Nematology,2003,V ol.5(3), 405-420

Baujardia mirabilis gen. n., sp. n.from pitcher plants andits phylogenetic position withinPanagrolaimidae (Nematoda: )

1; 2 1 Wim BERT ¤, Irma TANDINGAN DE LEY , Rita VAN DRIESSCHE , Hendrik SEGERS 3 and Paul DE LEY 2 1 Departmentof Biology,Ghent University, Ledeganckstraat 35, 9000 Gent, Belgium 2 Departmentof Nematology,University of California, Riverside, CA 92521,USA 3 FreshwaterLaboratory, Royal Belgium Institute for Natural Sciences, V autierstraat29, 1000 Brussels, Belgium Received:16 September2002; revised: 13 January2003 Acceptedfor publication:13 January2003

Summary – Measurements,line drawings and scanning electromicrographs are provided of Baujardiamirabilis gen.n., sp. n., isolated frompitcher  uidof Nepenthesmirabilis fromThailand. The new genus differs from all known in having two opposing andoffset spermatheca-like pouches at the junction of oviduct and uterus. It also differs from most known Rhabditida in having four cephalicsetae instead of papillae.Phylogenetic analysis of small subunit rDNA sequencedata robustly places the new genus within Panagrolaimidaeas asistertaxon to Panagrellus .Theseunusual nematodes resemble Panagrellus inbodysize (1.8-2.7 mm infemales, 1.3-1.9mm inmales) and in the monodelphic, prodelphic female reproductive system with thickened vaginal walls and prominent postvulvalsac. However, they differ from Panagrellus inthecharacters mentioned above, in their comparatively longer stegostom and intheshape of themale spicules. Because of its aberrant characters, inclusion of this new genus in Panagrolaimidae requires changes tothefamily diagnosis. Keywords – Nepenthes,newgenus, new species, Panagrellus ,phylogeny,, Thailand.

Duringa visitto Tung-Kai Botanical Garden in Trang Materials andmethods Province,near the Isthmus of Kra, SouthernThailand, oneof us (Hendrik Segers) sampledthe liquid con- SAMPLING tainedwithin pitchers of thecarnivorous plant Nepenthes mirabilis (Lour.)Druce, in order to obtain some of the Sampleswere collectedby Ž lteringthe content of peculiarrotifers recorded from suchhabitats. Except for some50 pitchers through a 50 ¹mplanktonnet and someunidentiŽ able bdelloids, no rotiferswere found,but were immediatelyŽ xedby adding one tenth of the thesamples contained abundant material of aremarkable samplevolume of concentratedformalin (36%) at ambient newnematode genus together with some diplogastrids. temperature. Nepenthesmirabilis was encounterednear Someof the unique morphological characters of this theend of thetrail through the primary swamp forest of newgenus make its placement difŽ cult. In order to ob- TungKai Botanical garden. A singlebulk sample was tainadditional information about the identity of the ne- collectedon 18 July 1999. Several additional samples, matodeand its position in relation to known taxa, we includinga samplepreserved in 70% ethanol, were conductedmolecular phylogenetic analyses based on the collectedon 13January2002. smallsubunit (SSU) ribosomalDNA. Inthis paper, we describethis new genus and species from Nepenthes and MORPHOLOGICALOBSERVATIONS presentan emended diagnosis of thefamily Panagrolaim- For lightmicroscopy, nematodes were processedto an- idae. hydrousglycerol (Seinhorst, 1959) and mounted on alu- miniumslides with double cover slips. Measurements and

* Correspondingauthor, e-mail: [email protected]

