Nematology 2003 Vol 5(3) 405-420

Baujardia mirabilis gen n sp n from pitcher plantsand its phylogenetic position within Panagrolaimidae

(Nematoda Rhabditida)Wim BERT 1curren Irma TANDINGAN DE LEY 2 Rita VAN DRIESSCHE 1

Hendrik SEGERS 3 and Paul DE LEY 2

1 Department of Biology Ghent University Ledeganckstraat35 9000 Gent Belgium2 Department of Nematology University of California Riverside CA 92521 USA

3 Freshwater Laboratory Royal Belgium Institute for Natural Sciences Vautierstraat 29 1000 Brussels Belgium

Received 16 September 2002 revised 13 January 2003Accepted for publication13 January 2003

Summary ndash Measurements line drawings and scanning electromicrographsare provided of Baujardia mirabilis gen n sp n isolatedfrom pitcher uid of Nepenthes mirabilis from Thailand The new genus differs from all known nematodes in having two opposingand offset spermatheca-like pouches at the junction of oviduct and uterus It also differs from most known Rhabditida in having fourcephalic setae instead of papillae Phylogenetic analysis of small subunit rDNA sequence data robustly places the new genus withinPanagrolaimidae as a sister taxon to Panagrellus These unusual nematodes resemble Panagrellus in body size (18-27 mm in females13-19 mm in males) and in the monodelphic prodelphic female reproductive system with thickened vaginal walls and prominentpostvulval sac However they differ from Panagrellus in the characters mentioned above in their comparatively longer stegostom andin the shape of the male spicules Because of its aberrant characters inclusion of this new genus in Panagrolaimidae requires changesto the family diagnosis

Keywords ndash Nepenthes new genus new species Panagrellus phylogeny taxonomy Thailand

During a visit to Tung-Kai Botanical Garden in TrangProvince near the Isthmus of Kra Southern Thailandone of us (Hendrik Segers) sampled the liquid con-tained within pitchers of the carnivorous plant Nepenthesmirabilis (Lour) Druce in order to obtain some of thepeculiar rotifers recorded from such habitats Except forsome unidenti able bdelloids no rotifers were found butthe samples contained abundant material of a remarkablenew nematode genus together with some diplogastridsSome of the unique morphological characters of thisnew genus make its placement dif cult In order to ob-tain additional information about the identity of the ne-matode and its position in relation to known taxa weconducted molecular phylogenetic analyses based on thesmall subunit (SSU) ribosomal DNA In this paper wedescribe this new genus and species from Nepenthes andpresent an emended diagnosis of the family Panagrolaim-idae

Corresponding author e-mail wimbertrugacbe

Materials and methods

SAMPLING

Samples were collected by ltering the content ofsome 50 pitchers through a 50 sup1m plankton net andwere immediately xed by adding one tenth of thesample volumeof concentratedformalin (36) at ambienttemperature Nepenthes mirabilis was encountered nearthe end of the trail through the primary swamp forest ofTung Kai Botanical garden A single bulk sample wascollected on 18 July 1999 Several additional samplesincluding a sample preserved in 70 ethanol werecollected on 13 January 2002

MORPHOLOGICAL OBSERVATIONS

For light microscopy nematodes were processed to an-hydrous glycerol (Seinhorst 1959) and mounted on alu-minium slides with double cover slips Measurements and

copy Koninklijke Brill NV Leiden 2003 405Also available online - wwwbrillnl

W Bert et al

illustrations were prepared from camera lucida line draw-ings using Olympus BX50 and BX51 DIC microscopesand the drawings were prepared using Illustratorreg 80software (Adobe Systems Mountain View CA USA)For scanning electron microscopic (SEM) observationglycerine-embedded nematodeswere rst transferred intoa drop of glycerine gradually transferred to distilled wa-ter over a period of 4 h then dehydrated in a 25 50 7595 and 100 ethanol series at 2 h intervals followed byan overnight dehydration in 100 ethanol Dehydratednematodes were critical-point dried with CO2 (Wergin1981) sputter coated with 20 nm gold and examined witha JEOL JSM-840 at 15 kV

In addition to slide voucher specimens morphologyof the new genus was recorded as video clips thatmimic multifocal observation through a light micro-scope following the Video Capture and Editing pro-cedures developed by De Ley and Bert (2002) Theresulting virtual specimens are available on the web(httpfacultyucreduraquopdeleyvceNepnephtml)

Terminology on stoma morphology was adapted fromDe Ley et al (1995) The papilla formula follows De Leyet al (1999) pre- and postcloacal papillae are separatedby lsquorsquo with precloacal papillae listed rst and the numberof pairs in each cluster of papillae separated from theadjacent cluster with a lsquo+rsquo The lsquoprsquo for phasmid pair isomitted as we did not observe any phasmids

MOLECULAR ANALYSIS

The nematodes were rehydrated in distilled water for1 h to remove ethanol DNA extraction and sequencingwere as described in Tandingan De Ley et al (2002)

The DNA sequence of Baujardia gen n (GenBank ac-cession number AF547385) was aligned with the pub-lished sequences of 21 other taxa A secondary structurealignment was constructed using Dedicated ComparativeSequence Editor (DCSE v34) (De Rijk amp De Wachter1993) based on the unweighted SSU rRNA model of Vande Peer et al (1998) derived from base-pairings detectedas compensatingmutations with equal weighting to stemsand loops Using Modeltest (Posada amp Crandall 1988)this alignment was analysed to determine the appropri-ate model of DNA substitution (GTR + I + 0 6 substi-tution types) and to estimate the substitution rates rela-tive base frequencies gamma distribution shape parame-ter and proportion of invariable sites

Using the parameter estimates obtained from Mod-eltest molecular phylogenetic relationships were recon-structed with neighbour joining (NJ) maximum parsi-

mony (MP) and maximum likelihood (ML) algorithmsimplemented in PAUP 4b10 (Swofford 2002) Duringthe analyses trees were rooted using Plectus Gaps werealways treated as missing data To estimate overall sup-port for monophyly NJ distance calculations were per-formed with 3000 bootstrap replications with correctionsfor multiple substitutionsusing Log Determinant distancemeasure (Lockhart et al 1994) MP heuristic analysiswas performed with 3000 bootstrap replicates and withoutbootstrappingwith 100 replicates of random branch addi-tion Finallya non-bootstrapheuristic ML search was alsoperformed with 100 replicates of random branch addi-tion The trees were then compared statistically using theKishino and Hasegawa (1989) pairwise tests with ML andMP optimality criteria and Templeton test with MP opti-mality criterion Cladograms were examined with TREE-VIEW 16 (Page 1996) and converted into graphic lesfor Adobe Illustratorreg 90

Baujardia mirabilis gen n sp n(Figs 1-4)

MEASUREMENTS

See Table 1

DESCRIPTION

Female

Body large (18-27 mm) almost straight after xa-tion Cuticle nely and faintly annulated 08 sup1m wideat midbody Lateral eld narrow (25 sup1m wide at mid-body) starting at end of procorpus and becoming incon-spicuous at anal region At midbody lateral eld form-ing a single protruding ridge anked by ne faint indis-tinct longitudinal lines Mouth opening partly covered bysix liplets each bearing a labial papilla Liplets continu-ous with six discrete lips separated by shallow groovesFour short cephalic setae 3-4 sup1m long placed at base ofsubventral and subdorsal lips Amphid opening slit likeencircled by a round shallow incisure and situated twodiscontinuous annuli posterior to setae Stoma triradiaterelatively wide along full length (on average one third aswide as head diam) Lengths of cheilostom gymnostom

The genus name is given in honor of Dr Pierre Baujard for hisoutstanding contributions to nematology It is to be regarded asfeminine in gender The speci c epithet refers to the biotope thisnematode inhabits ie the pitcher uid of Nepenthes mirabilis

406 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 1 Baujardia mirabilis gen n sp n female (holotype) A Neck region B Reproductive system showing granulated sperm cells(arrows) C Head region (supercial view) D Stoma E End of ovary oviduct spermatheca with two offset pouches (arrows) andbeginning of uterus (arrowheads) F Vulva-tail region G Entire female (Scale bars B G D 100 sup1m A F D 50 sup1m C D E D10 sup1m)

