useful for pro-ducing phylogenies especially in cases where morpholo-gical characters lead to ambiguous interpretation In re-cent years ribosomal RNA genes have attracted the at-tention of many systematists and evolutionists becauseof their functional importance their ease of amplificationand because they are assumed to record the evolutionaryhistory of the organism relatively faithfully (Lydeard etal 2000) In nematodes rRNA gene sequences were usedto infer the phylogeny of many groups including somephytoparasitic taxa (eg Al-Banna et al 1997 Blaxter etal 1998 Kaplan et al 2000 Subbotin et al 2001)

When assembled into the ribosome together with otherproteins ribosomal RNA is usually folded into a com-plicated secondary and tertiary structure Although someof these structures have been detected by X-ray crys-tallisation (Cate et al 1999) or by cryo-electron mi-croscopic reconstruction (Mueller et al 2000) most ofthe structures deposited in the public domain such asthe Antwerp database of large (De Rijk et al 1999)and small rRNA sequences (Van de Peer et al 1996)(httpwwwpsbugentberRNAindexhtm) and the com-parative RNA website (httpwwwrnaicmbutexasedu)(Cannone et al 2002) are derived from comparativeanalysis which generates the folding from the commoncompensatory substitutions and pairing patterns on manysequences The secondary structure of rRNA providesa very useful template for improved construction of se-quence alignments and critical for phylogenetic construc-tion (Kier 1995 Hickson et al 1996) Researchers have

achieved some success by using alignments refined withthe aid of the secondary structure and an optimised com-puter algorithm (Titus amp Frost 1996) Additionally sec-ondary structure can provide useful information for as-sessing the sequence in weighted parsimony or otherweighted methods Structural motifs may themselves con-tain information useful for phylogeny inference (Lydeardet al 2000)

In this paper we report on the first phylogenetic analy-ses of the family Longidoridae using molecular data col-lected from the LSU rRNA gene We also tried to providesystematists with persuasive molecular information to aidin the reconstruction of those taxa Species were sampledand used from all of the longidorid genera with the excep-tion of Longidoroides which was however synonymisedwith the genus Paralongidorus (Siddiqi et al 1993) astatus that is still questioned (Coomans 1996) Paraxiphi-dorus and the monotypic Australodorus

Materials and methods

TAXON SAMPLING

Nematode samples collected for this study togetherwith their authorities are listed in Table 1 and include23 species from the genus Longidorus two species fromthe genus Paralongidorus two species from the genusXiphidorus and 35 species from the genus XiphinemaThe majority of the populations were identified on thebasis of both morphometrics and morphology only forsome populations was the identification made on thebasis of general morphology These latter populations aremarked with an asterisk in Table 1

TOTAL DNA EXTRACTION

One juvenile or adult nematode was transferred into13 microl ddH2O and cut into two to five pieces witha sterilised scalpel Ten microl 2 times worm lysis buffer(20 mM Tris-HCl (pH 80) 100 mM KCl 30 mM Mg2Cl20 mM DTT 09 Tween 20) and 01 microl proteinaseK stock solution (20 mgml) was added to a 200 or500 microl microcentrifuge tube The nematode fragmentswere pipetted up in 99 microl ddH2O and added to thetube which was then briefly centrifuged and stored atminus70C for at least 10 min Subsequently each tubewas incubated at 65C for 1-2 h and the proteinase Kwas denatured at 95C for 10 min Finally the DNAsuspensions were cooled to 4C and stored at minus20C

112 Nematology

Phylogeny of Longidoridae

Table 1 Taxon sampling for longidorids and outgroup used in this study

Nematode species Code Locality of sample GenBank Source (ID)accession

Aporcelaimellus obtusicaudatus (Bastian 1865)Altherr 1968

AOFD Merelbeke Belgium AY601632 A CoomansSA Subbotin

Longidorus africanus Merny 1966 CA46 California USA AY601583 F LambertiL apulus Lamberti amp Bleve-Zacheo 1977 CAN23 Mola di Bari Italy AY601571 TC VrainL arthensis Brown Grunder Hooper Klingler

amp Kunz 1994CAN115 Suter Switzerland AY601570 TC Vrain

L athesinus Lamberti Coiro amp Agostinelli 1992 EU105 Italy AY601574 DJF BrownL attenuatus Hooper 1961 CAN17 Germany AY601572 TC VrainL breviannulatus Norton amp Hoffmann 1975 CAN268 Nebraska USA AY601576 TC VrainL caespiticola Hooper 1961 EU20 Scotland UK AY601567 DJF BrownL camelliae Zheng amp Brown 2000 EU130 Hangzhou China AY601585 J ZhengL carpathicus Liškovaacute Robbins amp Brown 1997 Carpa Kirchbichel Germany AF480072 T RubtsovaL diadecturus Eveleigh amp Allen 1982 CAN31 Elkins White river USA AY601584 TC VrainL edmundsi Hunt amp Siddiqi 1977 VE275 Caribbean sea beach Cuba AY601575 F LambertiL elongatus (de Man 1876) Micoletzky 1922 EU1 Ingraston Scotland UK AY601578 DJF BrownL euonymus Mali amp Hooper 1974 EU124 Zabagr Hungary AY601573 DJF BrownL goodeyi Hooper 1961 EU26 Peebles Scotland UK AY601581 DJF BrownL helveticus Lamberti Kunz Grunder Molinari

De Luca Agostinelli amp Radicci 2001SV46 Camenzuid Switzerland AY601566 F Lamberti

L intermedius Kozlowska amp Seinhorst 1979 Inter Planegg Germany AF480074 T RubtsovaL juvenilis Dalmasso 1969 CAN196 Moca Slovakia AY601579 TC VrainL latocephalus Lamberti Choleva amp Agostinelli BLUE Nylstrom South Africa AY601568 F Lamberti

1983L latocephalus CAN114 Greece AY601569 TC VrainL leptocephalus Hooper 1961 EU8 Scotland UK AY601580 DJF BrownL macrosoma Hooper 1961 LM1 Switzerland AY601565 J GrunderL piceicola Liškovaacute Robbins amp Brown 1997 EU112 Branisko Slovakia AY601577 DJF BrownL profundorum Hooper 1966 Prof Gandesbergen Germany AF480073 T RubtsovaL sturhani Rubtsova Subbotin Brown amp Moens Vise348 Augen Germany AF480071 T Rubtsova