© KoninklijkeBrill NV ,Leiden,2003 405 Alsoavailable online - www.brill.nl W. Bert et al. illustrationswere preparedfrom cameralucida line draw- mony(MP) andmaximum likelihood (ML) algorithms ingsusing Olympus BX50 and BX51 DIC microscopes implementedin P AUP* 4b10(Swofford, 2002).During andthe drawings were preparedusing Illustrator ® 8.0 theanalyses, trees were rootedusing Plectus. Gaps were software(Adobe Systems, Mountain View ,CA,USA). alwaystreated as missing data. T oestimateoverall sup- For scanningelectron microscopic (SEM) observation, portfor monophyly,NJ distancecalculations were per- glycerine-embeddednematodeswere Žrst transferredinto formedwith 3000 bootstrap replications with corrections adropof glycerine,gradually transferred to distilled wa- for multiplesubstitutions using Log Determinant distance terover a periodof 4h,thendehydrated in a 25,50, 75, measure(Lockhart et al.,1994).MP heuristicanalysis 95,and 100% ethanol series at 2hintervals,followed by was performedwith 3000 bootstrap replicates and without anovernight dehydration in 100% ethanol. Dehydrated bootstrappingwith 100 replicates of randombranch addi- nematodeswere critical-pointdried with CO 2 (Wergin, tion.Finally, a non-bootstrapheuristic ML searchwas also 1981),sputter coated with 20 nmgold,and examined with performedwith 100 replicates of random branch addi- aJEOL JSM-840at 15kV. tion.The trees were thencompared statistically using the Inaddition to slide voucher specimens, morphology Kishinoand Hasegawa(1989) pairwise tests with ML and ofthe new genus was recordedas video clips that MPoptimalitycriteria, and T empletontest with MP opti- mimicmultifocal observation through a lightmicro- malitycriterion. Cladograms were examinedwith TREE- scopefollowing the V ideoCapture and Editing pro- VIEW 1.6(Page, 1996) and converted into graphic Ž les ceduresdeveloped by De Leyand Bert (2002). The for AdobeIllustrator ® 9.0. resultingvirtual specimens are available on the web (http://faculty.ucr.edu/ pdeley/vce/Nep/nep.html). » * Terminologyon stoma morphology was adaptedfrom Baujardiamirabilis gen.n., sp. n. De Ley et al. (1995).The papilla formula follows De Ley (Figs 1-4) et al.(1999):pre- andpostcloacal papillae are separated by‘/’,withprecloacal papillae listed Ž rst,and the number MEASUREMENTS ofpairs in each cluster of papillae separated from the adjacentcluster with a ‘+’. The‘ p’for phasmidpair is See Table 1. omitted,as we didnot observe any phasmids. DESCRIPTION MOLECULARANALYSIS Female Thenematodes were rehydratedin distilled water for Bodylarge (1.8-2.7 mm), almoststraight after Ž xa- 1htoremove ethanol. DNA extractionand sequencing tion.Cuticle Ž nelyand faintly annulated, 0.8 ¹m wide were asdescribedin T andinganDe Ley et al. (2002). atmidbody. Lateral Ž eldnarrow (2.5 ¹mwideat mid- TheDNA sequenceof Baujardia gen.n. (GenBankac- body),starting at end of procorpusand becoming incon- cessionnumber: AF547385) was alignedwith the pub- spicuousat anal region. At midbody,lateral Ž eldform- lishedsequences of 21othertaxa. A secondarystructure inga singleprotruding ridge  ankedby Žne,faint, indis- alignmentwas constructedusing Dedicated Comparative tinctlongitudinal lines. Mouth opening partly covered by SequenceEditor (DCSE v.3.4)(De Rijk& De Wachter, sixliplets each bearing a labialpapilla. Liplets continu- 1993),based on theunweighted SSU rRNA modelof V an ouswith six discrete lips separated by shallow grooves. de Peer et al.(1998)derived from base-pairingsdetected Fourshort cephalic setae, 3-4 ¹mlong,placed at base of ascompensating mutations, with equal weighting to stems subventraland subdorsal lips. Amphid opening slit like, andloops. Using Modeltest (Posada & Crandall,1988) encircledby a roundshallow incisure and situated two thisalignment was analysedto determine the appropri- discontinuousannuli posterior to setae. Stoma triradiate, atemodel of DNA substitution(GTR +I+ 0, 6 substi- relativelywide along full length (on average one third as tutiontypes) and to estimate the substitution rates, rela- wideas headdiam.). Lengths of cheilostom,gymnostom tivebase frequencies, gamma distribution shape parame- ter,and proportion of invariablesites. * Thegenus name is given in honor of Dr Pierre Baujard for his Usingthe parameter estimates obtained from Mod- outstandingcontributions to nematology. It istobe regarded as eltest,molecular phylogenetic relationships were recon- femininein gender.The speciŽ c epithetrefers to the biotope this structedwith neighbour joining (NJ), maximumparsi- nematodeinhabits, i.e.,thepitcher  uidof Nepenthesmirabilis.

406 Nematology Baujardiamirabilis gen.n., sp. n. from pitcher plants

Fig. 1. Baujardiamirabilis gen.n., sp. n., female (holotype). A: Neckregion; B: Reproductivesystem showing granulated sperm cells (arrows);C: Headregion (superŽ cial view); D: Stoma;E: Endof ovary;oviduct, spermatheca with two offset pouches (arrows) and beginningof uterus (arrowheads); F: Vulva-tailregion; G: Entirefemale. (Scale bars: B, G 100 ¹m; A, F 50 ¹m; C, D, E D D D 10 ¹m.) Vol.5(3), 2003 407 W. Bert et al.

Fig. 2. Baujardiamirabilis gen.n., sp. n., male. A: Neckregion; B: Cloacalregion with copulatory papillae; C: Entiremale. (Scale bars: C 100 ¹m; A 50 ¹m; B 10 ¹m.) D D D

408 Nematology Baujardiamirabilis gen.n., sp. n. from pitcher plants

Fig. 3. Baujardiamirabilis gen.n., sp. n., SEM studiesof female.A: En face view;B: Lateralview head; C: Longitudinalsection of stomaregion; D: LateralŽ eld(at middle of body); E: Longitudinalsection at the vulval region; F: Vulvalateral view; G: Anusventral view;H: Middleof tail.Arrowheads: cephalic setae. Arrows: digitate stoma projections. (Scale bars: C, E,F 10 ¹m;A, B,D,G,H D 1 ¹m.) D

Vol.5(3), 2003 409 W. Bert et al.