Vol 5(3) 2003 407

W Bert et al

Fig 2 Baujardia mirabilis gen n sp n male A Neck region B Cloacal region with copulatory papillae C Entire male (Scalebars C D 100 sup1m A D 50 sup1m B D 10 sup1m)

408 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 3 Baujardia mirabilis gen n sp n SEM studies of female A En face view B Lateral view head C Longitudinal section ofstoma region D Lateral eld (at middle of body) E Longitudinal section at the vulval region F Vulva lateral view G Anus ventralview H Middle of tail Arrowheads cephalic setae Arrows digitate stoma projections (Scale bars C E F D 10 sup1m A B D G HD 1 sup1m)

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Fig 4 Baujardia mirabilis gen n sp n SEM studies of male A Longitudinal section of neck region B Longitudinal section of stomaregion showing the subventral sectors C Longitudinal section of stoma region showing the dorsal sector black arrow D projection ofsubventral sector white arrows D basal lamina D Cloacal and tail region lateral view E Cloacal region ventral view F Lateral eld (at middle of body) (Scale bars A B C D E D 10 sup1m F D 1 sup1m)

410 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 Morphometric data of two populations of Baujardia mirabilis gen n sp n Measurements are in sup1m and presented as meansect standard deviation (range)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

n 19 17 12L 2138 2316 sect 238 103 1694 sect 175 103 2113 sect 380 18

(1837-2745) (1350-1944) (1512-2550)a 347 31 sect 35 113 343 sect 41 118 325 sect 45 14

(22-36) (27-43) (23-37)b 57 62 sect 05 87 52 sect 05 96 63 sect 12 19

(53-74) (41-59) (44-77)c 7 75 sect 06 84 87 sect 11 124 66 sect 05 72

(62-89) (67-11) (61-75)c0 105 101 sect 1 101 63 sect 07 108 106 sect 23 21

(73-117) (46-75) (65-135)Maximum body diam 616 76 sect 15 198 50 sect 77 155 643 sect 132 21

(54-107) (36-68) (48-84)Stoma length 20 20 sect 08 41 18 sect 1 55 19 sect 17 87

(18-22) (16-20) (17-22)Head diam (at amphid) 133 13 sect 10 78 12 sect 08 71 12 sect 07 6

(12-15) (9-13) (11-13)Stoma lengthhead diam 15 15 sect 01 69 15 sect 02 104 16 sect 02 97

(13-17) (13-2) (14-18)Seta length 41 28 sect 06 23 27 sect 05 172 26 sect 04 17

(17-42) (17-35) (2-3)Amphid width 67 6 sect 11 19 54 sect 11 195 55 sect 09 17

(4-85) (3-7) (4-7)Neck length 375 372 sect 16 44 326 sect 19 58 336 sect 287 85

(337-402) (285-352) (294-378)Corpus length 252 253 sect 163 64 216 sect 15 69 219 sect 19 87

(222-280) (186-237) (189-250)Procorpus length 145 135 sect 117 87 117 sect 86 73 118 sect 116 98

(117-153) (97-127) (108-132)Metacorpus length 107 115 sect 11 95 984 sect 81 82 114 sect 91 79

(97-132) (78-112) (103-123)Isthmus length 60 57 sect 41 71 512 sect 44 85 538 sect 51 95

(51-62) (40-57) (48-61)Bulb length 425 46 sect 34 73 43 sect 53 123 40 sect 38 94

(40-52) (35-52) (35-46)Corpusisthmus 42 44 sect 04 87 42 sect 04 86 41 sect 03 83

(37-52) (34-48) (36-46)Excretory pore position 232 224 sect 22 98 199 sect 273 137 220 sect 255 12

(180-260) (155-252) (186-252)Nerve ring position 270 269 sect 207 77 237 sect 152 64 255 sect 303 12

(235-312) (210-237) (216-294)EP (as Neck length) 62 59 sect 42 71 615 sect 71 116 66 sect 56 85

(53-66) (48-73) (57-73)NR (as Neck length) 72 722 sect 34 48 73 sect 35 48 758 sect 68 89

(66-80) (65-79) (66-86)Tail length 305 307 sect 18 6 195 sect 154 79 317 sect 481 15

(275-345) (162-225) (243-378)

Vol 5(3) 2003 411

W Bert et al

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

Anal body diam 291 31 sect 3 98 31 sect 27 85 31 sect 83 27(26-40) (258-36) (22-50)

Rectum length 50 49 sect 42 86 47 sect 6 13(42-56) (38-56)

RectumAnal body diam 17 16 sect 02 128 16 sect 03 17(12-21) (11-2)

TailRectum 6 63 sect 05 77(55-73)

Gonad length 1250 1370 sect 187 136 1049 sect 110 105 1251 sect 327 26(1012-1700) (775-1206) (768-1635)

Vulva position 1625 1768 sect 179 101 1620 sect 335 21(1375-2062) (1104-2070)

Vulva body diam 466 57 sect 178 31 46 sect 87 19(42-107) (33-59)

Vagina length 266 29 sect 32 112 26 sect 4 15(25-35) (21-34)

Vulva-Anus distance 207 240 sect 737 306 175 sect 565 322(120-439) (102-270)

Vulva-AnusTail 07 08 sect 02 286 06 sect 02 35(04-14) (03-08)

Postvulval sac length 121 126 sect 106 84 115 sect 154 13(103-149) (93-141)

Postvulval sac length 26 23 sect 05 202 25 sect03 12vulva body diam (12-3) (2-29)

Postvulval stalk length 29 34 sect 53 153 34 sect 64 19(26-44) (23-40)

Postvulval pouch length 91 91 sect 112 123 81 sect 11 14(75-120) (70-101)

Vaginal cell cluster length 20 17 sect 22 126 15 sect 26 18(15-21) (9-17)

Vaginal cell cluster diam 108 13 sect 2 161 9 sect 24 25(10-18) (6-12)

No of eggs in uterus 20 sect 10 491 20 sect 84 42(4-34) (8-31)

V 76 764 sect 21 27 763 sect 3 4(72-81) (72-81)

Gonad (as L) 585 59 sect 28 47 584 sect 55 95(55-64) (51-66)

Vagina (as Vulva 57 531 sect 141 265 59 sect 132 22body diam) (24-74) (40-76)

T 621 sect 45 73(52-74)

Spicule length 57 sect 38 67(50-62)

SpiculeAnal body diam 18 sect 02 118(15-21)

Gubernaculum length 24 sect 22 89(20-28)

412 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

GubernaculumSpicule 04 sect 0 89(04-05)

Spicate tail portion 972 sect 84 84(85-117)

Spicate tailTail length 05 sect 01 141(04-07)

source of specimens for DNA extraction males were not recovered

and stegostom in ratio 1 1 2 Cheilostom without dis-tinct sclerotisations Stegostom divided into two parts insectioned specimens observed with SEM Posterior partapparently with seven denticles or digitate projections ca2 sup1m long reclining against and posteriorly connectedto stoma wall Projections as observed with SEM in twospecimens and consistent with light microscope obser-vations on three females positioned as follows three indorsal sector and two in each subventral sector (the sec-ond projection in the right subventral sector was presum-ably sliced through in Fig 4B) Dorsal gland opening atbase of these projections Basal lamina visible betweengymnostom and stegostom (arrows in Fig 4C) Neck re-gion forming one fth to one seventh of body Pharynxdivided into a cylindrical corpus comprising on average68 of neck length a short isthmus and an oval bulbuswith transverse valves Cardia well developedNerve ringat transition of corpus-isthmus Excretory pore 15-80 sup1manterior to nerve ring wide but often dif cult to nd be-cause of its lack of sclerotisation Deirid not found In-testine with wide lumen demarcated by refractive liningmicrovilli visible anterior to rectum Rectum long withlarge sphincter cells at its beginningReproductivesystemmonodelphic prodelphicwith posteriorly re exed ovaryOviduct always with two distinct nuclei located at ex-ure between ovary and uterus Two opposing and offsetspermatheca-like pouches present at junction of oviductand uterus Each pouch with one distinct terminal cell itslining with a membranous appearance Granulated spermcells very large (15-34 sup1m long) and often compressedinto serial clusters erratically dispersed throughoututerusoften alternating with developing eggs Vulva a trans-verse cleft with overhanging anterior vulval lip locatedat 75 of body length Vagina with thickened walls andsurrounded by strong muscles Prominent postvulval sac

with anterior stalk and posterior pouchTransition vagina-postvulvalsac guarded by a cluster of four cells consistingof two pairs lying side by side Anus a broad curved slitin ventral view Tail elongate-conical with ne tip Irreg-ular pattern of lines substituting annulation over a shortdistance at middle of tail as seen by SEM No phasmidsor phasmid-likestructures observed with SEM or light mi-croscope