2001Paralongidorus maximus (Buumltschli 1874)

Siddiqi 1964Max592 Harrier Sand Germany AF480083 T Rubtsova

Paralongidorus sp CAN201 Bosaka H1 SL Czech Republic AY601582 TC VrainXiphidorus minor Rashid Coomans amp Sharma 1986 VE269 Amazon forest Venezuela AY601612 F LambertiXiphidorus sp CAN248 Argentina AY601611 TC VrainXiphinema abrantinum Roca amp Pereira 1991 CAN223 Portugal AY601625 TC VrainX americanum Cobb 1913 PE24 Pennsylvania USA AY601599 F LambertiX americanum XA1 USA AY601591 DJF BrownX bakeri Williams 1961 CAN27 Fayetteville USA AY601623 TC VrainX basirilowast group sp EU125 San Jose Cuba AY601629 DJF BrownX basirilowast group sp EU126 San Jose Cuba AY601630 DJF BrownX brasilienselowast Lordello 1951 EU41 Para State Brazil AY601616 DJF BrownX brevicollum Lordello amp Da Costa 1961 Xb1 South Africa AY601601 A CoomansX brevicollum EU132 Beijing China AY601604 J ZhengX brevicollum EU29 Brazil AY601605 DJF BrownX brevisicum Lamberti Bravo Agostinelli

amp Lemos 1994EU5 Braga Portugal AY601610 L Poiras

X bricolense Ebsary Vrain amp Graham 1989 CAN39 Winfield BC Canada AY601594 TC VrainX bricolense PE18 Pennsylvania USA AY601596 F LambertiX californicum Lamberti amp Bleve-Zacheo 1979 CA54 Kearney CA USA AY601592 F Lamberti

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Table 1 (Continued)


X chambersi Thorne 1939 AB3 Lee county AL USA AY601617 F LambertiX coxi Tarjan 1964 GG10 Jenkil Tsloud GG USA AY601631 F LambertiX dentatum Sturhan 1978 EU111 Branisko Slovakia AY601627 DJF BrownX diversicaudatum (Micoletzky 1927) Thorne 1939 EU7 Forest Braga Portugal AY601624 DJF BrownX diffusum Lamberti amp Bleve-Zacheo 1979 CAN162 South Africa AY601600 TC VrainX elongatum Schuurmans Stekhoven amp

Teunissen 1938CAN24 Israel AY601618 TC Vrain

X georgianum Lamberti amp Bleve-Zacheo 1979 GG14 Jenkil Tsloud GG USA AY601586 F LambertiX incognitum Lamberti amp Bleve-Zacheo 1979 PE42 Pennsylvania USA AY601597 F LambertiX index Thorne amp Allen 1950 EU25 Argentina AY601628 DJF BrownX insigne Loos 1949 EU131 Hangzhou China AY601619 J ZhengX italiae Meyl 1953 BAR1 Italy AY601613 F LambertiX pachtaicum (Tulaganov 1938) Kirjanova 1951 T48 Castelnuovo Berarjengo Italy AY601606 F LambertiX pachtaicum M21 Albata Moldova AY601607 L PoirasX pacificum Ebsary Vrain amp Graham 1989 GG15 Pike GG USA AY601590 F LambertiX pachydermum Sturhan 1983 EU109 Portugal type locality AY601608 DJF BrownX pyrenaicumDalmasso 1969 EU121 Cyprus AY601626 DJF BrownX radicicola Goodey 1936 V1273 Chursquomomray Vietnam AY601622 CN NguyenX rivesi Dalmasso 1969 PE20 Pennsylvania USA AY601588 F LambertiX rivesi PE1 Pennsylvania USA AY601589 F LambertiX santos Lamberti Lemos Agostinelli amp

drsquoAddabo 1993CAN224 Portugal AY601587 TC Vrain

X savanicola Luc amp Southey 1980 CAN72 Dakar Senegal AY601620 TC VrainX setariae Luc 1958 EU27 Brazil AY601621 DJF BrownX simile Lamberti Choleva amp Agostinelli 1983 M5 Anenii Nou Moldova AY601609 L PoirasX taylori Lamberti Ciancio Agostinelli amp Coiro EU117 Spa Slovakia AY601602 DJF Brown

1992X taylori TN1 Treuna Italy AY601603 F LambertiX thornei Lamberti amp Golden 1986 CO3 Colorado USA AY601595 F LambertiX thornei OR4 Molella Oregon USA AY601593 F LambertiX utahense Lamberti amp Bleve-Zacheo 1979 CO5 Colorado USA AY601598 F LambertiX vuittenezi Luc Lima Weischer amp Flegg 1964 EU123 Zabagr Hungary AY601614 DJF BrownXiphinema sp EU110 Portugal AY601615 DJF Brown

Populations identified on the basis of general morphology

until use No additional purification was required forsubsequent PCR procedure

PCR AMPLIFICATION

The D2 and D3 expansion regions of the large sub-unit rDNA were amplified using the primers D2A (5prime-ACAAGTACCGTGAGGGAAAGTTG-3prime) and D3B (5prime-TCGGAAGGAACCAGCTACTA-3prime) The cycling profileof the PCR was 94C for 3 min 35 cycles of 94C for 30 s54C for 40 s and 72C for 1 min followed by an exten-sion at 72C for 10 min PCR products were visualised

under UV after separation in a 1 agarose gel and stain-ing with ethidium bromide The fragments were recoveredfrom the gel by excision and purified with Gel purificationkit (Qiagen-Westburg Leusden The Netherlands)

SEQUENCING AND SEQUENCE ANALYSES

A direct sequencing strategy was used for the ampli-fied product DNA fragments were sequenced using theBigDye Terminator v31 Cycle Sequencing Ready Reac-tion Kit according to the manufacturerrsquos instructions (PEApplied Biosystems Foster City CA USA) The final se-

114 Nematology


quences were determined by an ABI prism 377 geneticanalyser (Applied Biosystems) The GenBank accessionnumbers for sequences are given in Table 1 Sequenceswere assembled and edited with BioEdit (Hall 1999)