Fig. 4. Baujardiamirabilis gen.n., sp. n.,SEM studiesof male.A: Longitudinalsection of neck region; B: Longitudinalsection of stoma regionshowing the subventral sectors; C: Longitudinalsection of stomaregion showing the dorsal sector ,blackarrow projectionof subventralsector ,whitearrows basallamina; D: Cloacaland tail region, lateral view; E: Cloacalregion, ventral view; D F: Lateral D Želd(at middle of body).(Scale bars: A, B,C,D,E 10 ¹m; F 1 ¹m.) D D

410 Nematology Baujardiamirabilis gen.n., sp. n. from pitcher plants

Table 1. Morphometricdata of twopopulations of Baujardiamirabilis gen.n., sp. n. Measurements are in ¹mandpresented as: mean standarddeviation (range). § 1999Population (T ype) 2002Population * Holotype Female CV Male CV Female CV paratypes paratypes paratypes n 19 17 12 L 2138 2316 238 10.3 1694 175 10.3 2113 380 18 § § § (1837-2745) (1350-1944) (1512-2550) a 34.7 31 3:5 11.3 34:3 4:1 11.8 32:5 4:5 14 § § § (22-36) (27-43) (23-37) b 5.7 6:2 0:5 8.7 5:2 0:5 9.6 6:3 1:2 19 § § § (5.3-7.4) (4.1-5.9) (4.4-7.7) c 7 7:5 0:6 8.4 8:7 1:1 12.4 6:6 0:5 7.2 § § § (6.2-8.9) (6.7-11) (6.1-7.5) c 10.5 10:1 1 10.1 6:3 0:7 10.8 10:6 2:3 21 0 § § § (7.3-11.7) (4.6-7.5) (6.5-13.5) Maximumbody diam. 61.6 76 15 19.8 50 7:7 15.5 64:3 13:2 21 § § § (54-107) (36-68) (48-84) Stomalength 20 20 0:8 4.1 18 1 5.5 19 1:7 8.7 § § § (18-22) (16-20) (17-22) Headdiam. (at amphid) 13.3 13 1:0 7.8 12 0:8 7.1 12 0:7 6 § § § (12-15) (9-13) (11-13) Stomalength/ headdiam. 1.5 1:5 0:1 6.9 1:5 0:2 10.4 1:6 0:2 9.7 § § § (1.3-1.7) (1.3-2) (1.4-1.8) Seta length 4.1 2:8 0:6 23 2:7 0:5 17.2 2:6 0:4 17 § § § (1.7-4.2) (1.7-3.5) (2-3) Amphidwidth 6.7 6 1:1 19 5:4 1:1 19.5 5:5 0:9 17 § § § (4-8.5) (3-7) (4-7) Neck length 375 372 16 4.4 326 19 5.8 336 28:7 8.5 § § § (337-402) (285-352) (294-378) Corpuslength 252 253 16:3 6.4 216 15 6.9 219 19 8.7 § § § (222-280) (186-237) (189-250) Procorpuslength 145 135 11:7 8.7 117 8:6 7.3 118 11:6 9.8 § § § (117-153) (97-127) (108-132) Metacorpuslength 107 115 11 9.5 98:4 8:1 8.2 114 9:1 7.9 § § § (97-132) (78-112) (103-123) Isthmuslength 60 57 4:1 7.1 51:2 4:4 8.5 53:8 5:1 9.5 § § § (51-62) (40-57) (48-61) Bulb length 42.5 46 3:4 7.3 43 5:3 12.3 40 3:8 9.4 § § § (40-52) (35-52) (35-46) Corpus/isthmus 4.2 4:4 0:4 8.7 4:2 0:4 8.6 4:1 0:3 8.3 § § § (3.7-5.2) (3.4-4.8) (3.6-4.6) Excretorypore position 232 224 22 9.8 199 27:3 13.7 220 25:5 12 § § § (180-260) (155-252) (186-252) Nervering position 270 269 20:7 7.7 237 15:2 6.4 255 30:3 12 § § § (235-312) (210-237) (216-294) EP(as% Necklength) 62 59 4:2 7.1 61:5 7:1 11.6 66 5:6 8.5 § § § (53-66) (48-73) (57-73) NR(as% Necklength) 72 72:2 3:4 4.8 73 3:5 4.8 75:8 6:8 8.9 § § § (66-80) (65-79) (66-86) Tail length 305 307 18 6 195 15:4 7.9 317 48:1 15 § § § (275-345) (162-225) (243-378)

Vol.5(3), 2003 411 W. Bert et al.

