Journal of Nematology 42(4):359–369. 2010. Ó The Society of Nematologists 2010. Molecular rDNA phylogeny of Telotylenchidae Siddiqi, 1960 and evaluation of tail termini

L. K. CARTA,A.M.SKANTAR,Z.A.HANDOO

Abstract: Three stunt , leviterminalis, T. dubius and T. claytoni were characterized with segments of small subunit 18S and large subunit 28S rDNA sequence and placed in molecular phylogenetic context with other polyphyletic taxa of Telotylenchidae. Based upon comparablysized phylogenetic breadth of outgroupsand ingroups, the 28S rDNA contained three times the number of phylogenetically informative alignmentcharacters relative to the alignment total compared to the larger 18S dataset even though there were fewer than half the number of taxa represented. Tail shapes and hyaline termini were characterized for taxa within these subfamily trees, and variability discussed for some related species. In 18S trees, similar terminal tail thickness was found in awell-supportedclade of three Tylenchorhynchus:broad-tailed T. leviterminalis branched outside relatively narrow-tailed T. claytoni and T. nudus.Terminal tail thickness within Merliniinae, Telotylenchinae and related taxa showed amosaic distribution. Thick-tailed Trophurus, Macrotrophurus and putative Paratrophurus did not group together in the 18S tree. Extremely thickened tail termini arose at least once in Amplimerlinius and Pratylenchoides among ten species of Merliniinae plus three Pratylenchoides,and three times within twelve taxa of Telotylenchinae and Trophurinae. Conflicting generic and family nomenclaturebased on characters such as pharyngeal overlap are discussedinlight of current molecular phylogeny.Contrary to some expectations from current , Telotylenchus and Tylenchorhynchus cf. robustus did not cluster with three Tylenchorhynchus spp. Twoputative species of Neodolichorhynchus failed to group together,and two populations of Scutylenchus quadrifer demonstratedasmuch or greater genetic distance between them than among three related species of Merlinius. Key words: character analysis, evolutionary convergence, morphology, nomenclature, phylogeny,stunt nematode, systematics, tail, taxonomy, Tylenchorhynchus.

Stuntnematodes (Tylenchorhynchussensu lato)and them areoften notdiscrete. Molecularphylogenetic relatives within theTelotylenchidae Siddiqi, 1960 are analyses areneededtoevaluatecompeting taxonomic extremely common in the rhizosphere of nativeand schemesand thecharactersonwhich they arebased cultivated plants. Becauseofthe large number of stunt before anynew namescan be readilyaccepted. These nematode species, taxonomists have been motivated to phylogeniescan provideanindependent meanstoun- simplify identification into more manageable generic derstand characterdistributionthatimpacts stability units, butanunusual number of confusingand com- of higher taxonomiccategories. Molecularsequences peting systemsbased on differentcharacter priorities also provideimportant informationongenetic variation nowexist.Since an earliercomprehensivereviewofTy- of populationswithinmorpho-species, andevaluating lenchorhynchus sensulato (Allen,1955),various taxonomic whether similar species that lack males should be synon- designations forstunt nematode genera have been pro- ymized with species that have them. Tied to morphology, posed, from Merlinius, Quinisulcius and Uliginotylenchus similarity searches of sequences areespecially useful when listed in thecompendiumofTarjan, 1973 througha competing generic and subfamily names areused in the currentassemblageoffive genera within Merliniinae literature,asiscurrently the case forTelotylenchidae. Siddiqi, 1971,twelvegenerainTelotylenchinae Siddiqi, An isolate of Tylenchorhynchusleviterminalis (Siddiqi, 1960,and sixjunior synonyms under Tylenchorhynchus Mukherjee and Dasgupta, 1982) Siddiqi, 1986 was itself (Siddiqi,2000).These taxa were basedondifferent identified by us from aforeignplant interception, and hierarchiesofcharacterssuchaspharyngeal glandover- there are no recordsofthis species’ existence in North lap, number of linesinthe lateralfield, male genitalia, America.ITS rDNA sequences are availableinGenBank andmajor differencesinfemaleterminal tail features for T. leviterminalis (Chenetal., 2006),but comparable (Jairajpuriand Hunt,1984; Gomez-Barcinaetal.,1992). sequences from other relatives are lacking.Among However, some of thenewer genericnames of stunt species of Tylenchorhynchus,ithas arelatively thicktail, nematodesand relativeshavebeenignored in recent but this character has not previously been examined in compendiafor ease of practicalidentification (Fortuner relation to thetails of other family members in amor- andLuc,1987; Brzeskiand Dolinski,1998; Handoo, phology-independent molecularphylogenetic context. 2000), andthe morphologicalcharacterstodistinguish Alimited number of telotylenchine taxa were included in recent small subunit (SSU) 18S (Holterman et al., 2006; Meldal et al., 2007; Holterman et al., 2009; van Received for publication January11, 2008. Megen et al., 2009) and large subunit (LSU) 28S trees Nematology Laboratory, USDA, ARS, HenryA.Wallace Beltsville Agricultural Research Center,Bldg. 011A, Room 165B, Beltsville, MD 20705-2350. (Subbotinetal., 2006),demonstrating that Telotylen- We thank Donna Ellington, Maria Hult, Jennifer Kramer and Sharon Ochs chinae and Merliniinae arepolyphyletic. They also dem- for excellent technical assistance. We thank Michael Petrillo, USDA-APHIS, San Francisco for T. leviterminalis and Stephen Fraedrich, USDA Forest Service, onstratedsupport for theMerliniidae(Siddiqi,1971) Athens, GA for Tylenchorhynchusclaytoni.Mentionofatrade name or commer- Ryss, 1993, an amended family generally possessing cial product in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. deirids (except in Scutylenchus)that includes Merlinii- Department of Agriculture. nae and Pratylenchoides. They also supported the Telo- E-mail:[email protected] This paper was edited by Dr.Gregor Yeates. tylenchidaeSiddiqi, 1960/syn. Tylenchorhynchidae Eliava, 359 360 Journal of Nematology,Volume 42, No. 4, December 2010