SECONDARY STRUCTURE CONSTRUCTION AND

ANALYSES

The secondary structure model of the D2 region wasinferred with the aid of Mfold (Zuker et al 1999) and theVienna RNA package (Hofacker et al 2003) The ViennaRNA package was also used to aid in the constructionof the secondary structure template of the D3 region byimposing constraints based on the predicted secondarystructures of Caenorhabditis elegans (Maupas 1900)Osche 1952 (Ellis et al 1986) Drosophila melanogasterMeigen 1930 (Tautz et al 1988) and Xenopus laevisDaudin 1803 (Clark et al 1984) The variability of thesecondary structure templates was analysed by tree editdistance comparisons as implemented in the Vienna RNApackage

PHYLOGENY INFERENCE

Alignment

A sequence alignment was made with ClustalX 18(Thompson et al 1997) using default parameters (gapopening penalty = 1555 and gap extension penalty =666) followed by manual editing in BioEdit (Hall 1999)based on the secondary structure templates The analyseswere based on this manually optimised alignment

Phylogenetic analyses

The datasets of the D2 and D3 sequences were analysedwith PAUP4b10 (Swofford 2002) As D2 and D3 re-gions may evolve at different rates and with different his-torical records of evolution the homogeneity test (Farriset al 1994) was used to measure the incongruence be-tween the two regions so as to decide whether to performanalyses on the combined dataset Homogeneity of nu-cleotide compositions was given by χ2 statistics imple-mented in PAUP

Different phylogenetic methods have different strengthsand weaknesses the use of multiple methods increases theconfidence of the inferred phylogeny Trees were there-fore constructed using the algorithms of maximum par-simony (MP) and maximum likelihood (ML) methods asimplemented in PAUP Weighted parsimony was also per-formed on the combined D2 and D3 dataset (a weight of

1 was assigned to nucleotides in ambiguously aligned re-gions and a weight of 3 to those in robustly aligned re-gions) The weight strategies referred to the secondarystructure model of D2 and D3 regions A weight of 1 wasassigned to nucleotides in regions for which the homolo-gous nucleotides are difficult to be defined and a weightof 3 to those in homologous regions

The MP method was used with heuristic search treebisection and reconnection (TBR) swapping algorithmand ten random additions of sequences One hundred non-parametric bootstrap replicates (BS) were analysed withheuristic search algorithm Decay indices (DI) (Bremer1994) were calculated by Autodecay (Ericsson 2001)

The appropriate ML model was selected by the LogLikelihood ratio test (LRT) implemented in the softwareModeltest (Posada amp Crandall 1998) Nested modelswere evaluated by LRT and the best model was selectedfor the ML method The starting tree for LRT was ob-tained by the neighbour joining (NJ) method or the bestMP tree The heuristic search with the TBR swappingalgorithm and random sequence addition The TBR waslimited to 10 000 trees due to the extended computationtime Eleven searches were performed Eleven ML treeswere compared using the Shimodaira-Hasegawa (SH) testwith resampling estimated log-likelihood (RELL) approx-imation (Shimodaira amp Hasegawa 1999) The selectedbest tree topology (with the highest log likelihood value)was tested by the Swofford-Hillis (SOWH) test (Goldmanet al 2000) using parametric bootstrap methods Onehundred parametric bootstrap replicates were produced bySeqGen implementing Monte Carlo simulation (Rambautamp Grassly 1997) based on the ML estimated parametersof the given topology

A tree based on the RNA secondary structure was con-structed from tree edit distances The secondary structurecomparison was reduced to a comparison of the orderedlabelled trees that were used to represent the secondarystructures as proposed by Shapiro and Zhang (1990) us-ing the NJ method The tree edit distances matrix wascomputed using Vienna RNA package Confidence as-sessments for the MP analyses branch and topology sup-ports were performed by non-parametric bootstrap analy-sis Decay indices were calculated for each branch Be-cause of the computational limitations encountered withML we could not perform extensive non-parametric boot-strap analyses on the big data sets We chose Bayesianinference (BI) analyses to estimate posterior probabilities(BPP) for the phylogenetic relationships inferred by MLanalysis MrBayes 20 (Huelsenbeck amp Ronquist 2001)

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was used for the analysis in combination with the modelpreviously identified by Modeltest Bayesian analysis wasimplemented with random starting trees four incremen-tally heated Markov chains and 10 times 106 generations andsampling Markov chains at 100 generation intervals Treesobtained before the stationary point were discarded aslsquoburn-inrsquo samples Analyses were performed three timesto avoid local optima traps (Huelsenbeck amp Ronquist2001)

An alternative phylogenetic hypothesis test was car-ried out by imposing constraints on the MP analyses Forthese analyses species were forced into groups accordingto the phylogeny of the family as inferred from morpho-logical data (Coomans 1985 1996) The trees obtainedwith these constraints were tested by implementing SH-test (Shimodaira amp Hasegawa 1999) with likelihood set-ting previously generated from the Modeltest The testedhypothetical monophyletic groups included the subfam-ilies Longidorinae and Xiphinematinae and the generaXiphidorus Xiphinema and Paralongidorus

Results and discussion

SECONDARY STRUCTURE OF D2 AND D3 EXPANSION

REGION

Secondary structures of the expansion region wereinferred for each species The general secondary structureof the D2 region of longidorids was composed of threelong stem and loop structures (Fig 1A) some variationswere observed (data not shown) The D3 structure wasrather conserved across the species studied (Fig 1B)Variation was found in the D4_1 stem and loop regionof D3 structure which were absent in several Longidorusspecies

PHYLOGENY

The base composition of the D2 and D3 expansionregions did not reveal high heterogeneity between thespecies No significant differences in base compositionwere observed in the D2 region (χ2 = 20949 df = 219P = 067) or the D3 region (χ2 = 5305 df = 219 P =10) Partition homogeneity analyses (Farris et al 1994)resulted in P = 022 which supported the analyses ofthe combined D2 and D3 regions The g1 statistic for theD2 and D3 datasets was minus035 and minus029 respectivelyindicating that the data sets contain good phylogeneticsignal (Hillis amp Huelsenbeck 1992) Statistic g1 was

calculated by evaluating the tree length distribution of10 000 random trees the g1 for the combined datasetequalled minus037