Table 1. (Continued). 1999Population (T ype) 2002Population * Holotype Female CV Male CV Female CV paratypes paratypes paratypes

Analbody diam. 29.1 31 3 9.8 31 2:7 8.5 31 8:3 27 § § § (26-40) (25.8-36) (22-50) Rectumlength 50 49 4:2 8.6 47 6 13 § § (42-56) (38-56) Rectum/Analbody diam. 1.7 1:6 0:2 12.8 1:6 0:3 17 § § (1.2-2.1) (1.1-2) Tail/Rectum 6 6:3 0:5 7.7 § (5.5-7.3) Gonadlength 1250 1370 18713.6 1049 11010.5 1251 327 26 § § § (1012-1700) (775-1206) (768-1635) Vulvaposition 1625 1768 179 10.1 1620 335 21 § § (1375-2062) (1104-2070) Vulvabody diam. 46.6 57 17:8 31 46 8:7 19 § § (42-107) (33-59) Vaginalength 26.6 29 3:2 11.2 26 4 15 § § (25-35) (21-34) Vulva-Anusdistance 207 240 73:7 30.6 175 56:5 32.2 § § (120-439) (102-270) Vulva-Anus/Tail 0.7 0:8 0:2 28.6 0:6 0:2 35 § § (0.4-1.4) (0.3-0.8) Postvulvalsac length 121 126 10:6 8.4 115 15:4 13 § § (103-149) (93-141) Postvulvalsac length/ 2.6 2:3 0:5 20.2 2.5 0:3 12 § § vulvabody diam. (1.2-3) (2-2.9) Postvulvalstalk length 29 34 5:3 15.3 34 6:4 19 § § (26-44) (23-40) Postvulvalpouch length 91 91 11:2 12.3 81 11 14 § § (75-120) (70-101) Vaginalcell cluster length 20 17 2:2 12.6 15 2:6 18 § § (15-21) (9-17) Vaginalcell cluster diam. 10.8 13 2 16.1 9 2:4 25 § § (10-18) (6-12) No.of eggsin uterus 20 10 49.1 20 8:4 42 § § (4-34) (8-31) V 76 76:4 2:1 2.7 76:3 3 4 § § (72-81) (72-81) Gonad(as % L) 58.5 59 2:8 4.7 58:4 5:5 9.5 § § (55-64) (51-66) Vagina(as % Vulva 57 53:1 14:1 26.5 59 13:2 22 § § body diam.) (24-74) (40-76) T 62:1 4:5 7.3 § (52-74) Spiculelength 57 3:8 6.7 § (50-62) Spicule/Analbody diam. 1:8 0:2 11.8 § (1.5-2.1) Gubernaculumlength 24 2:2 8.9 § (20-28)

412 Nematology Baujardiamirabilis gen.n., sp. n. from pitcher plants

Table 1. (Continued). 1999Population (T ype) 2002Population * Holotype Female CV Male CV Female CV paratypes paratypes paratypes

Gubernaculum/Spicule 0:4 0 8.9 § (0.4-0.5) Spicatetail portion 97:2 8:4 8.4 § (85-117) Spicatetail/ Taillength 0:5 0:1 14.1 § (0.4-0.7)

* sourceof specimensfor DNA extraction;males were not recovered. andstegostom in ratio 1 :1:2.Cheilostom without dis- withanterior stalk and posteriorpouch. Transition vagina- tinctsclerotisations. Stegostom divided into two parts in postvulvalsac guarded by aclusterof fourcells consisting sectionedspecimens observed with SEM. Posteriorpart oftwopairs lying side by side.Anus a broad,curved slit apparentlywith seven denticles or digitateprojections ca inventralview .Tailelongate-conical with Ž netip.Irreg- 2 ¹mlong,reclining against, and posteriorly connected ularpattern of lines substituting annulation over a short to,stoma wall. Projections as observed with SEM intwo distanceat middle of tail,as seenby SEM.Nophasmids specimensand consistent with light microscope obser- orphasmid-likestructures observed with SEM or lightmi- vationson three females positioned as follows: three in croscope. dorsalsector and two in each subventral sector (the sec- ondprojection in the right subventral sector was presum- Male ablysliced through in Fig. 4B). Dorsal gland opening at Generalmorphology similar to female. Body 1.3- baseof these projections. Basal lamina visible between 1.9mm long,arcuate ventrad and especially so in pos- gymnostomand stegostom (arrows inFig. 4C). Neck re- teriorregion, with tail usually curved into 1.5 coils. Cu- gionforming one Ž fthto one seventh of body. Pharynx ticleŽ nelyannulated, 0.8 ¹mwideat midbody. Lat- dividedinto a cylindricalcorpus comprising on average eralŽ eldindistinct, with several discontinuous lines, ex- 68%of neck length, a shortisthmus and an oval bulbus tendingslightly posterior to cloacal opening, 3 ¹m wide withtransverse valves. Cardia well developed. Nerve ring atmidbody. Neck region comprising 20-25% of body attransition of corpus-isthmus.Excretory pore 15-80 ¹m length.Excretory pore from 80 ¹manteriorto nerve anteriorto nerve ring, wide but often difŽ cult to Ž ndbe- ring to 6 ¹mposteriorto nerve ring. Reproductive tract causeof its lack of sclerotisation. Deirid not found. In- occupying50-75% of body length, monorchic, dextral, testinewith wide lumen demarcated by refractivelining, testisventrally re exed for 36-120 ¹m. Vesiculaseminalis microvillivisible anterior to rectum. Rectum long, with containinglarge granulated spermatocytes. Spicules mas- largesphincter cells at its beginning. Reproductive system sive,arcuate, each with complex angular manubrium and monodelphic,prodelphic, with posteriorly re exed ovary. welldeveloped velum. Demarcation of shaft and velum Oviductalways with two distinct nuclei, located at  ex- stronglysclerotised, spicule tip not divided. Gubernacu- urebetween ovary and uterus. T woopposing and offset lumwithventral triangular extension and prominent crura spermatheca-likepouches present at junction of oviduct whichmay protrude from cloacalaperture. Papilla for- anduterus. Each pouch with one distinct terminal cell, its mula: 1 1 1=1 2 1.Seven precloacal papillae: C C C C liningwith a membranousappearance. Granulated sperm Žrst pairsubventral on cuticular sockets at about two cellsvery large (15-34 ¹mlong)and often compressed analbody diam. anterior to cloaca, second pair lateral, intoserial clusters erratically dispersed throughout uterus, lessthan one anal body diam. before cloaca, third pair oftenalternating with developing eggs. V ulvaa trans- subventralon cuticular sockets at level of precloacal lip versecleft with overhanging anterior vulval lip located andone medioventral papilla located on highersocket at at75% of bodylength. V aginawith thickened walls and samelevel as second precloacal pair .Fourpapilla pairs surroundedby strong muscles. Prominent postvulval sac ontail: Ž rst andfourth pair lateral, situated respectively