Male

General morphology similar to female Body 13-19 mm long arcuate ventrad and especially so in pos-terior region with tail usually curved into 15 coils Cu-ticle nely annulated 08 sup1m wide at midbody Lat-eral eld indistinct with several discontinuous lines ex-tending slightly posterior to cloacal opening 3 sup1m wideat midbody Neck region comprising 20-25 of bodylength Excretory pore from 80 sup1m anterior to nervering to 6 sup1m posterior to nerve ring Reproductive tractoccupying 50-75 of body length monorchic dextraltestis ventrally re exed for 36-120sup1m Vesicula seminaliscontaining large granulated spermatocytes Spicules mas-sive arcuate each with complex angular manubrium andwell developed velum Demarcation of shaft and velumstrongly sclerotised spicule tip not divided Gubernacu-lum with ventral triangular extension and prominent crurawhich may protrude from cloacal aperture Papilla for-mula 1 C 1 C 1=1 C 2 C 1 Seven precloacal papillae rst pair subventral on cuticular sockets at about twoanal body diam anterior to cloaca second pair lateralless than one anal body diam before cloaca third pairsubventral on cuticular sockets at level of precloacal lipand one medioventral papilla located on higher socket atsame level as second precloacal pair Four papilla pairson tail rst and fourth pair lateral situated respectively

Vol 5(3) 2003 413

W Bert et al

at ca one and two anal body diam posterior to cloacaSecond and third papilla pair close to each other subven-trally with second pair more pronounced Tail elongate-conical narrowing sharply at midpoint and with ne tailtip No phasmids observed with SEM or light micro-scope

TYPE LOCALITY

Pitchers of Nepenthes mirabilis (Lour) Druce in Tung-Kai Botanical Garden Trang Province Southern Thai-land

TYPE MATERIAL

Holotype female (slide MDNC 4021) two paratype fe-males (slides MDNC 4022 4023) and four paratype males(slides MDNC 4024 4025)deposited in the nematodecol-lection of the Zoology Museum Department of BiologyGhent University Belgium four paratype females andfour paratype males on slides with UCR-NC AccessionNo 30059-30063 in the nematode collection of the De-partment of Nematology University of California River-side USA three paratype females and two paratype maleson UCNC slide numbers 5234-5237 in the nematode col-lection of the Department of Nematology University ofCalifornia Davis USA two paratype females and twoparatype males in the nematode type collection of CABIBioscience Egham UK

DIAGNOSIS AND RELATIONSHIPS

Baujardia mirabilis gen n sp n differs from allknown nematodes in having two opposing and offsetspermatheca-like pouches at the junction of oviduct anduterus It also differs from nearly all known Rhabditidain having four cephalic setae instead of papillae in thepresence of six liplets and in the apparent absence ofphasmids

This new genus resembles Panagrellus Thorne 1938in the long body size in the elongate-conical tail and inthe monodelphic prodelphic female reproductive systemwith posteriorlyplaced vulva thickenedvaginalwalls andprominent postvulval sac However Baujardia gen ndiffers from Panagrellus in the characters mentionedabove in the comparatively longer stegostom and inthe more robust male spicules with complex angularmanubrium and undivided tip

The liplets cephalic setae and lack of phasmids arecharacters reminiscent of the genus Teratocephalus de

Man 1876 in the family Teratocephalidae but our newgenus differs from this family in numerous respects egthe amphidial apertures are located on the lips there is adistinct differentiationbetween the pharyngealcorpus andisthmus and the female has an elongate reproductive sys-tem Another taxon in which liplets and setae occur onthe lip region is the family Chambersiellidae Howevermembers of this family have distinct labial setae moreposteriorly located amphids a much narrower stegostomand the tail is usually equipped with a hook-like mucroOverall the mixture of characters resembling membersof different families precludes unequivocal placement ofthe new genus on morphological grounds alone In viewof the molecular analysis discussed below we concludethat it belongs in the infraorder Panagrolaimomorphasu-perfamily Panagrolaimoideaand family PanagrolaimidaeThe morphological diagnosis of Panagrolaimidae mustbe revised signi cantly to accommodate our new genusand the new information on some other panagrolaimsrecently described by Stock et al (2002) As a resultvery few unequivocal diagnostic characters currently re-main for Panagrolaimidae especially when compared tofamilies Cephalobidae Chambersiellidae or Brevibucci-dae (we consider the latter two to be incertae sedis ndash seeDe Ley amp Blaxter 2002) Molecular data are clearly es-sential as an additional source of diagnostic characters inthese nematodes

MOLECULAR PHYLOGENY

The nearly complete SSU sequences of the 22 in-cluded taxa consisted of 1491 (Rhabditophanes sp) to1611 (Panagrolaimus sp) bp with an intermediate valuefor Baujardia mirabilis gen n sp n (1587 bp) Thesecondary structure alignment comprised 1769 positionsof which 971 (55) were variable including 715 (40)parsimony-informative characters The A + T content ofBaujardia gen n was 53

Table 2 summarises the bootstrap support for andmonophyly of clades in the obtained trees NJ MP andML trees consistently support (100 bootstrap) a sistertaxon relationship between the new genus and Panagrel-lus redivivus (L 1767) Goodey 1945 (cf clade A Figs 5-8) Panagrolaimus sp has an uncertain position relativeto these two genera it is placed within the same clade inthe NJ and ML trees while the MP trees place it closerto the strongly supported sister taxa Halicephalobus gin-givalis (Stefanski 1954) Andraacutessy 1984 and Turbatrixaceti (Muumlller 1783) Peters 1927 (clade B) Consistentmaximal support was also given to sister taxa Plectonchus

414 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 2 Monophyly (X D yes X D no) and bootstrap support () for clades

Clades NJ MP1 MP2 MP majority ML(Fig 5) (Fig 6) (Fig 7) rule (Fig 6) (Fig 8)

Clade A(Panagrolaimus (Baujardia (Panagrellus redivivus 57 X X 52 X

AF036599 Panagrellus redivivus AF083007)))(Baujardia (Panagrellus redivivus AF036599 100 X X 100 X

Panagrellus redivivus AF083007))(Panagrolaimus sp (Halicephalobus gingivalis X X X 52 X

Turbatrix aceti))

Clade B(Halicephalobus gingivalis Turbatrix aceti) 86 X X 79 X

Clade C(Plectonchus hunti Panagrobelus stammeri) 100 X X 100 X(Clade C (Clade A Clade B)) (DPanagrolaimidae) 100 X X 100 XMonophyly of Strongyloidoidea (Steinernema X X X 51 X

carpocapsae (Rhabditophanes Strongyloides stercolaris))Monophyly of Panagrolaimoidea and Strongyloidoidea X X X except X X

(DInfraorder Panagrolaimomorpha1 SteinernemaRhabditomorpha1 (DRhabditina) as sister taxon to X X

Panagrolaimomorpha1

Panagrolaimomorpha1 as sister X Xtaxon to Cephalobomorpha1 Tylenchomorpha1

1De Ley and Blaxter (2002)

hunti Stock De Ley De Ley Mundo-Ocampo Baldwinamp Nadler 2002 and Panagrobelus stammeri Ruumlhm 1956(clade C) and to a basal position for this clade withinPanagrolaimidae In other respects our results are com-parable to the SSU rDNA sequence analysis of Feacutelix etal (2000) for example the position of Brevibucca withinRhabditida is uncertain variously assuming a positionat the base of either infraorder Cephalobomorpha Pana-grolaimomorpha or Rhabditomorpha (De Ley amp Blaxter2002) Steinernema carpocapsae (Weiser 1955) WoutsMraacutedaggercek Gerdin amp Bedding 1982 is another nematodewhose position is unclear both in our trees and thoseof Feacutelix et al (2000) but analysis with a more fo-cused dataset con rms its placement in a monophyleticsuperfamily Strongyloidoidea (Dorris et al 2002) Asthe purpose of our study was to explore phylogeneticaf nities between Baujardia gen n and its closest rel-atives we limited the number of sequences included inour dataset and it is therefore not surprising that resolu-tion of relationships among higher taxa remains unclearin our trees (eg aphelenchs are not found to be mono-phyletic)