The ML and MP analyses of the D2 dataset andthe combined dataset produced similar tree topologiesAnalyses of the D3 dataset did not resolve all lineages al-though they also recovered the strongly supported cladesinferred from D2 alone and from the combined dataset(data not shown)

MP analysis of the combined dataset resulted in 5154maximum parsimonious trees with a tree length of 2732A consensus tree of the 5154 equally scored trees ispresented in Figure 2 Bootstrap values and decay indicescalculated for the consensus trees are added to thecorresponding nodes The tree topology obtained from theweighted MP analysis was the same as the one obtainedfrom unweighted MP analysis (data not shown)

Maximum likelihood analyses were performed on thecombined datasets The tree topology obtained from thecombined dataset is similar to that of the MP trees Theselected model was GTR + + I (general time reversibleplus gamma rates and proportion of invariable sites)Eleven heuristic searches resulted in 11 ML trees thatwere compared by the Kishino-Hasegawa tests (KH-test)The 11 topologies were not significantly different Thetree with the highest likelihood score (lnL = minus1299865)was selected as the default best ML tree (Fig 3A)

The NJ tree inferred from the tree edit distance ofthe secondary structures maintained clades statisticallystrongly supported in the ML and MP analyses (Fig 3B)

POSITION OF GENERA

Both the ML tree (Fig 3A) and MP tree (Fig 2) showedthe same phylogenetic clades The monophyly of thegenus Longidorus was strongly supported with 10 BPPfor the tree inferred by ML analysis and 92 BS in theMP analysis

Non-X americanum-group species and X america-num-group species (both classified in the genus Xiphi-nema) were strongly supported as two isolated clades (10BPP for non-X americanum-group and 092 for X ame-ricanum-group in the ML analysis and 100 BS for thenon-X americanum-group and 53 for the X america-num-group in the MP analysis) The analysis of the ITS1-rRNA by Ye et al (2004) also yielded trees with two sep-arate clades within Xiphinema The non-X americanum-group was supported as a sister clade to Longidorus (098BPP in ML analysis and 69 BS in MP analysis)

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Fig 2 Phylogeny of the Longidoridae inferred from maximum parsimony analysis of the sequences of the D2 and D3 expansion regionsof the LSU rRNA gene Bootstrap support (more than 50) and decay index (starting with d) are shown in the MP consensus tree

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Table 2 Results of the SH-tests for different topologies and alternative hypotheses

Topologies and hypothesis tested minusLnL Difference of minusLnL P value

ML tree 1299865 best ndashMP tree 1302252 2388 049Distance tree based on secondary structurea 1400306 100441 000

Bayesian consensus treeb 1304884 5020 029All Xiphinema species constrained into one group 1306422 6558 0109Paralongidorus constrained to be a group outside of Longidorus 1318411 18546 0002

Xiphidorus constrained to be a group outside of all Xiphinema species 1319879 20014 0001

P lt 005 indicates the significant differences between the two inferred tree topologya Distance tree inferred from the tree edit distance of secondary structurea The 50 majority consensus tree obtained in the result of BI analysis

The two Xiphidorus species were grouped together with10 BPP in the ML analysis and 100 BS in the MPanalysis Instead of a closer position to Longidorus theXiphidorus species were grouped with the X america-num-group species (073 in ML analysis and 58 BS inMP analysis)

The clade composed of the two Paralongidorus specieswas strongly supported (10 BPP in ML analysis and100 BS in MP analysis) It clustered as an internal cladeof the genus Longidorus confirming the results of thephylogenetic analysis made by Rubtsova et al (2001)

Our analyses of both the sequence and secondary struc-ture based phylogenies support four major clades withinthe Longidoridae i) the Longidorus clade (including Pa-ralongidorus) ii) the clade composed of the non-X ame-ricanum-group species of the genus Xiphinema iii) the Xamericanum-group clade and iv) the Xiphidorus clade

Recently published analyses of ITS1-rRNA (Ye et al2004) and 18S rRNA (Oliveira et al 2004) also revealedtwo distinct major groups within the genus Xiphinemaand are fully congruent with the results of our analysisThe phylogenetic testing of our D2D3 tree (Table 2) didnot refute the monophyly of the genus Xiphinema eventhough it was split into two major clades (P = 0109)The genus Paralongidorus was rejected as a valid taxon(P = 0002) The genus Xiphidorus was rejected as agroup outside of genus Xiphinema (P = 0001)

MOLECULAR EVOLUTION OF THE SECONDARY

STRUCTURE

Although the tree generated from the tree edit distancesof the secondary structure shared the major clades withtrees obtained from ML and MP analyses it neverthelessdiffered significantly (P = 000) from the ML tree in-

ferred from the sequence data (Table 2) Remarkable dif-ferences were observed for the positions of two species(X radicicola ndash V1273 X brasiliense ndash EU41) belong-ing to the non-X americanum-group (Figs 2 3A) whichin the secondary structure tree were grouped with Longi-dorus species because their derived secondary structuresin stem loop C5 of the D2 region were similar to those inseveral Longidorus species (Fig 3B) These derived struc-tures presumably represent convergence The notable de-rived feature of the D3 region was the loss of the D4_1stem-loop structure in several Longidorus species Eightspecies that had lost the D4_1 structure were distributed inthree clades obtained with sequence analyses six speciesforming the strongly supported clade including L car-pathicus L elongatus L piceicola L intermedius L ju-venilis and L leptocephalus (098 BPP in ML analysis)one species L profundorum was positioned with L at-tenuatus in one clade supported with 089 BPP in MLanalysis and one species L latocephalus in the cladecontaining L caespiticola (Fig 3A) We also noted thatsix species from the above mentioned three clades wereclustered into one clade in the NJ tree inferred from thesecondary structure distance matrix (Fig 3B) while twospecies (L piceicola and L intermedius) from the cladeincluding L carpathicus and L leptocephalus were posi-tioned with L goodeyi in another clade because of theirderived C5 stem-loop structure in the D2 expansion re-gion (Fig 3B) Additionally some minor differences ofstructural evolution resulted in changes of the positions ofseveral species inside the major clades (Fig 3) All discre-pancies described above may reflect differences of evolu-tionary rates between the nucleotide sequences and theirsecondary structures