Vol.5(3), 2003 413 W. Bert et al. at ca oneand two anal body diam. posterior to cloaca. Man,1876 in the family Teratocephalidae, but our new Secondand third papilla pair close to each other subven- genusdiffers from thisfamily in numerous respects, e.g., trallywith second pair more pronounced. T ailelongate- theamphidial apertures are located on thelips, there is a conical,narrowing sharply at midpointand with Ž netail distinctdifferentiation between the pharyngealcorpus and tip.No phasmids observed with SEM orlight micro- isthmusand the female has an elongatereproductive sys- scope. tem.Another taxon in which liplets and setae occur on thelip region is the family Chambersiellidae. However, TYPELOCALITY membersof this family have distinct labial setae, more posteriorlylocated amphids, a muchnarrower stegostom Pitchers of Nepenthesmirabilis (Lour.)Druce in Tung- andthe tail is usually equipped with a hook-likemucro. KaiBotanical Garden, Trang Province, Southern Thai- Overall,the mixture of characters resembling members land. ofdifferentfamilies precludes unequivocal placement of thenew genus on morphological grounds alone. In view TYPEMATERIAL ofthe molecular analysis discussed below, we conclude thatit belongs in theinfraorder Panagrolaimomorpha, su- Holotypefemale (slide MDNC 4021),two paratype fe- perfamilyPanagrolaimoidea and family Panagrolaimidae. males(slides MDNC 4022,4023) andfourparatype males Themorphological diagnosis of Panagrolaimidae must (slidesMDNC 4024,4025)deposited in the nematodecol- berevised signiŽ cantly to accommodate our new genus lectionof theZoology Museum, Department of Biology, andthe new information on some other panagrolaims GhentUniversity, Belgium; four paratype females and recentlydescribed by Stock et al.(2002).As aresult, fourparatype males on slides with UCR-NC Accession veryfew unequivocaldiagnostic characters currently re- No.30059-30063 in the collection of the De- mainfor Panagrolaimidae,especially when compared to partmentof Nematology,University of California,River- familiesCephalobidae, Chambersiellidae or Brevibucci- side,USA; threeparatype females and two paratype males dae(we considerthe latter two to be incertaesedis – see onUCNC slidenumbers 5234-5237 in the nematode col- De Ley& Blaxter,2002). Molecular data are clearly es- lectionof the Department of Nematology, University of sentialas anadditionalsource of diagnosticcharacters in California,Davis, USA; twoparatype females and two thesenematodes. paratypemales in the nematode type collection of CABI Bioscience,Egham, UK. MOLECULARPHYLOGENY