BIOLOGY

We assume that the pitchers of Nepenthes mirabilis arethe natural habitat of Baujardia mirabilis gen n sp nconsidering that the species was found alive in severalpitchers These pitchers contained a relatively diversefauna dominated by mosquito larvae midges and thenew genus in addition to a few dipteran and coleopteranlarvae The most abundant prey insects in the pitcherswere ants and diverse ying hymenopterans Apparentlya diverse assemblage of organisms lives in associationwith the pitchers of this Nepenthes species This mayrepresent an ecologically complex community similarto the better-known association inhabiting Sarraceniapitchers (eg Kitching 2001 Greeney 2001) Possiblythe nematodes have a phoretic relationship with oneor more insect species Closely related genera are alsocapable of withstanding harsh environments Turbatrixaceti (the vinegar eelworm) Panagrellus redivivus Psilusiae and P nepenthicola inhabit vinegar bookbinderrsquospaste beer lters and pitcher plants respectively

Not much is known about the occurrence of nematodesin pitcher plants and they were not even mentioned in

Vol 5(3) 2003 415

W Bert et al

Fig 5 Neighbour joining tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupnumbers next to branches are percentage bootstrap support values from corresponding50 majority-ruleconsensus tree obtained withNJ analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within the Panagrolaimidae

a reference work of the Nepenthes-inhabiting fauna byThienemann (1933) More extensive study is needed toinvestigate the life cycle and biochemistry of this unusualnew genus Moreover pitcher plants and other epiphytesare clearly a promising source of surprising new nematodetaxa

Discussion

The nematofauna of pitcher plants in Indonesia wasstudied by Menzel (1922) who reported the genera Plec-tus Dorylaimus Rhabditis and Diplogaster He verybrie y described a new species Anguillula nepenthicolaMenzel 1922 which he considered as the only true inhab-itant of pitcher plants and not an accidental recovery An-guillula nepenthicola together with several other speciesincluding Panagrellus pycnus Thorne 1938 was laterplaced in the newly erected genus Turbator by Goodey

(1943) which later (Goodey 1945) became a junior syn-onym of Panagrellus

As P nepenthicola was described from the same habi-tat and similar geographical region as our new genus wehave brie y examined Menzelrsquos (1922) very limited de-scription to justify proposal of a new species for our ma-terial especially in the absence of original illustrations forP nepenthicola Both species are comparable in length(18-27 vs 23-29 mm for P nepenthicola) and in vulvaposition (average of 76 vs 77 for P nepenthicola) Therelative tail length is however clearly longer in our ne-matodes being on average 125 of the vulva-anus dis-tance vs only half the vulva-anus distance length in P ne-penthicola the male tail is on average twice the spiculalength vs three times the spicula length in P nepenthicolathe female c value is 62-89 vs 9-10 in P nepenthicolaand the male c value is 67-11 vs 12-15 in P nepenthicolaThe identity of P nepenthicolabecame somewhat clearerwith the redescription by Micoletzky and Menzel (1928)although only the male tail was illustrated The most ap-

416 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 6 First of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup numbers next to branches are bootstrap values from corresponding 50 majority-ruleconsensus tree obtained with MP analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within thePanagrolaimidae Broken line in the bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to theothers (Figs 5 8)

parent similarities of the new genus with the redescribedP nepenthicolaare the muscular vagina and long postvul-val sac (103-149 vs 78-150 for P nepenthicola) Howeverthe spicules of our new genus and species have more an-gular manubria and their tips are not bi d (an indistinctbi d tip can be observed on the original illustration of Pnepenthicola) Furthermore the male tail is more stronglycurved compared to P nepenthicola and the number andpositioning of the genital papillae is different The differ-ence in tail curvature however may be due to cold xa-tion of our nematodes

The genital system of the new genus is panagrolaimidand shows similarities with the morphology of the femalegonad of Panagrellus redivivus (according to results ob-tained after extraction of the genital system by Geraertet al 1980) As in P redivivus the exure of the gen-ital system is formed by the oviduct and a pair of dis-tinct oviduct cells are observed at the spermatheca side

However the two opposing and offset spermatheca-likepouches observed here represent a unique feature amongstdescribed nematodes These pouches are probably glan-dular rather than having a function in sperm storage assuggested by the unusually large size of the sperm cellsand their occurrence throughout the uterus of gravid fe-males Possibly each compressed cluster of sperm orig-inated from successive copulations as multiple matingsper female might be more frequent in a limited and liquidenvironment Alternatively the movement of eggs couldcause the break-up of single clusters of sperm

On morphological grounds alone it was not immedi-ately apparent in which family Baujardiagen n shouldbeplaced The molecular analysis however robustly placesthe new genus as a sister taxon of Panagrellus within thePanagrolaimidae As a matter of fact the morphologicalsimilarities with Panagrellus as discussed above are rel-atively obvious a posteriori but were not evident before

Vol 5(3) 2003 417

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Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

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Fig 2 Baujardia mirabilis gen n sp n male A Neck region B Cloacal region with copulatory papillae C Entire male (Scalebars C D 100 sup1m A D 50 sup1m B D 10 sup1m)

408 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 3 Baujardia mirabilis gen n sp n SEM studies of female A En face view B Lateral view head C Longitudinal section ofstoma region D Lateral eld (at middle of body) E Longitudinal section at the vulval region F Vulva lateral view G Anus ventralview H Middle of tail Arrowheads cephalic setae Arrows digitate stoma projections (Scale bars C E F D 10 sup1m A B D G HD 1 sup1m)

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Fig 4 Baujardia mirabilis gen n sp n SEM studies of male A Longitudinal section of neck region B Longitudinal section of stomaregion showing the subventral sectors C Longitudinal section of stoma region showing the dorsal sector black arrow D projection ofsubventral sector white arrows D basal lamina D Cloacal and tail region lateral view E Cloacal region ventral view F Lateral eld (at middle of body) (Scale bars A B C D E D 10 sup1m F D 1 sup1m)

410 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 Morphometric data of two populations of Baujardia mirabilis gen n sp n Measurements are in sup1m and presented as meansect standard deviation (range)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

n 19 17 12L 2138 2316 sect 238 103 1694 sect 175 103 2113 sect 380 18

(1837-2745) (1350-1944) (1512-2550)a 347 31 sect 35 113 343 sect 41 118 325 sect 45 14

(22-36) (27-43) (23-37)b 57 62 sect 05 87 52 sect 05 96 63 sect 12 19

(53-74) (41-59) (44-77)c 7 75 sect 06 84 87 sect 11 124 66 sect 05 72

(62-89) (67-11) (61-75)c0 105 101 sect 1 101 63 sect 07 108 106 sect 23 21

(73-117) (46-75) (65-135)Maximum body diam 616 76 sect 15 198 50 sect 77 155 643 sect 132 21

(54-107) (36-68) (48-84)Stoma length 20 20 sect 08 41 18 sect 1 55 19 sect 17 87

(18-22) (16-20) (17-22)Head diam (at amphid) 133 13 sect 10 78 12 sect 08 71 12 sect 07 6

(12-15) (9-13) (11-13)Stoma lengthhead diam 15 15 sect 01 69 15 sect 02 104 16 sect 02 97

(13-17) (13-2) (14-18)Seta length 41 28 sect 06 23 27 sect 05 172 26 sect 04 17

(17-42) (17-35) (2-3)Amphid width 67 6 sect 11 19 54 sect 11 195 55 sect 09 17

(4-85) (3-7) (4-7)Neck length 375 372 sect 16 44 326 sect 19 58 336 sect 287 85

(337-402) (285-352) (294-378)Corpus length 252 253 sect 163 64 216 sect 15 69 219 sect 19 87

(222-280) (186-237) (189-250)Procorpus length 145 135 sect 117 87 117 sect 86 73 118 sect 116 98

(117-153) (97-127) (108-132)Metacorpus length 107 115 sect 11 95 984 sect 81 82 114 sect 91 79

(97-132) (78-112) (103-123)Isthmus length 60 57 sect 41 71 512 sect 44 85 538 sect 51 95

(51-62) (40-57) (48-61)Bulb length 425 46 sect 34 73 43 sect 53 123 40 sect 38 94