120 Nematology


Fig 4 A Tree derived from the phylogeny of the genus Xiphinema proposed by Coomans et al (2001) B Tree containing Xiphinemaspecies (except for X americanum-group) derived from the molecular ML tree

Fig 5 A Tree derived from the phylogeny of Xiphinema americanum-group proposed by Lamberti and Ciancio (1993) B Treecontaining the Xiphinema americanum-group species derived from the molecular ML tree

CORRELATION WITH MORPHOLOGICAL CHARACTERS

AND GROUPS

Longidorus and Paralongidorus

The only interesting correspondence between morpho-logical characters and phylogenetic trees inferred frommolecular data is the grouping of Longidorus species co-incident with similarity in the amphid structure as previ-ously noticed by Rubtsova et al (2001) (Fig 3A) Twogroups were observed in the tree One group included Lcaespiticola L helveticus and L macrosoma with funnelshaped amphid pouches (Type 1 see Fig 3A) the othergroup included L africanus L apulus L arthensis Lathesinus L attenuatus L breviannulatus L carpathi-cus L edmundsi L elongatus L euonymus L inter-medius L juvenilis L leptocephalus L piceicola L pro-fundorum and L sturhani with symmetrically (Type 2) orL euonymus and L goodeyi with asymmetrically (Type 3)lobed amphids The remaining species L camelliae L di-

adecturus (Type 5) and L latocephalus did not form onegroup although L camelliae and L latocephalus share thesame Type 4 amphid pouch The amphids with a stirrup-shaped pouch were only found in species of the genusParalongidorus which formed a separate group withinLongidorus Examining more species of Paralongidorusshould reveal the distribution of the other amphid shapesreported for the genus

Mapping of amphid types on the tree suggests that Type3 and Type 4 appeared several times during evolution ofthe genus Longidorus The correspondence implies thatevolution of some molecules may be synchronous with theevolution of some morphological characters even whenthere is no obvious morphogenetic link between bothThis synchronous evolution facilitates the recovery of cor-rect phylogeny based on both molecular and morpholo-gical analyses especially for extant taxa lacking informa-tive fossil records such as nematodes

Vol 7(1) 2005 121

Y He et al

Xiphinema and Xiphidorus

The phylogeny of the genus Xiphinema was constructedby Coomans et al (2001) based on 44 morphologicalcharacters To facilitate the analyses the authors subdi-vided the sampled species into several groups accordingto tail shape The tree topology obtained from our analy-ses is very close to the topology obtained by Coomans etal (2001) as redrawn here in Figure 4A Congruent group-ings included those of X dentatum and X pyrenaicum thegroup X dentatum X pyrenaicum X index and X diver-sicaudatum the group including X coxi and X basiri andthe large group including all species cited above as well asX bakeri X setariae and X radicicola The positions forother species also corresponded fairly well between bothanalyses However the two topologies differed distinctlyin the position of the X americanum-group A reasonableexplanation is that the evolutionary rates of the D2 and D3expansion regions of these species are not synchronisedwith the evolution of the selected morphological charac-ter (tail shape)

Giving consideration to all Xiphinema species and ex-cluding those of the X americanum-group we conducteda KH-test (with RELL approximation) between two de-rived trees One tree was derived from the ML tree basedon the combined D2 and D3 dataset (Fig 3A) the otherwas derived from the tree shown in Figure 4 The re-sult does not show significant differences between the twotopologies (P lt 005 P = 0001)

Examining more species of the genus Xiphidorus mayreveal the distribution of the three reported amphid shapeswithin the genus besides the cup-shaped amphid pouch(Type 4) observed in Xiphidorus minor

The Xiphinema americanum lineage

In our analyses the X americanum lineage appears asa clade close to Xiphidorus albeit with low support frombootstrap analyses (Fig 2) Within the lineage we ob-served two groups well supported in our analyses Thefirst group (X americanum subgroup) included X ame-ricanum X brevicollum and several virus vector speciesthe second group (X pachtaicum subgroup) included Xpachtaicum X pachydermum and X brevisicum an am-phimictic species (Lamberti et al 2000) In comparisonwith cluster analysis based on morphological characters(Lamberti amp Ciancio 1993) an analysis that resulted inthe subdivision of the X americanum-group into the Xbrevicollum X americanum X taylori X pachtaicumand X lamberti subgroups our results merged the X tay-lori subgroup into the X brevicollum subgroup itself part

of the X americanum subgroup (Fig 5) As we did nothave a species from the X lamberti subgroup at our dis-posal we could not infer position and consequently therelationships of this subgroup remain unclear

Conclusion

This is the first extensive study using large subunitrDNA molecular data to infer the phylogeny of the fam-ily Longidoridae Our analysis revealed four major groupswithin Longidoridae Longidorus the X americanum-group other Xiphinema species and Xiphidorus Thegenus Paralongidorus was clustered as an internal groupof the genus Longidorus and ML testing rejected the va-lidity of this genus Although the result of the alternativephylogenetic hypotheses testing (Table 2) did not refutethe monophyly of the genus Xiphinema the species ofthis genus were split into two distinct clades in all treesThe genus Xiphinema was originally described by Cobbin 1913 (Lamberti et al 2000) the type species being Xamericanum itself a member of course of the X ame-ricanum-group If additional analyses of other genes re-veal the same phylogenetic pattern in the distribution ofXiphinema sl species then it may become necessary torestrict Xiphinema to the species of the X americanum-group and establish one or more additional genera alongthe lines hypothesised by Lamberti and Bleve Zacheo(1979) for the remainder of the species Comparativeanalysis revealed that sequence-based vs structural phy-logenies can lead to different results and are not alwayscongruent

Acknowledgement

This research was partly funded by the Commissionof the European Union Contract N SMT1506 for theproject Xiphinema americanum-group virus-vector ne-matodes development of a diagnostic protocol

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BREMER K (1994) Branch support and tree stability Cladis-tics 10 295-304

122 Nematology


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

CANNONE JJ SUBRAMANIAN S SCHNARE MN COL-LETT JR DrsquoSOUZA LM DU Y FENG B LIN NMADABUSI LV MULLER KM PANDE N SHANGZ YU N amp GUTELL RR (2002) The comparative RNAweb (CRW) site An online database of comparative sequenceand structure information for ribosomal intron and otherRNAs BioMed Central Bioinformatics 3