1964 that includes members of Tylenchorhynchinae Eliava, 1964, Trophurinae Paramonov,1967, Macro- trophurinae Fotedar and Handoo, 1978, and Teloty- lenchinae Siddiqi, 1960 as reviewed in Siddiqi (2000). However,information from these broad molecular trees, each with relativelyfew taxa,was insufficient for inferences about morphologicalcharacters within the subfamilies to be made. Therefore, we generated both LSU and SSU rDNA sequences for T. leviterminalis and two othercommon species, T. claytoni Steiner,1937and Bitylenchus dubius (Bu¨tschli, 1873) Filipjev,1934 [= T. dubius (Bu¨tschli, 1873) Filipjev,1936] and constructed phylogenetic trees in order to investigate their genetic relationships and especially for tail character analysis. Thickened female tail terminiare immediately notice- able traits among tylenchid , so tail termini measurementsbased on specimensand literature were mapped onto atreefor an initiallookatcharacter dis- tributionand reliabilityastheyrelatetotaxonomyand nomenclature.

MATERIALS AND METHODS

Specimens: Tylenchorhynchusleviterminalis is found in Asia and was collected in late 2001 from soil originating in Vietnam and relayed via APHIS in February2002 to the USDA Nematology Laboratoryfor species identifi- cation. Tylenchorhynchus claytoni was found in soil from sorghuminTrenton, SC in April 2005. Bitylenchus du- bius originated from soil under acool season perennial bunch grass (probable orchard grass, Dactylisglomerata) at the base of asycamore (Platanus occidentalis)tree in Beltsville, MD.Specimens were identified and imaged with high-power light microscopy beforeprocessing for PCR. Microscopy: Tail images were taken with aZeiss Ultra- phot III (Carl Zeiss, Inc., Jena, Germany, and Baltimore Instrument Company, Baltimore, MD,USA) using Dif- ferential InterferenceContrast (DIC) optics, and re- corded withaToshiba IKTU CCDcamera (Toshiba Corp., Japan) (Fig. 1). Tail drawings representingtaxa FIG.1. Bitylenchus and Tylenchorhynchus female tails, lateral view A) used in 18S trees (Fig. 2) were made from original and B. dubius. B, C) T. claytoni.D)T. leviterminalis.Scale Bar =10mm. other descriptions(Allen, 1955; Caveness, 1958; Loof, 1958;Loof, 1956,Loof, 1959,Loof, 1963,Loof, 1978; Thorne,1949; Tylenchorhynchus cf. robustus,Paratrophurus PCR andsequencing: Multipleadults were collected sp., and Sauertylenchus maximus measures made from web and identified for B. dubius and T. claytoni, and asingle vouchers at http://nematode.unl.edu/). Images were female wasused for T. leviterminalis.Nematodes were scannedand uniformlysized usingHyperSnap-DXver. mechanically disrupted in 20 mlofextraction buffer as 5.60.00(Hyperionics,Inc., Murrysville, PA)and Photo- described by Thomasetal. (1997), and then stored in Shop ver. CS (Adobe SystemsInc., SanJose, CA). The PCRtubes at –80oCuntil needed. Extracts were prepared percentofhyalinetailterminuslengthtototal tail length by incubating the tubes at 60oCfor 60 min, followed by wascalculatedfromthese drawings and/or literature 95oCfor 15 min to deactivate the proteinase Kand and coded as moderately thick (+) $ 4%, thick+$9%, or centrifuged briefly prior to use in PCR. For 28S, each very thick ++ ($ 20%) (Fig.2)and assignedtotree 25 mlreaction contained 1unit Platinum Taq(In- branches in tree Figures 3through 5. Taxonomic cate- vitrogen, Carlsbad, CA), 1X reaction buffer [20 mM gories and synonyms with thenomenclature of Siddiqi Tris-HCl pH 8.4,50mMKCl, 2.5 mM MgCl2], 0.2 mM (2000)used in this work arealsogiven in thetables. dNTP mix, 0.8 mMprimersD2A (5’-ACAAGTACCGTG Stuntnematode phylogenyand tail morphology: Carta et al. 361