DIAGNOSISANDRELATIONSHIPS Thenearly complete SSU sequencesof the 22 in- cludedtaxa consisted of 1491 ( Rhabditophanes sp.) to Baujardiamirabilis gen.n., sp. n. differs from all 1611 (Panagrolaimus sp.)bp, with an intermediatevalue knownnematodes in having two opposing and offset for Baujardiamirabilis gen.n., sp. n. (1587 bp). The spermatheca-likepouches at the junction of oviduct and secondarystructure alignment comprised 1769 positions, uterus.It also differs from nearlyall known Rhabditida ofwhich 971 (55%) were variableincluding 715 (40%) inhaving four cephalic setae instead of papillae, in the parsimony-informativecharacters. The A +Tcontentof presenceof six liplets, and in the apparent absence of Baujardia gen.n. was 53%. phasmids. Table2 summarisesthe bootstrap support for, and Thisnew genus resembles Panagrellus Thorne,1938 monophylyof, clades in the obtained trees. NJ, MP,and inthe long body size, in the elongate-conical tail and in MLtreesconsistently support (100% bootstrap) a sister themonodelphic, prodelphic female reproductive system taxonrelationship between the new genus and Panagrel- withposteriorly placed vulva, thickened vaginal walls and lusredivivus (L.,1767)Goodey,1945 ( cf cladeA, Figs5- prominentpostvulval sac. However, Baujardia gen. n. 8). Panagrolaimus sp.has an uncertain position relative differs from Panagrellus inthe characters mentioned tothese two genera: it is placedwithin the same clade in above,in the comparatively longer stegostom, and in theNJ andML trees,while the MP treesplace it closer themore robust male spicules with complex angular tothe strongly supported sister taxa Halicephalobusgin- manubriumand undivided tip. givalis (Stefanski,1954) Andrá ssy, 1984 and Turbatrix Theliplets, cephalic setae and lack of phasmids are aceti (Müller, 1783) Peters, 1927 (clade B). Consistent charactersreminiscent of the genus Teratocephalus de maximalsupport was alsogiven to sister taxa Plectonchus

414 Nematology Baujardiamirabilis gen.n., sp. n. from pitcher plants

Table 2. Monophyly ( yes, X no)and bootstrap support (%) forclades. X D D Clades NJ MP1 MP2 MPmajorityML (Fig.5) (Fig. 6) (Fig. 7) rule(Fig. 6) (Fig. 8) Clade A (Panagrolaimus , (Baujardia (Panagrellusredivivus 57 X X 52 X AF036599, Panagrellusredivivus AF083007))) (Baujardia (Panagrellusredivivus AF036599, 100 X X 100 X Panagrellusredivivus AF083007)) (Panagrolaimus sp. (Halicephalobusgingivalis , X X X 52 X Turbatrixaceti )) Clade B (Halicephalobusgingivalis, ) 86 X X 79 X Clade C (Plectonchushunti, P anagrobelusstammeri ) 100 X X 100 X (CladeC (CladeA, CladeB)) ( Panagrolaimidae) 100 100 D X X X Monophylyof Strongyloidoidea ( Steinernema X X X 51 X carpocapsae (Rhabditophanes , Strongyloidesstercolaris )) Monophylyof Panagrolaimoidea and Strongyloidoidea X X X except X X ( InfraorderPanagrolaimomorpha 1/ / Steinernema D Rhabditomorpha 1/ ( Rhabditina)as sistertaxon to D X X Panagrolaimomorpha 1/ 1 Panagrolaimomorpha / as sister X X taxonto Cephalobomorpha 1/ ,Tylenchomorpha 1/

1/DeLey and Blaxter (2002). hunti Stock,De Ley,De Ley,Mundo-Ocampo, Baldwin BIOLOGY &Nadler,2002 and Panagrobelusstammeri Rühm, 1956 (cladeC); andto a basalposition for thisclade within Weassumethat the pitchers of Nepenthesmirabilis are Panagrolaimidae.In other respects, our results are com- thenatural habitat of Baujardiamirabilis gen.n., sp. n., parableto the SSU rDNA sequenceanalysis of Fé lix et consideringthat the species was foundalive in several al.(2000),for examplethe position of Brevibucca within pitchers.These pitchers contained a relativelydiverse Rhabditidais uncertain, variously assuming a position fauna,dominated by mosquito larvae, midges and the atthe base of eitherinfraorder Cephalobomorpha, Pana- newgenus, in additionto afew dipteranand coleopteran grolaimomorphaor Rhabditomorpha(De Ley& Blaxter, larvae.The most abundant prey insects in the pitchers 2002). Steinernemacarpocapsae (Weiser,1955) W outs, were antsand diverse  yinghymenopterans. Apparently, Mrácek,† Gerdin & Bedding,1982 is another nematode adiverseassemblage of organisms lives in association whoseposition is unclear, both in our trees and those withthe pitchers of this Nepenthes species.This may of Félix et al.(2000),but analysis with a morefo- representan ecologically complex community similar cuseddataset conŽ rms itsplacement in a monophyletic tothe better-known association inhabiting Sarracenia superfamilyStrongyloidoidea (Dorris et al., 2002). As pitchers (e.g.,Kitching,2001; Greeney, 2001). Possibly thepurpose of our study was toexplore phylogenetic thenematodes have a phoreticrelationship with one afŽnities between Baujardia gen.n. and its closest rel- ormore insect species. Closely related genera are also atives,we limitedthe number of sequences included in capableof withstanding harsh environments; Turbatrix ourdataset and it is therefore not surprising that resolu- aceti (thevinegar eelworm), Panagrellusredivivus , P. tionof relationships among higher taxa remains unclear silusiae and P.nepenthicola inhabitvinegar, bookbinder’ s inour trees ( e.g.aphelenchsare not found to be mono- paste,beer Ž ltersand pitcher plants, respectively. phyletic). Notmuch is known about the occurrence of nematodes inpitcher plants and they were noteven mentioned in

Vol.5(3), 2003 415 W. Bert et al.