CATE JH YUSUPOV MM YUSUPOVA GZ EARNESTTN amp NOLLER HF (1999) X-ray crystal structures of70S ribosomal functional complexes Science 285 2095-2104

CHEN Q HOOPER DJ LOOF PAA amp XU J (1997)A revised polytomous key for the identification of speciesof the genus Longidorus Micoletzky 1922 (Nematoda Dory-laimoidea) Fundamental and Applied Nematology 20 15-28

CLARK CG TAGUE BW WARE VC amp GERBI SA(1984) Xenopus laevis 28S ribosomal RNA a secondarystructure model and its evolutionary and functional implica-tions Nucleic Acids Research 12 6197-6220

COBB NA (1913) New nematode genera found inhabitingfresh water and non-brackish soils Journal of the WashingtonAcademy of Sciences 3 432-444

COOMANS A (1985) A phylogenetic approach to the classi-fication of the Longidoridae (Nematoda Dorylaimida) Agri-culture Ecosystems and Environment 12 335-354

COOMANS A (1996) Phylogeny of the Longidoridae RussianJournal of Nematology 4 51-60

COOMANS A HUYS R HEYNS J amp LUC M (2001)Character analysis phylogeny and biogeography of thegenus Xiphinema Cobb 1913 (Nematoda Longidoridae) An-nalen Zooumllogische Wetenschappen Koninklijk Museum voorMidden-Afrika Tervuren Belgieuml 287 1-239

DE RIJK P ROBBRECHT E DE HOOG S CAERS AVAN DE PEER Y amp DE WACHTER R (1999) Database onthe structure of large subunit ribosomal RNA Nucleic AcidsResearch 27 174-178

ELLIS RE SULSTON JE amp COULSON AR (1986) TherDNA of C elegans sequence and structure Nucleic AcidsResearch 14 2345-2364

ERIKSSON Y (2001) AutoDecay ver 50 Bergius FoundationRoyal Swedish Academy of Sciences Stockholm

FARRIS JS KAumlLLERSJOuml M KLUGE AG amp BULT C(1994) Testing significance of incongruence Cladistics 10315-319

GOLDMAN N ANDERSON JP amp RODRIGO AG (2000)Likelihood-based tests of topologies in phylogenetics Syste-matic Biology 49 652-670

HALL TA (1999) BioEdit a user-friendly biological se-quence alignment editor and analysis program for Windows9598NT Nucleic Acids Symposium Series 41 95-98

HICKSON RE SIMON C COOPER A SPICER GSSULLIVAN J amp PENNY D (1996) Conserved sequencemotifs alignment and secondary structure of the third do-main of animal 12SrRNA Molecular Biology and Evolution13 150-169

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

HOFACKER IL (2003) Vienna RNA secondary structureserver Nucleic Acids and Research 31 3429-3431

HUELSENBECK JP amp RONQUIST F (2001) MrBAYESBayesian inference of phylogeny Bioinformatics 17 754-755

KAPLAN DT THOMAS WK FRISSE LM SARAH J-L STANTON JM SPEIJER PR MARIN DH amp OP-PERMAN CH (2000) Phylogenetic analysis of geographi-cally diverse Radopholus similis via rDNA sequence revealsa monomorphic motif Journal of Nematology 32 134-142

KIER KM (1995) Use of rRNA secondary structure inphylogenetic studies to identify homologous positions Anexample of alignment and data presentation from the frogsMolecular Phylogenetics and Evolution 4 314-330

LAMBERTI F amp BLEVE ZACHEO T (1979) Studies onXiphinema americanum sensu lato with descriptions of fif-teen new species (Nematoda Longidoridae) NematologiaMediterranea 7 51-106

LAMBERTI F amp CIANCIO A (1993) Diversity of Xiphinemaamericanum-group species and hierarchical cluster analysisof morphometrics Journal of Nematology 25 332-343

LAMBERTI F MOLINARI S MOENS M TAYLOR CEamp BROWN DJF (2000) The Xiphinema americanum-group I Putative species their geographical occurrence anddistribution and regional polytomous identification keys forthe group Russian Journal of Nematology 8 65-84

LAMBERTI F MOLINARI S MOENS M amp BROWN DJF(2002) The Xiphinema americanum-group II Morphometricrelationships Russian Journal of Nematology 10 99-112

LIMA MB (1965) Studies on species of the genus Xiphinemaand other nematodes PhD Thesis University of LondonUK 165 pp

LOOF PAA amp LUC M (1990) A revised polytomous keyfor the identification of species of the genus Xiphinema Cobb1913 (Nematoda Longidoridae) with exclusion of the Xamericanum-group Systematic Parasitology 16 35-66

LYDEARD C HOLZNAGEL WE SCHNARE MN ampGUTELL RR (2000) Phylogenetic analysis of molluscanmitochondrial LSU rDNA sequences and secondary struc-tures Molecular Phylogenetics and Evolution 15 83-102

MUELLER F SOMMER I BARANOV P MATADEEN RSTOLDT M WOumlHNERT J GOumlRLACH M VAN HEELM amp BRIMACOMBE R (2000) The 3D arrangement of

Vol 7(1) 2005 123

Y He et al

the 23S and 5S rRNA in the Escherichia coli 50S ribosomalsubunit based on a cryo-electron microscopic reconstructionat 75 Aring resolution Journal of Molecular Biology 298 35-59

OLIVEIRA CMG HUumlBSCHEN J BROWN DJF FER-RAZ LCCB WRIGHT F amp NEILSON R (2004) Phy-logenetic relationships among Xiphinema and Xiphidorus ne-matode species from Brazil inferred from 18S rDNA se-quences Journal of Nematology 36 153-159

POSADA D amp CRANDALL KA (1998) Modeltest testingthe model of DNA substitution Bioinformatics 14 817-818

RAMBAUT A amp GRASSLY NC (1997) Seq-Gen an applica-tion for the MonteCarlo simulation of DNA sequences evo-lution along phylogenetic trees Computational and AppliedBioscience 13 235-238

RUBTSOVA TV SUBBOTIN SA BROWN DJF ampMOENS M (2001) Description of Longidorus sturhani spn (Nematoda Longidoridae) and molecular characterizationof several longidorid species from western Europe RussianJournal of Nematology 9 127-136