FIG.2.Tail terminal drawings of Merliniidae sensu Ryss, 1993 and Telotylenchidae sensu Siddiqi, 2000 (syn. Tylenchorhynchidae Eliava, 1964). Drawings based on literature, web vouchers of the Powers lab at UniversityofNebraska,USDANC slides and Fig. 1. Coding of terminal tail thickening for verythick ($ 20%) ++, for thick +($9%), and (+) for moderately thick terminal tail ($ 4%) are given for the %ratio of hyaline tail length/tail length: Psilenchus hilarulus <1%-(Thorne, 1949), Pratylenchoides ritteri 22.5% ++ (Sher,1970), Pratylenchoides magnicauda 19%+(+)(Baldwinetal.,1983), Merliniusmicrodorus 2.6% (+)(Geraert, 1966), Merliniusbrevidens 3% (+) (Allen,1955), Merliniusjoctus 16%+ (Thorne andMalek,1968), Nagelusleptus 11 -16%+(Thorne,1949, http://nematode.unl.edu/nagle4.jpg,Powersetal.,1983), Nagelus obscurus 4.3% (+)(Allen, 1955), Nagelusalpinus 3.7% (+)(Allen, 1955), Scutylenchusquadrifer 9.3–13.7%+(Loof, 1978), Amplimerlinius macrurus 20%++(USDANC slideG-3022), Amplimerlinius icarus 27%++(USDANC slideG3024), Tylenchorhynchusrobustus 11.3%+(Thorne and Malek,1968), Tylenchorhynchusleviterminalis 9.4-16%+, 10.4 -16.2% +(Loof,1959),and Tylenchorhynchus claytoni 12.5 -25% +(+), Macrotrophurusarbusticola 46.7%++(Loof,1958), Bitylenchus dubius 4.4%(+), Telotylenchusventralis 6% (+)(Loof,1963), Sauertylenchus maximus 11 -14% +(http://nematode.unl.edu/tymax15.jpg, Allen, 1955), Neodolichorhynchusmicrophasmis 7.7% (+)(Loof, 1959), Neodolichorhynchus lamelliferus 3.1% (+)(Allen, 1955), Quinisulcius acutus 4.5-17%+(Allen,1955, http://nematode.unl.edu/quina9. jpg), Trophurusimperialis 30%++(Loof,1956), Trophurusminnesotensis 21%++(Caveness, 1958), Trophurussculptus 25%++(Loof,1956), Tylenchorhynchus cf. robustus 7% (+)(http://nematode.unl.edu/tylerob3.jpg), Paratrophurus sp.13% +(http://nematode.unl.edu/patrop. htm).Scale Bar=10 mm.

AGGGAAAGTTG-3’) and D3B(5’-TCGGAAGGAACCA PCR products were visualized with UV illumination GCTACTA-3’), and 5 mlnematode extract. Cycling was after ethidium bromide staining. DNA was excised from performedasdescribed in De Ley et al. (2005). Partial the gelsand purified with the QIAquickGel Extraction 18S sequencewas amplified in two overlappingseg- Kit (Qiagen, Valencia, CA). Clean PCR products were ments, using the primers SSU-550F (5’-GGCAAGTCT sequenced directly at the UniversityofMaryland Center GGTGCCAGCAGCC-3’) witheukR(10) (5’-TGATCCT for Biosystems Research. DNAsequences were assem- CCTGCAGGTTCACCTAC-3’), and SSU-385F (5’-CGG bled using Sequencher 4.7 (Genecodes, Ann Arbor, TGGTTATAACGGGTAACGGAG-3’) with18S-R-1108R MI). DNA sequences were analyzedusing the BLASTN (5’-CCACTCCTGGTGGTGCCCTTCC-3’)(more infor- megablast program optimizedfor highly similar se- mation availableathttp://nematol.unh.edu/protocols. quences, http://www.ncbi.nlm.nih.gov/blast/Blast.cgi. php).Reactions were assembledin25mland included Sequences were submitted to GenBank under accession 1unit DyNAzyme polymerase (MJResearch,Waltham, numbers T. leviterminalis 18S (EU368585), Bitylenchus

MA), 1X reaction buffer including 1.5 mM MgCl2, dubius 18S (EU368586), T. claytoni 18S (EU368587), 0.63 mMeachprimer, 0.2mMdNTPmix,and 2.5 ml T. claytoni 28S D2-D3 (EU368589), B. dubius 28S D2-D3 template DNA. Cyclingconditionsfor 18Sconsisted of (EU368590), and T. leviterminalis 28S D2-D3 (EU368591). 1cycle of 94oCfor 2min,followedby40cyclesof94oC Phylogenetic Reconstruction: To construct 18Strees,Gen- for30sec,58oCfor 30 sec, and72oCfor 2min,and Bank SSUrDNAsequences were collectedfor various finishing with 1cycle of 72oCfor 10 min. genera and species of Telotylenchinae and outgroups 362 Journal of Nematology,Volume 42, No. 4, December 2010

TABLE 1. SSU 18S rDNA GenBank Sequences for Merliniinae, Telotylenchinae,Macrotrophurinae, (Radopholinae) and Outgroups in Fig. 3, 4Trees.

Accession #Nematode Species and Strain Bp #SequenceReference AJ966511 Tylenchulus semipenetranse 1740 Meldal et al., 2007 AJ966471 Hemicycliophoraconidae 1764 Meldal et al., 2007 EF025336 sp.e 1723 Ye et al., 2007 DQ912919 longicaudatuse 1725 Zeng et al., 2007 AY284593 Psilenchus cf. hilaruluse 1710 Holterman et al., 2006 AY919271 Psilenchus hilaruluse 634 Powers et al., 2005 AJ966494 aberranse 1765 Meldal et al., 2007 FJ969114 Amplimerlinius macrurusa 1731 van Megen et al., 2009 EU306351 Amplimerlinius icarusa 1764 Bert et al., 2008 EU306352 Bitylenchus dubiusb (T) 1746 Bert et al., 2008 AY284595 Macrotrophurus arbusticola c 1714 Holterman et al., 2006 AY284597 Merlinius brevidensa 1709 Holterman et al., 2006 FJ969128 Merlinius joctusa 1731 van Megen et al., 2009 AY919184 Merlinius cf. microdorusa 634 Powers et al., 2005 AY146449 Nagelus alpinusa, g (M) 634 Mullin, 2004 EU306350 Nagelus obscurusa 1760 Bert et al., 2008 AY919217 Nagelus leptusa 634 Powers et al., 2005 AY284598 Neodolichorhynchus lamelliferusa 1598 Holterman et al., 2006 AY593903 Neodolichorhynchus microphasmisa (T) 837 Holterman et al., 2006 AY919229 Paratrophurus sp.b 635 Powers et al., 2005 FJ969137 Pratylenchoides sp.d 1732 van Megen et al., 2009 AF202157 Pratylenchoidesmagnicaudad 1643 Fe´lix et al., 2000 AJ966497 Pratylenchoidesritteri d 1831 Meldal et al., 2007 DQ080517 Quinisulciush acutus 634 Powers et al., 2005 AY993979 Sauertylenchus maximusb (T) 1766 Meldal et al., 2007 AY284599 Scutylenchusi quadrifera 1598 Holterman et al., 2006 AY993977 Scutylenchusi quadrifer (G)a 1765 Meldal et al., 2007 AY593905 Telotylenchush ventralisb 1743 Holterman et al., 2006 FJ969144 Trophurus imperialisb 1743 van Megen et al., 2009 AY146555 Trophurus minnesotensisb 635 Mullin, 2004 DQ080547 Tylenchorhynchus cf. robustusb 1695 Powers et al., 2005 EU368587 Tylenchorhynchus claytoni b, f 1338 Skantar,Carta, Handoo EU368585 Tylenchorhynchus leviterminalisb, f 1407 Skantar,Carta, Handoo DQ080546 Tylenchorhynchus nudusb 634 Powers et al., 2005