Fig. 5. Neighbourjoining tree based on secondarystructure alignment of smallsubunit rDNA. Plectus wasdesignated as outgroup; numbersnext to branchesare percentage bootstrap support values from corresponding 50% majority-rule consensus tree obtained with NJanalysisof 3000 bootstrap replicates. A, BandC indicatethe three obtained clades within the P anagrolaimidae.

areferencework of the Nepenthes-inhabitingfauna by (1943),which later (Goodey, 1945) became a juniorsyn- Thienemann(1933). More extensive study is needed to onym of Panagrellus . investigatethe life cycle and biochemistry of thisunusual As P.nepenthicola was describedfrom thesame habi- newgenus. Moreover, pitcher plants and other epiphytes tatand similar geographical region as our new genus, we areclearly a promisingsource of surprisingnew nematode havebrie y examinedMenzel’ s (1922)very limited de- taxa. scriptionto justify proposal of anewspecies for ourma- terial,especially in the absence of originalillustrations for P.nepenthicola .Bothspecies are comparable in length Discussion (1.8-2.7 vs 2.3-2.9mm for P.nepenthicola )andin vulva position(average of 76 vs 77% for P.nepenthicola ). The relativetail length is, however, clearly longer in our ne- Thenematofauna of pitcher plants in Indonesia was matodes,being on average 125% of the vulva-anus dis- studiedby Menzel(1922), who reported the genera Plec- tance vs onlyhalf the vulva-anus distance length in P. ne- tus, Dorylaimus, Rhabditis and Diplogaster. He very penthicola;themale tail is on average twice the spicula briey describeda newspecies; Anguillulanepenthicola length vs threetimes the spiculalength in P.nepenthicola ; Menzel,1922 which he consideredas the only true inhab- the female c valueis 6.2-8.9 vs 9-10 in P.nepenthicola itantof pitcherplants and not an accidental recovery. An- and the male c valueis 6.7-11 vs 12-15 in P.nepenthicola . guillulanepenthicola ,togetherwith several other species, Theidentity of P.nepenthicola becamesomewhat clearer including Panagrelluspycnus Thorne,1938, was later withthe redescription by Micoletzkyand Menzel (1928) placedin the newly erected genus Turbator by Goodey althoughonly the male tail was illustrated.The most ap-

416 Nematology Baujardiamirabilis gen.n., sp. n. from pitcher plants

Fig. 6. Firstof two best trees obtained with Maximum P arsimonyanalysis based on secondary structure alignment of small subunit rDNA. Plectus wasdesignated as outgroup; numbers next to branches are bootstrap values from corresponding 50% majority-rule consensustree obtained with MP analysisof 3000 bootstrap replicates. A, BandC indicatethe three obtained clades within the Panagrolaimidae.Broken line in the bracket for clade A denotesparaphyletic position of Panagrolaimus inthis tree compared to the others(Figs 5, 8).

parentsimilarities of thenew genus with the redescribed However,the two opposing and offset spermatheca-like P.nepenthicola arethe muscular vagina and long postvul- pouchesobserved here represent a uniquefeature amongst valsac (103-149 vs 78-150 for P.nepenthicola ). However, describednematodes. These pouches are probably glan- thespicules of ournew genus and species have more an- dular,rather than having a functionin sperm storage, as gularmanubria and their tips are not biŽ d (anindistinct suggestedby the unusually large size of thesperm cells biŽd tipcan be observedon theoriginal illustration of P. andtheir occurrence throughout the uterus of gravid fe- nepenthicola ).Furthermore,the male tail is more strongly males.Possibly each compressed cluster of sperm orig- curvedcompared to P.nepenthicola andthe number and inatedfrom successivecopulations, as multiple matings positioningof thegenital papillae is different.The differ- perfemale might be morefrequent in a limitedand liquid encein tailcurvature, however, may be dueto coldŽ xa- environment.Alternatively, the movement of eggscould tionof ournematodes. causethe break-up of singleclusters of sperm. Thegenital system of thenew genus is panagrolaimid Onmorphological grounds alone, it was notimmedi- andshows similarities with the morphology of the female atelyapparent in which family Baujardiagen.n.should be gonad of Panagrellusredivivus (accordingto results ob- placed.The molecular analysis, however, robustly places tainedafter extraction of the genital system by Geraert thenew genus as asistertaxon of Panagrellus within the et al.,1980).As in P.redivivus ,the exureof the gen- Panagrolaimidae.As amatterof fact,the morphological italsystem is formed by the oviduct and a pairof dis- similaritieswith Panagrellus ,asdiscussedabove, are rel- tinctoviduct cells are observed at the spermatheca side. ativelyobvious aposteriori ,butwere notevident before

Vol.5(3), 2003 417 W. Bert et al.