(40-52) (35-52) (35-46)Corpusisthmus 42 44 sect 04 87 42 sect 04 86 41 sect 03 83

(37-52) (34-48) (36-46)Excretory pore position 232 224 sect 22 98 199 sect 273 137 220 sect 255 12

(180-260) (155-252) (186-252)Nerve ring position 270 269 sect 207 77 237 sect 152 64 255 sect 303 12

(235-312) (210-237) (216-294)EP (as Neck length) 62 59 sect 42 71 615 sect 71 116 66 sect 56 85

(53-66) (48-73) (57-73)NR (as Neck length) 72 722 sect 34 48 73 sect 35 48 758 sect 68 89

(66-80) (65-79) (66-86)Tail length 305 307 sect 18 6 195 sect 154 79 317 sect 481 15

(275-345) (162-225) (243-378)

Vol 5(3) 2003 411

W Bert et al

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

Anal body diam 291 31 sect 3 98 31 sect 27 85 31 sect 83 27(26-40) (258-36) (22-50)

Rectum length 50 49 sect 42 86 47 sect 6 13(42-56) (38-56)

RectumAnal body diam 17 16 sect 02 128 16 sect 03 17(12-21) (11-2)

TailRectum 6 63 sect 05 77(55-73)

Gonad length 1250 1370 sect 187 136 1049 sect 110 105 1251 sect 327 26(1012-1700) (775-1206) (768-1635)

Vulva position 1625 1768 sect 179 101 1620 sect 335 21(1375-2062) (1104-2070)

Vulva body diam 466 57 sect 178 31 46 sect 87 19(42-107) (33-59)

Vagina length 266 29 sect 32 112 26 sect 4 15(25-35) (21-34)

Vulva-Anus distance 207 240 sect 737 306 175 sect 565 322(120-439) (102-270)

Vulva-AnusTail 07 08 sect 02 286 06 sect 02 35(04-14) (03-08)

Postvulval sac length 121 126 sect 106 84 115 sect 154 13(103-149) (93-141)

Postvulval sac length 26 23 sect 05 202 25 sect03 12vulva body diam (12-3) (2-29)

Postvulval stalk length 29 34 sect 53 153 34 sect 64 19(26-44) (23-40)

Postvulval pouch length 91 91 sect 112 123 81 sect 11 14(75-120) (70-101)

Vaginal cell cluster length 20 17 sect 22 126 15 sect 26 18(15-21) (9-17)

Vaginal cell cluster diam 108 13 sect 2 161 9 sect 24 25(10-18) (6-12)

No of eggs in uterus 20 sect 10 491 20 sect 84 42(4-34) (8-31)

V 76 764 sect 21 27 763 sect 3 4(72-81) (72-81)

Gonad (as L) 585 59 sect 28 47 584 sect 55 95(55-64) (51-66)

Vagina (as Vulva 57 531 sect 141 265 59 sect 132 22body diam) (24-74) (40-76)

T 621 sect 45 73(52-74)

Spicule length 57 sect 38 67(50-62)

SpiculeAnal body diam 18 sect 02 118(15-21)

Gubernaculum length 24 sect 22 89(20-28)

412 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

GubernaculumSpicule 04 sect 0 89(04-05)

Spicate tail portion 972 sect 84 84(85-117)

Spicate tailTail length 05 sect 01 141(04-07)

source of specimens for DNA extraction males were not recovered

and stegostom in ratio 1 1 2 Cheilostom without dis-tinct sclerotisations Stegostom divided into two parts insectioned specimens observed with SEM Posterior partapparently with seven denticles or digitate projections ca2 sup1m long reclining against and posteriorly connectedto stoma wall Projections as observed with SEM in twospecimens and consistent with light microscope obser-vations on three females positioned as follows three indorsal sector and two in each subventral sector (the sec-ond projection in the right subventral sector was presum-ably sliced through in Fig 4B) Dorsal gland opening atbase of these projections Basal lamina visible betweengymnostom and stegostom (arrows in Fig 4C) Neck re-gion forming one fth to one seventh of body Pharynxdivided into a cylindrical corpus comprising on average68 of neck length a short isthmus and an oval bulbuswith transverse valves Cardia well developedNerve ringat transition of corpus-isthmus Excretory pore 15-80 sup1manterior to nerve ring wide but often dif cult to nd be-cause of its lack of sclerotisation Deirid not found In-testine with wide lumen demarcated by refractive liningmicrovilli visible anterior to rectum Rectum long withlarge sphincter cells at its beginningReproductivesystemmonodelphic prodelphicwith posteriorly re exed ovaryOviduct always with two distinct nuclei located at ex-ure between ovary and uterus Two opposing and offsetspermatheca-like pouches present at junction of oviductand uterus Each pouch with one distinct terminal cell itslining with a membranous appearance Granulated spermcells very large (15-34 sup1m long) and often compressedinto serial clusters erratically dispersed throughoututerusoften alternating with developing eggs Vulva a trans-verse cleft with overhanging anterior vulval lip locatedat 75 of body length Vagina with thickened walls andsurrounded by strong muscles Prominent postvulval sac

with anterior stalk and posterior pouchTransition vagina-postvulvalsac guarded by a cluster of four cells consistingof two pairs lying side by side Anus a broad curved slitin ventral view Tail elongate-conical with ne tip Irreg-ular pattern of lines substituting annulation over a shortdistance at middle of tail as seen by SEM No phasmidsor phasmid-likestructures observed with SEM or light mi-croscope

Male

General morphology similar to female Body 13-19 mm long arcuate ventrad and especially so in pos-terior region with tail usually curved into 15 coils Cu-ticle nely annulated 08 sup1m wide at midbody Lat-eral eld indistinct with several discontinuous lines ex-tending slightly posterior to cloacal opening 3 sup1m wideat midbody Neck region comprising 20-25 of bodylength Excretory pore from 80 sup1m anterior to nervering to 6 sup1m posterior to nerve ring Reproductive tractoccupying 50-75 of body length monorchic dextraltestis ventrally re exed for 36-120sup1m Vesicula seminaliscontaining large granulated spermatocytes Spicules mas-sive arcuate each with complex angular manubrium andwell developed velum Demarcation of shaft and velumstrongly sclerotised spicule tip not divided Gubernacu-lum with ventral triangular extension and prominent crurawhich may protrude from cloacal aperture Papilla for-mula 1 C 1 C 1=1 C 2 C 1 Seven precloacal papillae rst pair subventral on cuticular sockets at about twoanal body diam anterior to cloaca second pair lateralless than one anal body diam before cloaca third pairsubventral on cuticular sockets at level of precloacal lipand one medioventral papilla located on higher socket atsame level as second precloacal pair Four papilla pairson tail rst and fourth pair lateral situated respectively

Vol 5(3) 2003 413

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at ca one and two anal body diam posterior to cloacaSecond and third papilla pair close to each other subven-trally with second pair more pronounced Tail elongate-conical narrowing sharply at midpoint and with ne tailtip No phasmids observed with SEM or light micro-scope

TYPE LOCALITY

Pitchers of Nepenthes mirabilis (Lour) Druce in Tung-Kai Botanical Garden Trang Province Southern Thai-land

TYPE MATERIAL

Holotype female (slide MDNC 4021) two paratype fe-males (slides MDNC 4022 4023) and four paratype males(slides MDNC 4024 4025)deposited in the nematodecol-lection of the Zoology Museum Department of BiologyGhent University Belgium four paratype females andfour paratype males on slides with UCR-NC AccessionNo 30059-30063 in the nematode collection of the De-partment of Nematology University of California River-side USA three paratype females and two paratype maleson UCNC slide numbers 5234-5237 in the nematode col-lection of the Department of Nematology University ofCalifornia Davis USA two paratype females and twoparatype males in the nematode type collection of CABIBioscience Egham UK

DIAGNOSIS AND RELATIONSHIPS

Baujardia mirabilis gen n sp n differs from allknown nematodes in having two opposing and offsetspermatheca-like pouches at the junction of oviduct anduterus It also differs from nearly all known Rhabditidain having four cephalic setae instead of papillae in thepresence of six liplets and in the apparent absence ofphasmids