Synonyms given with these accessions: (G) = Geocenamus,(M) = Merlinius (T) = Tylenchorhynchus. Bp =base pair or nucleotide. aMerliniinae. bTelotylenchinae. cMacrotrophurinae. dPratylenchidae (Radopholinae). eOutgroups. fOriginal sequences. gSynonym Merliniusalpinus (Powers et al., 1983). hJunior synonyms of Tylenchorhynchus (Fortuner and Luc, 1987). iGisolate of Scutylenchus quadrifer listed as Geocenamus quadrifer (Andra´ssy,1954) Brzeski, 1991 in agreement with Brzeski (1991) who considered Scutylenchus a junior synonym, but this species was not includedwithin arecent 12-species key of Geocenamus (Chitambar and Ferris, 2005).

(Table 1.). Outgroups included Tylenchulussemipenetrans and Hunt, 1984, Paratrophurus sp. Arias, 1970, Pratylen- Cobb,1913, Hemicycliophora conida Thorne,1955, Do- choidesmagnicauda (Thorne, 1935) Baldwin, Luc and lichodorus sp. Cobb,1914, Belonolaimus longicaudatus Rau, Bell1983, Pratylenchoides ritteri Sher,1970, Pratylenchoides 1958, and Psilenchus hilarulus de Man, 1921. Ingroup sp. (Thorne, 1935) Baldwin, Luc and Bell1983, Quini- taxa included Amplimerlinius macrurus (Goodey, 1932) sulcius acutus (Allen, 1955) Siddiqi, 1971, Sauertylenchus Siddiqi 1976, Amplimerliniusicarus (Wallace and Greet, maximus (Allen,1955) Siddiqi, 2000, Scutylenchus quad- 1964) Siddiqi 1976, Bitylenchusdubius, Macrotrophurus rifer (Andrassy, 1954)Siddiqi,1979, Telotylenchusventralis arbusticola Loof, 1958, Merliniusbrevidens (Allen, 1955) Loof,1963, Trophurusimperialis Loof,1956, Trophurus Siddiqi,1970, Merliniusjoctus (Thorne, 1949) Sher minnesotensis (Caveness, 1958)Caveness, 1959, Tylencho- 1974, Merlinius cf. microdorus (Geraert, 1966) Siddiqi, rhynchus nudus Allen, 1955,and Tylenchorhynchus cf. ro- 1970, Nagelusalpinus (Allen, 1955) Siddiqi, 1979, bustus Thorne andMalek,1968. Cf.was used to designate Nagelus obscurus (Allen, 1955) Powers, Baldwin apopulationassimilar to avalid speciesbut notidenti- and Bell, 1983, Nagelus leptus (Allen, 1955) Siddiqi, fiedassuchwithcertainty. 1979, Neodolichorhynchus(Mulkorhynchus) lamelliferus (de To construct a28S tree, Tylenchorhynchusleviterminalis, Man, 1880) Volkova, 1993, Neodolichorhynchus(Neo- T. claytoni,and Bitylenchus dubius sequences were as- dolichorhynchus)microphasmis (Loof, 1959) Jairajpuri sembled with the following taxa having LSUrDNAD2-D3 Stuntnematode phylogenyand tail morphology: Carta et al. 363

TABLE 2. LSU 28S rDNA Genbank Sequences for Merliniinae, Telotylenchinae,Macrotrophurinae, Pratylenchidae (Radopholinae) and Outgroups in Fig. 5Tree.

Accession #Nematode Species and Strain Bp #SequenceSource AY780972 Tylenchulussemipenetranse 547 Subbotin et al., 2005 AY780973 Hemicycliophora typicae 542 Subbotin et al., 2005 DQ915803 Belonolaimus longicaudatuse 723 Zen et al., 2007 DQ838803 Dolichodorus mediterraneuse 755 Jimenez Guirado et al., 2007 DQ328716 Psilenchus sp.e 655 Subbotin et al, 2006 AM412741 Nacobbus aberranse,d 316 Vovlas et al., 2007 DQ328714 Amplimerlinius icarusa 653 Subbotin et al, 2006 EU368590 Bitylenchus dubius (T) b,f 654 Skantar,Handoo, Carta DQ328708 Macrotrophurus arbusticolac 662 Subbotin et al, 2006 DQ328715 Nagelus leptusa 652 Subbotin et al, 2006 DQ328709 Trophurus sculptusb 671 Subbotin et al, 2006 EU368589 Tylenchorhynchus claytonib,f 661 Skantar,Carta, Handoo EU368591 Tylenchorhynchus leviterminalisb,f 660 Skantar,Carta, Handoo