Fig. 7. Secondof twobest trees obtained with Maximum P arsimonyanalysis based on secondarystructure alignment of small subunit rDNA. Plectus wasdesignated as outgroup. A, BandC indicatethe three obtained clades within the P anagrolaimidae.Broken line in thebracket for clade A denotesparaphyletic position of Panagrolaimus inthis tree compared to the others (Figs 5, 8). themolecular analysis. Morphological characters such as dricalor with valveless median bulb, terminal bulb val- spermathecastructure, stoma organisation, the presence vate.Female reproductive system monodelphic, prodel- ofliplets, setae and amphids are generally considered phic.Spermatheca usually absent or axial, rarely offset tobe appropriate to characterise a family.However, the asinCephalobidae. Uterus-oviduct junction exceptionally placementby molecularanalysis of thismorphologically withtwo offset spermatheca-like pouches. Postvulval sac deviantnew genus illustrates the inconsistency of these presentor absent. Female tail conical with pointed tip. characterswithin the family Panagrolaimidae. As acon- Maleswithout bursa, with one unpaired papilla and Ž ve sequence,a morphologicalredeŽ nition of this family is toseven pairs of genital papillae, arrangement variable. required. Spiculesoften with wide velum, gubernaculum often with posteriorlyexpanded outline. PanagrolaimidaeThorne, 1937 LISTOFGENERA

EMENDEDDIAGNOSIS Anguilluloides Rühm, 1956 Brevistoma Mukhina,1981 Lipregion without probolae or Ž mbriateprocesses. Halicephalobus Timm, 1956 Labialsensilla papilliform, cephalic sensilla papilliform Körner, 1954 D orrarelysetiform. Cuticula Ž nelyannulated (annules usu- Phytorhabditis Lordello& De Oliveira,1963 D ally <1 ¹mwide).Stoma fairly wide anteriorly, stegos- Panagrellus Thorne,1938 tomusually tapering, comprising between 50 and66% of Anguillula de Man, 1910 pro parte D stomalength, never with distinct subdivisions of itslining. Neocephalobus Steiner,1936 pro parte D Pharynxwith cylindrical procorpus, metacorpus cylin- Turbator Goodey,1943 D 418 Nematology Baujardiamirabilis gen.n., sp. n. from pitcher plants

Fig. 8. MaximumLikelihood tree based on secondarystructure alignment of smallsubunit rDNA. Plectus wasdesignated as outgroup. A,BandC indicatethe three obtained clades within the P anagrolaimidae.Lineages of taxaare based on proposed classiŽ cation scheme byDeLey and Blaxter (2002).

Tylorhabdus Sukul, 1971 References D Panagrobelus Thorne,1939 Panagrobelium Andrássy, 1984 Panagrolaimus Fuchs, 1930 DE LEY, I.T., DE LEY, P., BALDWIN, J.G., MUNDO- Plectonchus Fuchs, 1930 OCAMPO, M. & NADLER,S.A.(1999).Three new species Nothacrobeles Procephalobus Steiner,1934 of (Nemata:Cephalobidae) from the Mojave Desert,California. Journalof Nematology 31,482-497. Tricephalobu sSteiner,1936 DE LEY, P. & BERT,W.(2002). Video capture and editing as Pseudorhabditis Kreis, 1929 D atoolfor the storage, distribution and illustration of morpho- Trilabiatus Goodey,1963 D logicalcharacters of nematodes. Journalof Nematology 34, Turbatrix Peters,1927 296-302. DE LEY, P. & BLAXTER,M.L.(2002).Systematic position and phylogeny.In: Lee, D.L. (Ed.). Thebiology of nematodes . Acknowledgements London,T aylor& Francis,pp. 1-30. DE LEY, P., VAN DE VELDE, M.C., MOUNPORT, D., BAU- Theauthors thank P .Pholpunthin,S. Chittapunand S. JARD, P. & COOMANS,A.(1995).Ultrastructure of the stomain Cephalobidae, Panagrolaimidae and Rhabditidae, Kitichateof theDepartment of Biology,Prince of Songkla witha proposalfor a revisedstoma terminology in Rhabdi- University,Hat Y ai,Thailand for organisingŽ eldtrips and tida(Nematoda). Nematologica 41,153-182. for providingthe identiŽ cation of the Nepenthes species. DE RIJK, P. & DE WACHTER,R.(1993).DCSE v.2.54, WethankJ. Vaneteren and A. Vierstraetefor theDNA aninteractive tool for sequence alignment and secondary sequencingand alignment. The support of G.Borgoniein structureresearch. ComputerApplications in theBiosciences severalaspects of thisstudy is greatlyappreciated. W eare 9,735-740[software]. alsograteful to M.Yoder,L. Mundo,and S. Esfahanifor DORRIS, M., VINEY, M. & BLAXTER ,M.(2002).Molecular assistancewith the production of virtualspecimens. phylogeneticanalysis of the genus Strongyloides and related

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