SCHIMODAIRA H amp HASEGAWA M (1999) Multiple com-parisons of Log-Likelihoods with applications to phyloge-netic inference Molecular Biology and Evolution 16 1114-1116

SHAPIRO BA amp ZHANG K (1990) Comparing multipleRNA secondary structures using tree comparison Computa-tional and Applied Bioscience 6 309-318

SIDDIQI MR BAUJARD P amp MOUNPORT D (1993) De-scriptions of Paratylenchus pernoxius sp n and Paralongi-dorus duncani sp n from Senegal and the synonymizationof Longidoroides with Paralongidorus Afro-Asian Journal ofNematology 3 81-89

SUBBOTIN SA VIERSTRAETE A DE LEY P ROWE JWAEYENBERGE L MOENS M amp VANFLETEREN JR(2001) Phylogenetic relationships within the cyst-formingnematodes (Nematoda Heteroderidae) based on analysisof sequences from the ITS regions of ribosomal DNAMolecular Phylogenetics and Evolution 21 1-16

SWOFFORD DL (2002) PAUP Phylogenetic Analysis UsingParsimony (and Other Methods) Version 4 SunderlandMA USA Sinauer Associates 142 pp

TARJAN AC (1969) Variation within the Xiphinema ameri-canum group (Nematoda Longidoridae) Nematologica 15241-252

TAUTZ D HANCOCK JM WEBB DA TAUTZ C ampDOVER GA (1988) Complete sequences of the rRNAgenes of Drosophila melanogaster Molecular Biology andEvolution 5 366-376

TAYLOR CE amp BROWN DJF (1997) Nematode vectorsof plant viruses Wallingford Oxon UK CAB International286 pp

THOMPSON JD GIBSON TJ PLEWNIAK F JEANMOU-GIN F amp HIGGINS DG (1997) The ClustalX windowsinterface flexible strategies for multiple sequence alignmentaided by quality analysis tools Nucleic Acids Research 244876-4882

TITUS TA amp FROST DR (1996) Molecular homologyassessment and phylogeny in the lizard family Opluridae(Squamata Iguania) Molecular Phylogenetics and Evolution6 49-62

VAN DE PEER Y NICOLAIuml S DE RIJK P amp DE WACHTERR (1996) Database on the structure of small ribosomalsubunit RNA Nucleic Acids Research 24 86-91

WILCOX TP ZWICKL DJ HEATH TA amp HILLIS DM(2002) Phylogenetic relationships of the dwarf boas and acomparison of Bayesian and bootstrap measures of phylo-genetic support Molecular Phylogenetics and Evolution 25361-371

YE W SZALANSKI AL amp ROBBINS AT (2004) Phylo-genetic relationships and genetic variation in Longidorus andXiphinema species (Nematoda Longidoridae) using ITS1 se-quences of nuclear ribosomal DNA Journal of Nematology36 14-19

ZUKER M MATHEWS DH amp TURNER DH (1999) Al-gorithms and thermodynamics for RNA secondary structureprediction A practical guide In Barciszewski J amp ClarkBFC (Eds) RNA biochemistry and biotechnology NATOASI Series Kluwer Academic Publishers pp 11-43

124 Nematology

Y He et al

mists encountered many morphological varieties amongpopulations studied Lima (1965) and Tarjan (1969) sug-gested that X americanum is a complex of several speciesSince then 51 nominal species have been placed in thisgroup (Lamberti et al 2000) The traditional identifica-tion of these species by morphology and morphometricsis very difficult because many of the characters used over-lap Moreover questions concerning the true phylogenybehind such a complicated group and the position of thespecies within this group have been raised (Lamberti etal 2002) So far phylogenetic analyses of Longidoridaeare few The phylogeny based on selected morphologicalcharacters of the genus Xiphinema was analysed in greatdetail by Coomans et al (2001) Rubtsova et al (2001)used a molecular approach to investigate the phylogeneticposition of a few species from the genera Longidorus andParalongidorus Recently the phylogeny of longidoridswas inferred from ITS1 analyses (Ye et al 2004) and 18SrDNA sequences (Oliveira et al 2004)

Molecular systematic approaches are useful for pro-ducing phylogenies especially in cases where morpholo-gical characters lead to ambiguous interpretation In re-cent years ribosomal RNA genes have attracted the at-tention of many systematists and evolutionists becauseof their functional importance their ease of amplificationand because they are assumed to record the evolutionaryhistory of the organism relatively faithfully (Lydeard etal 2000) In nematodes rRNA gene sequences were usedto infer the phylogeny of many groups including somephytoparasitic taxa (eg Al-Banna et al 1997 Blaxter etal 1998 Kaplan et al 2000 Subbotin et al 2001)

When assembled into the ribosome together with otherproteins ribosomal RNA is usually folded into a com-plicated secondary and tertiary structure Although someof these structures have been detected by X-ray crys-tallisation (Cate et al 1999) or by cryo-electron mi-croscopic reconstruction (Mueller et al 2000) most ofthe structures deposited in the public domain such asthe Antwerp database of large (De Rijk et al 1999)and small rRNA sequences (Van de Peer et al 1996)(httpwwwpsbugentberRNAindexhtm) and the com-parative RNA website (httpwwwrnaicmbutexasedu)(Cannone et al 2002) are derived from comparativeanalysis which generates the folding from the commoncompensatory substitutions and pairing patterns on manysequences The secondary structure of rRNA providesa very useful template for improved construction of se-quence alignments and critical for phylogenetic construc-tion (Kier 1995 Hickson et al 1996) Researchers have

achieved some success by using alignments refined withthe aid of the secondary structure and an optimised com-puter algorithm (Titus amp Frost 1996) Additionally sec-ondary structure can provide useful information for as-sessing the sequence in weighted parsimony or otherweighted methods Structural motifs may themselves con-tain information useful for phylogeny inference (Lydeardet al 2000)

In this paper we report on the first phylogenetic analy-ses of the family Longidoridae using molecular data col-lected from the LSU rRNA gene We also tried to providesystematists with persuasive molecular information to aidin the reconstruction of those taxa Species were sampledand used from all of the longidorid genera with the excep-tion of Longidoroides which was however synonymisedwith the genus Paralongidorus (Siddiqi et al 1993) astatus that is still questioned (Coomans 1996) Paraxiphi-dorus and the monotypic Australodorus