Synonymsgiven with these accessions:(G) = Geocenamus,(T) = Tylenchorhynchus. Bp =basepair or nucleotide. aMerliniinae. bTelotylenchinae. cMacrotrophurinae. dPratylenchidae (Nacobbinae). eOutgroups. fOriginal sequences. sequences from GenBank in Table 2: Tylenchulus semi- into the MrBayes plugin whichran 1.1 million chains penetrans, Hemicycliophora typica de Man, 1921, Belono- with Burnin =110,000. Tree structures in Figs. 3–5 are laimus longicaudatus, Dolichodorus mediterraneus Jime´nez based on the RAxML phylogeny, withbranch support Guirado et al., 2006, (Thorne,1935) values above 50% given for ML followed by those for BI, Thorne and Allen, 1944, Amplimerlinius icarus, Trophu- and ML parameters given in figure legends. Bootstrap russculptus Loof, 1956, Macrotrophurus arbusticola,and proportions (BP) that represent ‘true’ clades with 95% Nagelus leptus. confidenceintervals occur above 70%, alevel consid- Alignments were made with ClustalW2 (Larkin et al., ered robust support,withmoderate support between 2007) checked by eye for consistency of conserved po- 50-70%.Maximum likelihood BPsare mostly lowerthan sitions, and edited in GeneDoc (Nicholas et al., 1997). Bayesian Posteriorprobability (BPP)scoresthatuse all Initially the alignment was run through PAUP*4b10 data rather than subsamples (reviewedinZander, 2004). (Swofford, 2002).Heuristic simple and bootstrapped Maximum Parsimony (MP) searches were conducted RESULTS AND DISCUSSION employing tree bisection-reconnection(TBR) branch swapping, and accelerated transformation (ACCTRAN) The morphology of male and female heads and tails character-state optimization.The computationally- of B. dubius (Fig. 1A), T. claytoni (Fig. 1B,C),and T. intensive, probabilistic Maximum likelihood(ML) leviterminalis (Fig. 1D)were consistent with original method is less affected by sampling error and infers descriptions and revisions (Steiner,1937; Thorne, 1949; better trees than distance or parsimony methods Siddiqi et al., 1982; Golden et al., 1987; Vovlasand (Swofford et al., 1996),soMLtrees are presented in Cheng, 1988). figures. Alignments were subjected to ModelTest ver. Bitylenchus dubius had acrenate tail tip that was not 3.7 (Posada and Crandall,2001) as implementedin always easy to see at certain planes of focus(Fig. 1A). Geneious Pro ver.4.7 (Biomatters, Auckland, New The hyalinetail ranged from 8to12%, n=6ofthe tail Zealand; Drummondetal., 2009). The Akaike in- length in this population of B. dubius. While thetail formation criterion(AIC) for model selection wasused was not figured in the original description of Tylenchus rather than that of thelikelihood ratiotest (LRT) due dubius females (Bu¨tschli, 1873),itwas drawnlater to demonstrated superiority (Posada and Buckley, (Goodey, 1931) with ahyaline region 12% of the tail 2004) and because it is the standard within the Gene- length. iousmodule. Alignmentsinphylip format were run in Tylenchorhynchusclaytoni had asmooth tail tip, with web-based RAxML (Stamatakis et al., 2008) with100 the hyaline tail region ranging from 12 to 25%, n=6, bootstrap runs and maximumlikelihoodestimateof of which the terminal cuticle represented 4to7%of 25 per site rate categories.The alignment was also the tail length;hyaline depositsofone (Fig. 1B)or subjected to Bayesian inference (BI) analysis with the two layers (Fig. 1C)can also be seen. Most of the tail MrBayes (Huelsenbeck and Ronquist, 2001) plugin for variation alreadydescribed in otherpopulations of Geneious. ModelTest parameters were used for input T. claytoni involved theshape, number of annules and 364 Journal of Nematology,Volume 42, No. 4, December 2010

FIG.3.Maximum Likelihood (ML) best SSU 18S tree of Telotylenchinae and Macrotrophurinae as implemented in RAxML, including Bayesian Inference (BI) clade support. Clade support percentages for ML are followed by BI near nodes, with __ representing the absence of acorresponding value. The 1731 position ClustalW alignment had 346 distinct alignmentpatterns. Likelihoodoffinal tree evaluated and optimized under GAMMA, Final ML Optimization Likelihood:-4777.415574, Model Information: alpha: 0.178052,Tree-Length: 0.738249. The percentage of hyaline tail terminus length to total tail length coded as moderatelythick (+) $ 4%, thick + $ 9%, or verythick ++ ($ 20%). their proximity to the tip (Golden et al., 1987), and not monyinformative characters, yielding eight trees from the quality of internal tissue as viewed withDIC. The aheuristic search. ModelTest found ModelTamura-Nei currently studied population of T. claytoni had relatively (TrN) +I+G,withnst =6,gammashape =0.591,and thicker and more variable hyaline tail dimensions proportion of invariantsites (pinvar)=0.516.BIre- compared to other related taxa with tail variation drawn sulted in Log likelihood (LnL) mean =-5883.53, TL in theliterature, e.g. Tylenchorhynchus tritici Golden et al., mean =0.744,and alpha shape parameterofgamma 1987 (13.5to18%,n=5)(Golden et al., 1987), T. nudus distribution =0.196. (10.5to15.5%,n=3)(Loof,1959), T. areoterminalis Among the ingroupswithin the three outgroups in Siddiqi, 2008 (17to21%,n=2)(Siddiqi, 2008), Scuty- the Fig. 3MLtree there was abasal clade with 100% lenchus (= Merlinius) quadrifer (11.4to14.3%,n=3)and ML/96% BI support for (Tylenchorhynchus nudus +T. Merliniusrugosus (15.6to18.4%,n=3)(Loof,1978). claytoni)and T. leviterminalis branching just outside. For T. leviterminalis, the hyaline tail region repre- This grouphad asistergroup composed of the other sented 11.6 to 16%, n=6ofthe tail. A T. leviterminalis telotylenchid taxa: Sauertylenchusmaximus and Bitylenchus population from Chinahad abouta15%hyalinetail/tail dubius outside apolytomyofthree groupsof(Telotylenchus proportion basedonmedianvalues (Vovlas and Cheng, ventralis, Quinisulcius acutus, two species of Trophurus), 1988),and apopulation from Japan had a23to24% (Neodolichorhynchusmicrophasmis), and (Macrotrophurus hyaline tail region based on derived average measure- arbusticola, N. lamelliferus, Tylenchorhynchusrobustus/ ments and adrawing (Talavera et al., 2002). Paratrophurus sp.). These last two morphologically- Drawings plus relative measurements and codes of identified generahad identical sequencefor pop- tail terminifor theother taxa represented in molecular ulations with different hyaline tail dimensions. The BI phylogenetic trees are given in Fig. 2. tree (notshown)was somewhat different from the ML 18S Trees for Telotylenchinae and Macrotrophurinae tree in having Trophurus branching outside the three (Fig. 3): The three types of trees (MP, ML, and BI) had Tylenchorhynchus species (72%clade support), and Ty- slightly different topologies and only theMLtree is lenchorhynchus cf. robustus/Paratrophurus outside the shown in Fig. 3. MP analysis detected 209/1731 parsi- entireremaining ingroup (74%clade support). For all Stuntnematode phylogenyand tail morphology: Carta et al. 365