This new genus resembles Panagrellus Thorne 1938in the long body size in the elongate-conical tail and inthe monodelphic prodelphic female reproductive systemwith posteriorlyplaced vulva thickenedvaginalwalls andprominent postvulval sac However Baujardia gen ndiffers from Panagrellus in the characters mentionedabove in the comparatively longer stegostom and inthe more robust male spicules with complex angularmanubrium and undivided tip

The liplets cephalic setae and lack of phasmids arecharacters reminiscent of the genus Teratocephalus de

Man 1876 in the family Teratocephalidae but our newgenus differs from this family in numerous respects egthe amphidial apertures are located on the lips there is adistinct differentiationbetween the pharyngealcorpus andisthmus and the female has an elongate reproductive sys-tem Another taxon in which liplets and setae occur onthe lip region is the family Chambersiellidae Howevermembers of this family have distinct labial setae moreposteriorly located amphids a much narrower stegostomand the tail is usually equipped with a hook-like mucroOverall the mixture of characters resembling membersof different families precludes unequivocal placement ofthe new genus on morphological grounds alone In viewof the molecular analysis discussed below we concludethat it belongs in the infraorder Panagrolaimomorphasu-perfamily Panagrolaimoideaand family PanagrolaimidaeThe morphological diagnosis of Panagrolaimidae mustbe revised signi cantly to accommodate our new genusand the new information on some other panagrolaimsrecently described by Stock et al (2002) As a resultvery few unequivocal diagnostic characters currently re-main for Panagrolaimidae especially when compared tofamilies Cephalobidae Chambersiellidae or Brevibucci-dae (we consider the latter two to be incertae sedis ndash seeDe Ley amp Blaxter 2002) Molecular data are clearly es-sential as an additional source of diagnostic characters inthese nematodes

MOLECULAR PHYLOGENY

The nearly complete SSU sequences of the 22 in-cluded taxa consisted of 1491 (Rhabditophanes sp) to1611 (Panagrolaimus sp) bp with an intermediate valuefor Baujardia mirabilis gen n sp n (1587 bp) Thesecondary structure alignment comprised 1769 positionsof which 971 (55) were variable including 715 (40)parsimony-informative characters The A + T content ofBaujardia gen n was 53

Table 2 summarises the bootstrap support for andmonophyly of clades in the obtained trees NJ MP andML trees consistently support (100 bootstrap) a sistertaxon relationship between the new genus and Panagrel-lus redivivus (L 1767) Goodey 1945 (cf clade A Figs 5-8) Panagrolaimus sp has an uncertain position relativeto these two genera it is placed within the same clade inthe NJ and ML trees while the MP trees place it closerto the strongly supported sister taxa Halicephalobus gin-givalis (Stefanski 1954) Andraacutessy 1984 and Turbatrixaceti (Muumlller 1783) Peters 1927 (clade B) Consistentmaximal support was also given to sister taxa Plectonchus

414 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 2 Monophyly (X D yes X D no) and bootstrap support () for clades

Clades NJ MP1 MP2 MP majority ML(Fig 5) (Fig 6) (Fig 7) rule (Fig 6) (Fig 8)

Clade A(Panagrolaimus (Baujardia (Panagrellus redivivus 57 X X 52 X

AF036599 Panagrellus redivivus AF083007)))(Baujardia (Panagrellus redivivus AF036599 100 X X 100 X

Panagrellus redivivus AF083007))(Panagrolaimus sp (Halicephalobus gingivalis X X X 52 X

Turbatrix aceti))

Clade B(Halicephalobus gingivalis Turbatrix aceti) 86 X X 79 X

Clade C(Plectonchus hunti Panagrobelus stammeri) 100 X X 100 X(Clade C (Clade A Clade B)) (DPanagrolaimidae) 100 X X 100 XMonophyly of Strongyloidoidea (Steinernema X X X 51 X

carpocapsae (Rhabditophanes Strongyloides stercolaris))Monophyly of Panagrolaimoidea and Strongyloidoidea X X X except X X

(DInfraorder Panagrolaimomorpha1 SteinernemaRhabditomorpha1 (DRhabditina) as sister taxon to X X

Panagrolaimomorpha1

Panagrolaimomorpha1 as sister X Xtaxon to Cephalobomorpha1 Tylenchomorpha1

1De Ley and Blaxter (2002)

hunti Stock De Ley De Ley Mundo-Ocampo Baldwinamp Nadler 2002 and Panagrobelus stammeri Ruumlhm 1956(clade C) and to a basal position for this clade withinPanagrolaimidae In other respects our results are com-parable to the SSU rDNA sequence analysis of Feacutelix etal (2000) for example the position of Brevibucca withinRhabditida is uncertain variously assuming a positionat the base of either infraorder Cephalobomorpha Pana-grolaimomorpha or Rhabditomorpha (De Ley amp Blaxter2002) Steinernema carpocapsae (Weiser 1955) WoutsMraacutedaggercek Gerdin amp Bedding 1982 is another nematodewhose position is unclear both in our trees and thoseof Feacutelix et al (2000) but analysis with a more fo-cused dataset con rms its placement in a monophyleticsuperfamily Strongyloidoidea (Dorris et al 2002) Asthe purpose of our study was to explore phylogeneticaf nities between Baujardia gen n and its closest rel-atives we limited the number of sequences included inour dataset and it is therefore not surprising that resolu-tion of relationships among higher taxa remains unclearin our trees (eg aphelenchs are not found to be mono-phyletic)

BIOLOGY

We assume that the pitchers of Nepenthes mirabilis arethe natural habitat of Baujardia mirabilis gen n sp nconsidering that the species was found alive in severalpitchers These pitchers contained a relatively diversefauna dominated by mosquito larvae midges and thenew genus in addition to a few dipteran and coleopteranlarvae The most abundant prey insects in the pitcherswere ants and diverse ying hymenopterans Apparentlya diverse assemblage of organisms lives in associationwith the pitchers of this Nepenthes species This mayrepresent an ecologically complex community similarto the better-known association inhabiting Sarraceniapitchers (eg Kitching 2001 Greeney 2001) Possiblythe nematodes have a phoretic relationship with oneor more insect species Closely related genera are alsocapable of withstanding harsh environments Turbatrixaceti (the vinegar eelworm) Panagrellus redivivus Psilusiae and P nepenthicola inhabit vinegar bookbinderrsquospaste beer lters and pitcher plants respectively

Not much is known about the occurrence of nematodesin pitcher plants and they were not even mentioned in

Vol 5(3) 2003 415

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Fig 5 Neighbour joining tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupnumbers next to branches are percentage bootstrap support values from corresponding50 majority-ruleconsensus tree obtained withNJ analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within the Panagrolaimidae

a reference work of the Nepenthes-inhabiting fauna byThienemann (1933) More extensive study is needed toinvestigate the life cycle and biochemistry of this unusualnew genus Moreover pitcher plants and other epiphytesare clearly a promising source of surprising new nematodetaxa

Discussion

The nematofauna of pitcher plants in Indonesia wasstudied by Menzel (1922) who reported the genera Plec-tus Dorylaimus Rhabditis and Diplogaster He verybrie y described a new species Anguillula nepenthicolaMenzel 1922 which he considered as the only true inhab-itant of pitcher plants and not an accidental recovery An-guillula nepenthicola together with several other speciesincluding Panagrellus pycnus Thorne 1938 was laterplaced in the newly erected genus Turbator by Goodey

(1943) which later (Goodey 1945) became a junior syn-onym of Panagrellus

As P nepenthicola was described from the same habi-tat and similar geographical region as our new genus wehave brie y examined Menzelrsquos (1922) very limited de-scription to justify proposal of a new species for our ma-terial especially in the absence of original illustrations forP nepenthicola Both species are comparable in length(18-27 vs 23-29 mm for P nepenthicola) and in vulvaposition (average of 76 vs 77 for P nepenthicola) Therelative tail length is however clearly longer in our ne-matodes being on average 125 of the vulva-anus dis-tance vs only half the vulva-anus distance length in P ne-penthicola the male tail is on average twice the spiculalength vs three times the spicula length in P nepenthicolathe female c value is 62-89 vs 9-10 in P nepenthicolaand the male c value is 67-11 vs 12-15 in P nepenthicolaThe identity of P nepenthicolabecame somewhat clearerwith the redescription by Micoletzky and Menzel (1928)although only the male tail was illustrated The most ap-