Materials and methods

TAXON SAMPLING

Nematode samples collected for this study togetherwith their authorities are listed in Table 1 and include23 species from the genus Longidorus two species fromthe genus Paralongidorus two species from the genusXiphidorus and 35 species from the genus XiphinemaThe majority of the populations were identified on thebasis of both morphometrics and morphology only forsome populations was the identification made on thebasis of general morphology These latter populations aremarked with an asterisk in Table 1

TOTAL DNA EXTRACTION

One juvenile or adult nematode was transferred into13 microl ddH2O and cut into two to five pieces witha sterilised scalpel Ten microl 2 times worm lysis buffer(20 mM Tris-HCl (pH 80) 100 mM KCl 30 mM Mg2Cl20 mM DTT 09 Tween 20) and 01 microl proteinaseK stock solution (20 mgml) was added to a 200 or500 microl microcentrifuge tube The nematode fragmentswere pipetted up in 99 microl ddH2O and added to thetube which was then briefly centrifuged and stored atminus70C for at least 10 min Subsequently each tubewas incubated at 65C for 1-2 h and the proteinase Kwas denatured at 95C for 10 min Finally the DNAsuspensions were cooled to 4C and stored at minus20C

112 Nematology

















Vol 7(1) 2005 113

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Table 1 (Continued)










PCR AMPLIFICATION





114 Nematology




ANALYSES


PHYLOGENY INFERENCE

Alignment









Vol 7(1) 2005 115

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REGION


PHYLOGENY







POSITION OF GENERA



116 Nematology


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Vol 7(1) 2005 117

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118 Nematology


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Vol 7(1) 2005 119

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STRUCTURE



120 Nematology





AND GROUPS





Vol 7(1) 2005 121

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Conclusion


Acknowledgement


References



122 Nematology































Vol 7(1) 2005 123

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124 Nematology

















Vol 7(1) 2005 113

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Table 1 (Continued)










PCR AMPLIFICATION





114 Nematology




ANALYSES


PHYLOGENY INFERENCE

Alignment









Vol 7(1) 2005 115

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REGION


PHYLOGENY







POSITION OF GENERA



116 Nematology


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1S

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dary

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Vol 7(1) 2005 117

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118 Nematology


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obed

6

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rup-

shap

e7

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est

irru

p-sh

ape

(aft

erC

hen

etal

19

97)

Vol 7(1) 2005 119

Y He et al












STRUCTURE



120 Nematology





AND GROUPS





Vol 7(1) 2005 121

Y He et al








Conclusion


Acknowledgement


References



122 Nematology































Vol 7(1) 2005 123

Y He et al




















124 Nematology

Y He et al

Table 1 (Continued)










PCR AMPLIFICATION





114 Nematology




ANALYSES


PHYLOGENY INFERENCE

Alignment









Vol 7(1) 2005 115

Y He et al





REGION


PHYLOGENY







POSITION OF GENERA



116 Nematology


Fig

1S

econ

dary

stru

ctur

em

odel

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and

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Vol 7(1) 2005 117

Y He et al


118 Nematology


Fig

3

Phy

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hin

the

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ape

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erC

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etal

19

97)

Vol 7(1) 2005 119

Y He et al












STRUCTURE



120 Nematology





AND GROUPS





Vol 7(1) 2005 121

Y He et al








Conclusion


Acknowledgement


References



122 Nematology































Vol 7(1) 2005 123

Y He et al




















124 Nematology




ANALYSES


PHYLOGENY INFERENCE

Alignment









Vol 7(1) 2005 115

Y He et al





REGION


PHYLOGENY







POSITION OF GENERA



116 Nematology


Fig

1S

econ

dary

stru

ctur

em

odel

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and

D3

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nsio

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Vol 7(1) 2005 117

Y He et al


118 Nematology


Fig

3

Phy

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the

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19

97)

Vol 7(1) 2005 119

Y He et al












STRUCTURE



120 Nematology





AND GROUPS





Vol 7(1) 2005 121

Y He et al








Conclusion


Acknowledgement


References



122 Nematology































Vol 7(1) 2005 123

Y He et al




















124 Nematology

Y He et al





REGION


PHYLOGENY







POSITION OF GENERA



116 Nematology


Fig

1S

econ

dary

stru

ctur

em

odel

sof

the

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and

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nsio

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led

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tatd

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rtio

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siti

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Vol 7(1) 2005 117

Y He et al


118 Nematology


Fig

3

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onsh

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the

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19

97)

Vol 7(1) 2005 119

Y He et al












STRUCTURE



120 Nematology





AND GROUPS





Vol 7(1) 2005 121

Y He et al








Conclusion


Acknowledgement


References



122 Nematology































Vol 7(1) 2005 123

Y He et al




















124 Nematology


Fig

1S

econ

dary

stru

ctur

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odel

sof

the

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and

D3

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nsio

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Vol 7(1) 2005 117

Y He et al


118 Nematology


Fig

3

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19

97)

Vol 7(1) 2005 119

Y He et al












STRUCTURE



120 Nematology





AND GROUPS





Vol 7(1) 2005 121

Y He et al








Conclusion


Acknowledgement


References



122 Nematology































Vol 7(1) 2005 123

Y He et al




















124 Nematology

Y He et al


118 Nematology


Fig

3

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19

97)

Vol 7(1) 2005 119

Y He et al












STRUCTURE



120 Nematology





AND GROUPS





Vol 7(1) 2005 121

Y He et al








Conclusion


Acknowledgement


References



122 Nematology































Vol 7(1) 2005 123

Y He et al




















124 Nematology


Fig

3

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19

97)

Vol 7(1) 2005 119

Y He et al












STRUCTURE



120 Nematology





AND GROUPS





Vol 7(1) 2005 121

Y He et al








Conclusion


Acknowledgement


References



122 Nematology































Vol 7(1) 2005 123

Y He et al




















124 Nematology

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STRUCTURE



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AND GROUPS





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Conclusion


Acknowledgement


References



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AND GROUPS





Vol 7(1) 2005 121

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Conclusion


Acknowledgement


References



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Conclusion


Acknowledgement


References



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