FIG.4.Maximum Likelihoodbest SSU 18S tree for Merliniinae and Pratylenchoides spp. as implemented in RAxML, including Bayesian Inference (BI) clade support. Clade supportpercentages for ML are followed by BI near nodes, with __ representing the absence of acorre- sponding value. The 1777 position Clustal Walignment had 98 distinct alignment patterns. Likelihoodoffinal tree evaluated and optimized under GAMMA+P-Invar,MLOptimization Likelihood: -2580.137694, alpha: 0.020013,pinvar: 0.000117, Tree-Length: 0.105073. The per- centage of hyaline tail terminus length to total tail length coded as moderatelythick (+) $ 4%, thick + $ 9%, or very thick ++ ($ 20%). trees, Macrotrophurus did not group with Trophurus into nuscarvedfrom Tylenchorhynchus called Macrorhynchus aclade of taxa with long hyaline tails, nor did Teloty- Sultan,Singhand Sakhuja, 1991,basedoncoarsebody lenchus group with Tylenchorhynchus,aprediction from annulationsand continuous lipregion(Sultan et al., proposed synonymy of Telotylenchus (Fortuner and Luc, 1991). This scheme is notcongruent with thecurrent 1983).Both species of Neodolichorhynchus failed to tree topology since T. nudus makesamore likely andwell group together. Neodolichorhynchus is agenus charac- supportedclade with T. claytoni rather than with T. levi- terized by longitudinalridges outside thelateral field. terminalis, andputative T. robustus is farremoved from Onesubgenus Neodolichorhynchus (Neodolichorhynchus) mi- this cladeinFig.3.One discrete difference amongthese crophasmis was defined without abursal notchand lacking Tylenchorhynchus spp. is that T. robustus hasmorethan lateral vulval membranes,while Neodolichorhynchus (Mul- twicethe number of tailannules (40–50)compared to the korhynchus)lamelliferus had these features (Jairajpuri and other three Tylenchorhynchus (10–21)(in Handoo, 2000). Hunt,1984).Inlight of theirdiverged position relative to Bitylenchus Filipjev,1934 and Sauertylenchus Sher,1974 oneanother on theFig. 3tree, subgenus Mulkorhynchus were located on adjacent branches (Fig. 3), consistent lamelliferus might change rank in thefuture. with their lack of agubernaculum crest that is present In the 18S alignments, there were only 2nucleotide in Tylenchorhynchus and Paratrophurus (Gomez-Barcina differences (0.3% of sequence) between T. claytoni and et al., 1992; Siddiqi, 2000). T. nudus and between T. claytoni and T. leviterminalis. Trophurus Loof, 1956 was thefirst among Te- They all had similar tail thickness, but the T. levitermi- lotylenchidae to be defined by its enlarged hyalinetail nalis tail was wider. Therewere about 40 nucleotide region plus asingle gonad. Paratrophurus Arias, 1970 differences between T. leviterminalis and thepopulation had two female gonads, and its thicktail wasloosely similar to T. robustus. defined by ‘‘cuticle strongly swollen on tail terminus.’’ Tylenchorhynchus leviterminalis, T. nudus and T. robustus Variousdegrees of ovaryregression areoften associated butnot T. claytoni were included in aproposednew ge- with tail regression in Paratrophurus (Luc et al., 1987; 366 Journal of Nematology,Volume 42, No. 4, December 2010

FIG.5.Maximum Likelihood (ML) best LSU 28S tree for Telotylenchidae as implemented in RAxML, including Bayesian Inference (BI) clade support. Clade support percentages for ML are followed by BI near nodes, with __ representing the absence of acorresponding value. The 780 position ClustalW alignmenthad 412 distinct alignment patterns. GAMMA+P-Invar Model parameters, Final ML Optimization Likelihood: -4993.382411, alpha: 1.317998, invar: 0.323274,Tree-Length: 4.438284. The percentage of hyaline tail terminus length to total tail length coded as moderately thick (+) $ 4%, thick + $ 9%, or very thick ++ ($ 20%).