416 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 6 First of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup numbers next to branches are bootstrap values from corresponding 50 majority-ruleconsensus tree obtained with MP analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within thePanagrolaimidae Broken line in the bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to theothers (Figs 5 8)

parent similarities of the new genus with the redescribedP nepenthicolaare the muscular vagina and long postvul-val sac (103-149 vs 78-150 for P nepenthicola) Howeverthe spicules of our new genus and species have more an-gular manubria and their tips are not bi d (an indistinctbi d tip can be observed on the original illustration of Pnepenthicola) Furthermore the male tail is more stronglycurved compared to P nepenthicola and the number andpositioning of the genital papillae is different The differ-ence in tail curvature however may be due to cold xa-tion of our nematodes

The genital system of the new genus is panagrolaimidand shows similarities with the morphology of the femalegonad of Panagrellus redivivus (according to results ob-tained after extraction of the genital system by Geraertet al 1980) As in P redivivus the exure of the gen-ital system is formed by the oviduct and a pair of dis-tinct oviduct cells are observed at the spermatheca side

However the two opposing and offset spermatheca-likepouches observed here represent a unique feature amongstdescribed nematodes These pouches are probably glan-dular rather than having a function in sperm storage assuggested by the unusually large size of the sperm cellsand their occurrence throughout the uterus of gravid fe-males Possibly each compressed cluster of sperm orig-inated from successive copulations as multiple matingsper female might be more frequent in a limited and liquidenvironment Alternatively the movement of eggs couldcause the break-up of single clusters of sperm

On morphological grounds alone it was not immedi-ately apparent in which family Baujardiagen n shouldbeplaced The molecular analysis however robustly placesthe new genus as a sister taxon of Panagrellus within thePanagrolaimidae As a matter of fact the morphologicalsimilarities with Panagrellus as discussed above are rel-atively obvious a posteriori but were not evident before

Vol 5(3) 2003 417

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Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

Baujardia mirabilis gen n sp n from pitcher plants

Fig 3 Baujardia mirabilis gen n sp n SEM studies of female A En face view B Lateral view head C Longitudinal section ofstoma region D Lateral eld (at middle of body) E Longitudinal section at the vulval region F Vulva lateral view G Anus ventralview H Middle of tail Arrowheads cephalic setae Arrows digitate stoma projections (Scale bars C E F D 10 sup1m A B D G HD 1 sup1m)

Vol 5(3) 2003 409

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Fig 4 Baujardia mirabilis gen n sp n SEM studies of male A Longitudinal section of neck region B Longitudinal section of stomaregion showing the subventral sectors C Longitudinal section of stoma region showing the dorsal sector black arrow D projection ofsubventral sector white arrows D basal lamina D Cloacal and tail region lateral view E Cloacal region ventral view F Lateral eld (at middle of body) (Scale bars A B C D E D 10 sup1m F D 1 sup1m)

410 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 Morphometric data of two populations of Baujardia mirabilis gen n sp n Measurements are in sup1m and presented as meansect standard deviation (range)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

n 19 17 12L 2138 2316 sect 238 103 1694 sect 175 103 2113 sect 380 18

(1837-2745) (1350-1944) (1512-2550)a 347 31 sect 35 113 343 sect 41 118 325 sect 45 14

(22-36) (27-43) (23-37)b 57 62 sect 05 87 52 sect 05 96 63 sect 12 19

(53-74) (41-59) (44-77)c 7 75 sect 06 84 87 sect 11 124 66 sect 05 72

(62-89) (67-11) (61-75)c0 105 101 sect 1 101 63 sect 07 108 106 sect 23 21

(73-117) (46-75) (65-135)Maximum body diam 616 76 sect 15 198 50 sect 77 155 643 sect 132 21

(54-107) (36-68) (48-84)Stoma length 20 20 sect 08 41 18 sect 1 55 19 sect 17 87

(18-22) (16-20) (17-22)Head diam (at amphid) 133 13 sect 10 78 12 sect 08 71 12 sect 07 6

(12-15) (9-13) (11-13)Stoma lengthhead diam 15 15 sect 01 69 15 sect 02 104 16 sect 02 97

(13-17) (13-2) (14-18)Seta length 41 28 sect 06 23 27 sect 05 172 26 sect 04 17

(17-42) (17-35) (2-3)Amphid width 67 6 sect 11 19 54 sect 11 195 55 sect 09 17

(4-85) (3-7) (4-7)Neck length 375 372 sect 16 44 326 sect 19 58 336 sect 287 85

(337-402) (285-352) (294-378)Corpus length 252 253 sect 163 64 216 sect 15 69 219 sect 19 87

(222-280) (186-237) (189-250)Procorpus length 145 135 sect 117 87 117 sect 86 73 118 sect 116 98

(117-153) (97-127) (108-132)Metacorpus length 107 115 sect 11 95 984 sect 81 82 114 sect 91 79

(97-132) (78-112) (103-123)Isthmus length 60 57 sect 41 71 512 sect 44 85 538 sect 51 95

(51-62) (40-57) (48-61)Bulb length 425 46 sect 34 73 43 sect 53 123 40 sect 38 94

(40-52) (35-52) (35-46)Corpusisthmus 42 44 sect 04 87 42 sect 04 86 41 sect 03 83

(37-52) (34-48) (36-46)Excretory pore position 232 224 sect 22 98 199 sect 273 137 220 sect 255 12

(180-260) (155-252) (186-252)Nerve ring position 270 269 sect 207 77 237 sect 152 64 255 sect 303 12

(235-312) (210-237) (216-294)EP (as Neck length) 62 59 sect 42 71 615 sect 71 116 66 sect 56 85

(53-66) (48-73) (57-73)NR (as Neck length) 72 722 sect 34 48 73 sect 35 48 758 sect 68 89

(66-80) (65-79) (66-86)Tail length 305 307 sect 18 6 195 sect 154 79 317 sect 481 15

(275-345) (162-225) (243-378)

Vol 5(3) 2003 411

W Bert et al

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

Anal body diam 291 31 sect 3 98 31 sect 27 85 31 sect 83 27(26-40) (258-36) (22-50)

Rectum length 50 49 sect 42 86 47 sect 6 13(42-56) (38-56)

RectumAnal body diam 17 16 sect 02 128 16 sect 03 17(12-21) (11-2)

TailRectum 6 63 sect 05 77(55-73)

Gonad length 1250 1370 sect 187 136 1049 sect 110 105 1251 sect 327 26(1012-1700) (775-1206) (768-1635)

Vulva position 1625 1768 sect 179 101 1620 sect 335 21(1375-2062) (1104-2070)

Vulva body diam 466 57 sect 178 31 46 sect 87 19(42-107) (33-59)

Vagina length 266 29 sect 32 112 26 sect 4 15(25-35) (21-34)

Vulva-Anus distance 207 240 sect 737 306 175 sect 565 322(120-439) (102-270)

Vulva-AnusTail 07 08 sect 02 286 06 sect 02 35(04-14) (03-08)

Postvulval sac length 121 126 sect 106 84 115 sect 154 13(103-149) (93-141)

Postvulval sac length 26 23 sect 05 202 25 sect03 12vulva body diam (12-3) (2-29)

Postvulval stalk length 29 34 sect 53 153 34 sect 64 19(26-44) (23-40)

Postvulval pouch length 91 91 sect 112 123 81 sect 11 14(75-120) (70-101)

Vaginal cell cluster length 20 17 sect 22 126 15 sect 26 18(15-21) (9-17)

Vaginal cell cluster diam 108 13 sect 2 161 9 sect 24 25(10-18) (6-12)

No of eggs in uterus 20 sect 10 491 20 sect 84 42(4-34) (8-31)

V 76 764 sect 21 27 763 sect 3 4(72-81) (72-81)

Gonad (as L) 585 59 sect 28 47 584 sect 55 95(55-64) (51-66)

Vagina (as Vulva 57 531 sect 141 265 59 sect 132 22body diam) (24-74) (40-76)

T 621 sect 45 73(52-74)

Spicule length 57 sect 38 67(50-62)

SpiculeAnal body diam 18 sect 02 118(15-21)

Gubernaculum length 24 sect 22 89(20-28)

412 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

GubernaculumSpicule 04 sect 0 89(04-05)

Spicate tail portion 972 sect 84 84(85-117)