Kleynhans, 1992). Paratrophurusloofi Arias, 1970from scription of Tylenchorhynchusbursifer (pre-Paratrophurus wheat in Sevilla, Spain, had ahyalinetailregion rep- synonymy) showed a60% hyaline tail proportion (Loof, resenting 33% of the tail (Arias, 1970). This type species 1959).Tail terminimeasurements of other species as- had amuch shorter postanalintestinal sac than that in signed to the genus Paratrophurus (Arias, 1970) ranged asecondspecies, Paratrophurus acristylus Siddiqi and from 20 to 40% (Castillo et al., 1989; Kleynhans, 1992). Siddiqui, 1983. The latterwas described as having the These proportions overlap those of T. leviterminalis, the hyaline portion of the female tail terminus equaling 21 current population of T. claytoni, and other Tylencho- to 28% of tail length as wellasaprominent postanal rhynchus species with long hyaline tails such as T. clav- intestinal sac, afeaturealso characteristic of Bitylenchus icaudatus Seinhorst, 1963 (34.5%) (Seinhorst, 1963). spp. (Gomez-Barcina et al., 1992). Bitylenchus dubius in Voucherimages of female Paratrophurus spp. and Tylen- particular has verysimilar morphometrics to P. acristylus chorhynchus cf. robustus (Mullin, 2000a; Mullin,2000b) from Libya and Morocco (Castillo et al., 1989),but P. revealed differencesinthickness of hyalinetailtermini acristylus has asomewhatshorter,thicker tail and ter- (13% vs.7%) despitehavingidentical 18Ssequences. minus (B. dubius vs. P. acristylus:c=12to17vs.16to19, This situationillustrates thedifficulty in assigninggenera c’ =2.2 to 3.7 vs.2.3 to 2.7 in Brzeskiand Dolinski, 1998; to species or populations with tail retraction, and no Castillo et al., 1989), and lips not annulated or offset. firm phylogenetic conclusionscan be made until se- Except for the tail terminus, Paratrophurus spp. are very quences from definedspecies and type populations of similar to Tylenchorhynchus spp. (Castillo et al., 1989; Paratrophurus arecompared. Siddiqi,2000).Anevenmore forcefulargumentwas 18S trees for Merliniidae(Fig. 4): Threetypes of trees made for Paratrophurus synonymy on the basis of in- had slightlydifferenttopologies and only the ML tree is termediate length tails of Paratrophurus bursifer populations shown. MP analysisdetected 39/1777 parsimony in- extending into the range of those for Tylenchorhynchus formative characters yielding 28 trees from aheuristic spp. (Sturhan and Lisˇkova´, 2004).The original de- search employing TBR branch swapping. ModelTest Stuntnematode phylogenyand tail morphology: Carta et al. 367 selected GTR+I+G, gammashape=0.806, andpinvar = In this 28Stree, Macrotrophurus wasbasal to Bitylenchus 0.812. BI gave LogLikelihood (LnL)mean=-3279.557, dubius,bothofwhich formed asistergroup with Tylen- Tree length (TL) mean =0.117,and alphashape pa- chorhynchus claytoni and T. leviterminalis. Belonolaimus and rameterofgamma distribution =0.038. Dolichodorus were positioned betweenthese Telotylen- Aclade of Merlinius and Scutylenchus (92% support) chinae/Tylenchorhynchidae, dividing them from Nage- withthick tails formedasister clade to Pratylenchoides lus, Amplimerlinius (Merliniinae)and Psilenchus.Nacobbus spp.,and these three generaplus Amplimerlinius (very was included based on its appearance outside Teloty- thick tails) formed asister group to Nagelus (moder- lenchinae and Macrotrophurinae in arecent Bayesian ately thick to thicktails). This could be interpreted as tree (Holterman, 2009),sothe appearanceinthistree verythick tailsarisingfirst in Amplimerlinius,continuing of Macrotrophurus in the expected positionofNacobbus in Pratylenchoides and reverting to merely thick tails outside the other Telotylenchinae may be an artifact of in Merlinius and Scutylenchus.The two populations of insufficient taxon sampling. The topology of this tree Scutylenchus quadrifer demonstrated as muchorgreater was otherwise congruentwith those from the 18S trees, geneticdistance between them as among the three re- although thesparsetaxon representation does notpro- lated species of Merlinius, possibly due to cryptic spe- vide much informationfor taxonomicevaluation. ciation, different haplotypes or misidentification. Thick-tailed Trophurus and Macrotrophurus did not Nagelus alpinus (Siddiqi, 1979; Siddiqi,2000) is con- group together in any 18S tree or in the 28S tree. Re- sidered Merlinius by some, so it is significant that N. gardless of the variationintree topologies, thick and alpinus didnot appear within the well-supported Mer- thin tail terminialternatedwithin tree clades and at the linius and Scutylenchus clade.Relatively long hyaline tail species and genus level in these trees. From hyalinetail regions were noted in Nagelus leptus and related species measurements (Fig. 2), and tail termini designations on (sequences not available), having deirids at the part of thetrees,itappears that very thicktailterminihave the lateral field where there are six incisures, as op- arisen at leastthree timeswithinthisassemblageofTe- posed to the other Nagelus spp. with deirids at the lotylenchinaewith Trophurus,Macrotrophurus and Tylen- junction of four incisures (Powers et al., 1983). Ampli- chorhynchus claytoni (Fig.3)and once for Amplimerlinius merlinius spp.,characterizedbythickenedfemale tail and Pratylenchoides within this group of Merliniinae/ terminal cuticle and extended hyaline tail regions, also Merliniidae (Fig. 4). had deirids and six linesinthe lateral field (Siddiqi, Argumentsoverwhich morphological characters will 1976). Consistentwith similar morphology (Powersetal., be most reliable overtime underlie conflicting higher 1983) Amplimerlinius spp. branched just outside Nagelus taxonomiccategories.The original character of great- spp. est historical concern to stunt nematode taxonomy was 28S trees of Merliniidae and Telotylenchidae (Fig. 5): the degree of overlap, if any,ofthe pharyngeal glands Threetypes of trees had slightlydifferent topologies relative to theintestine(Thorne, 1949).Anumber of and only the ML tree is shown. MP analysis detected taxonomists have argued againstthe use of this char- 302/780 parsimonyinformative characters yielding a acter at the family and even genus level (Fortuner and singletree of length 1020, and CI =0.63. ModelTest Luc, 1987; Loof, 1987). It is interesting that Telotylenchus gavethe General time reversible model (GTR) +I+G, with overlapping glands and a Tylenchorhynchus-like face nst =6,gamma shape =1.2967, and pinvar =0.3307. BI pattern (Sher and Bell, 1975) was far removed from the gaveLnL mean =-5017.331, TL =3.262,and alpha cladein18S treescontaining Tylenchorhynchus,agenus shapeparameterofgamma distribution =0.435. composed of specieseitherlacking or possessing aslight In terms of information content measured by abso- overlap(e.g. T. clarus Allen, 1955). Also Pratylenchoides lute numbersofparsimonyinformative characters in ritteri hadalong glandoverlap(Fortuner andLuc,1987) these tree alignments, the28S tree had 31% more unlike thetaxathatsurroundeditinthe tree. parsimony informative characters than the 18S align- In summary, populations of thick-tailed Trophurus, ment for Telotylenchinae (Fig. 3) and ten timesmore Macrotrophurus andputative Paratrophurus did not than that in the Merliniinae alignment (Fig. 4). The group together in any moleculartree, and tail thickness parsimony informative characters dividedbythe total was mosaically distributed among species within Merli- alignment characters were 39% for the 28S Fig. 5 niinae andTelotylenchinae andrelated taxa,withex- alignment, 12% for the 18S Fig. 3alignment, and 2.3% tremethickness arisingatleast once in Merliniinaeand forthe Fig. 4alignment. Thereforethe 28S rDNA align- three timesinTelotylenchinae. However,more taxa ment contained at least threetimes thenumberofphy- and molecularcharacters are needed to better delin- logenetically informative alignmentcharacters relative to eate and support various groups represented by this the alignmenttotal compared to the larger 18S dataset. data set. Although it is currently amajor character for There is also broader taxonsampling for the 18S mole- differentiating generaofTrophurus, Paratrophurus, Telo- cule which is better forrevealing deeperphylogenetic tylenchoides Siddiqi, 1971,Meiodorus Siddiqi, 1976, and relationships than for these genus and species level Amplimerlinius (Siddiqi, 2000),the striking character of comparisons. athickened,retracted female tail terminus should be 368 Journal of Nematology,Volume 42, No. 4, December 2010 considered ahighly convergent, species-level feature. Drummond,A.J., Ashton, B., Cheung, M., Heled, J., Kearse, M., Otherwise, insufficient or inappropriate keys may be Moir,R., Stones-Havas,S., Thierer,T., and Wilson, A. 2009. Geneious v4.7. Available from http://www.geneious.com/. erroneously consulted for borderline populations sim- Fe´lix, M. A., de Ley,P., Sommer,R.J., Frisse, L., Nadler,S.A., ilar to Paratrophurus or thick-tailed Bitylenchus or Tylen- Thomas, W. K., Vanfleteren,J., and Sternberg, P. W. 2000. Evolution of chorhynchus.Itisimportant to initially identify stunt vulva development in the Cephalobina (Nematoda). Developmental nematodeswithinabroad framework. Newercomponent Biology 221:68–86. genera or possibly subgenera mightearnwider usage Fortuner,R., and Luc, M. 1987. Areappraisal of Tylenchina once theirrelatives fillinthe not-always obviousgaps (Nemata). 6. The family Whitehead, 1960. Revue de within the spectrumofcurrent sequences. If paraphyly Ne´matologie 10:183–202. Geraert, E. 1966. The systematic position of the families Tylen- continuestobeconfirmed withmoretaxa for ribosomal chulidae and Criconematidae. Nematologica12:362–368. genes andfor key taxa using othergenes, usage of Telo- Golden, A. M., Maqbool, M. A., and Handoo, Z. A. 1987. De- tylenchidaewill be inappropriate if taxonomy is to reflect scriptions of two new species of Tylenchorhynchus Cobb, 1913 (Nem- monophyleticgroups. WhetherTelotylenchidae persists atoda: ) with details on morphologyand variation of should have little effect on alphataxonomythough.The T. claytoni.Journal of Nematology 19:58–68. basic tension betweenpractical identification withstable Gomez-Barcina, A., Siddiqi, M. R., and Castillo, P. 1992. The genus Bitylenchus Filipjev,1934 (Nematoda: Tylenchida) with descriptions namesand more theoretical phylogenetics forrefining of two new species from Spain. Proceedings of the Helminthological taxonlimits(de Pinna, 1999) contributes to competing Society of Washington 59:96–110. namesfor stuntnematodes.Agreementonone system is Goodey,T.1931. The genus Anguillulina Gerv. and v. Ben., 1859, vel notlikely in the near future. Tylenchus Bastian, 1865. Journal of Helminthology 10:75–180. Handoo,Z.A.2000. Akey and diagnostic compendium to the species of the genus Tylenchorhynchus Cobb, 1913 (Nematoda: Belo- nolaimidae). Journal of Nematology 32:20–34. 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