Spicate tailTail length 05 sect 01 141(04-07)

source of specimens for DNA extraction males were not recovered

and stegostom in ratio 1 1 2 Cheilostom without dis-tinct sclerotisations Stegostom divided into two parts insectioned specimens observed with SEM Posterior partapparently with seven denticles or digitate projections ca2 sup1m long reclining against and posteriorly connectedto stoma wall Projections as observed with SEM in twospecimens and consistent with light microscope obser-vations on three females positioned as follows three indorsal sector and two in each subventral sector (the sec-ond projection in the right subventral sector was presum-ably sliced through in Fig 4B) Dorsal gland opening atbase of these projections Basal lamina visible betweengymnostom and stegostom (arrows in Fig 4C) Neck re-gion forming one fth to one seventh of body Pharynxdivided into a cylindrical corpus comprising on average68 of neck length a short isthmus and an oval bulbuswith transverse valves Cardia well developedNerve ringat transition of corpus-isthmus Excretory pore 15-80 sup1manterior to nerve ring wide but often dif cult to nd be-cause of its lack of sclerotisation Deirid not found In-testine with wide lumen demarcated by refractive liningmicrovilli visible anterior to rectum Rectum long withlarge sphincter cells at its beginningReproductivesystemmonodelphic prodelphicwith posteriorly re exed ovaryOviduct always with two distinct nuclei located at ex-ure between ovary and uterus Two opposing and offsetspermatheca-like pouches present at junction of oviductand uterus Each pouch with one distinct terminal cell itslining with a membranous appearance Granulated spermcells very large (15-34 sup1m long) and often compressedinto serial clusters erratically dispersed throughoututerusoften alternating with developing eggs Vulva a trans-verse cleft with overhanging anterior vulval lip locatedat 75 of body length Vagina with thickened walls andsurrounded by strong muscles Prominent postvulval sac

with anterior stalk and posterior pouchTransition vagina-postvulvalsac guarded by a cluster of four cells consistingof two pairs lying side by side Anus a broad curved slitin ventral view Tail elongate-conical with ne tip Irreg-ular pattern of lines substituting annulation over a shortdistance at middle of tail as seen by SEM No phasmidsor phasmid-likestructures observed with SEM or light mi-croscope

Male

General morphology similar to female Body 13-19 mm long arcuate ventrad and especially so in pos-terior region with tail usually curved into 15 coils Cu-ticle nely annulated 08 sup1m wide at midbody Lat-eral eld indistinct with several discontinuous lines ex-tending slightly posterior to cloacal opening 3 sup1m wideat midbody Neck region comprising 20-25 of bodylength Excretory pore from 80 sup1m anterior to nervering to 6 sup1m posterior to nerve ring Reproductive tractoccupying 50-75 of body length monorchic dextraltestis ventrally re exed for 36-120sup1m Vesicula seminaliscontaining large granulated spermatocytes Spicules mas-sive arcuate each with complex angular manubrium andwell developed velum Demarcation of shaft and velumstrongly sclerotised spicule tip not divided Gubernacu-lum with ventral triangular extension and prominent crurawhich may protrude from cloacal aperture Papilla for-mula 1 C 1 C 1=1 C 2 C 1 Seven precloacal papillae rst pair subventral on cuticular sockets at about twoanal body diam anterior to cloaca second pair lateralless than one anal body diam before cloaca third pairsubventral on cuticular sockets at level of precloacal lipand one medioventral papilla located on higher socket atsame level as second precloacal pair Four papilla pairson tail rst and fourth pair lateral situated respectively

Vol 5(3) 2003 413

W Bert et al

at ca one and two anal body diam posterior to cloacaSecond and third papilla pair close to each other subven-trally with second pair more pronounced Tail elongate-conical narrowing sharply at midpoint and with ne tailtip No phasmids observed with SEM or light micro-scope

TYPE LOCALITY

Pitchers of Nepenthes mirabilis (Lour) Druce in Tung-Kai Botanical Garden Trang Province Southern Thai-land

TYPE MATERIAL

Holotype female (slide MDNC 4021) two paratype fe-males (slides MDNC 4022 4023) and four paratype males(slides MDNC 4024 4025)deposited in the nematodecol-lection of the Zoology Museum Department of BiologyGhent University Belgium four paratype females andfour paratype males on slides with UCR-NC AccessionNo 30059-30063 in the nematode collection of the De-partment of Nematology University of California River-side USA three paratype females and two paratype maleson UCNC slide numbers 5234-5237 in the nematode col-lection of the Department of Nematology University ofCalifornia Davis USA two paratype females and twoparatype males in the nematode type collection of CABIBioscience Egham UK

DIAGNOSIS AND RELATIONSHIPS

Baujardia mirabilis gen n sp n differs from allknown nematodes in having two opposing and offsetspermatheca-like pouches at the junction of oviduct anduterus It also differs from nearly all known Rhabditidain having four cephalic setae instead of papillae in thepresence of six liplets and in the apparent absence ofphasmids

This new genus resembles Panagrellus Thorne 1938in the long body size in the elongate-conical tail and inthe monodelphic prodelphic female reproductive systemwith posteriorlyplaced vulva thickenedvaginalwalls andprominent postvulval sac However Baujardia gen ndiffers from Panagrellus in the characters mentionedabove in the comparatively longer stegostom and inthe more robust male spicules with complex angularmanubrium and undivided tip

The liplets cephalic setae and lack of phasmids arecharacters reminiscent of the genus Teratocephalus de

Man 1876 in the family Teratocephalidae but our newgenus differs from this family in numerous respects egthe amphidial apertures are located on the lips there is adistinct differentiationbetween the pharyngealcorpus andisthmus and the female has an elongate reproductive sys-tem Another taxon in which liplets and setae occur onthe lip region is the family Chambersiellidae Howevermembers of this family have distinct labial setae moreposteriorly located amphids a much narrower stegostomand the tail is usually equipped with a hook-like mucroOverall the mixture of characters resembling membersof different families precludes unequivocal placement ofthe new genus on morphological grounds alone In viewof the molecular analysis discussed below we concludethat it belongs in the infraorder Panagrolaimomorphasu-perfamily Panagrolaimoideaand family PanagrolaimidaeThe morphological diagnosis of Panagrolaimidae mustbe revised signi cantly to accommodate our new genusand the new information on some other panagrolaimsrecently described by Stock et al (2002) As a resultvery few unequivocal diagnostic characters currently re-main for Panagrolaimidae especially when compared tofamilies Cephalobidae Chambersiellidae or Brevibucci-dae (we consider the latter two to be incertae sedis ndash seeDe Ley amp Blaxter 2002) Molecular data are clearly es-sential as an additional source of diagnostic characters inthese nematodes

MOLECULAR PHYLOGENY

The nearly complete SSU sequences of the 22 in-cluded taxa consisted of 1491 (Rhabditophanes sp) to1611 (Panagrolaimus sp) bp with an intermediate valuefor Baujardia mirabilis gen n sp n (1587 bp) Thesecondary structure alignment comprised 1769 positionsof which 971 (55) were variable including 715 (40)parsimony-informative characters The A + T content ofBaujardia gen n was 53

Table 2 summarises the bootstrap support for andmonophyly of clades in the obtained trees NJ MP andML trees consistently support (100 bootstrap) a sistertaxon relationship between the new genus and Panagrel-lus redivivus (L 1767) Goodey 1945 (cf clade A Figs 5-8) Panagrolaimus sp has an uncertain position relativeto these two genera it is placed within the same clade inthe NJ and ML trees while the MP trees place it closerto the strongly supported sister taxa Halicephalobus gin-givalis (Stefanski 1954) Andraacutessy 1984 and Turbatrixaceti (Muumlller 1783) Peters 1927 (clade B) Consistentmaximal support was also given to sister taxa Plectonchus

414 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 2 Monophyly (X D yes X D no) and bootstrap support () for clades

Clades NJ MP1 MP2 MP majority ML(Fig 5) (Fig 6) (Fig 7) rule (Fig 6) (Fig 8)

Clade A(Panagrolaimus (Baujardia (Panagrellus redivivus 57 X X 52 X

AF036599 Panagrellus redivivus AF083007)))(Baujardia (Panagrellus redivivus AF036599 100 X X 100 X

Panagrellus redivivus AF083007))(Panagrolaimus sp (Halicephalobus gingivalis X X X 52 X

Turbatrix aceti))

Clade B(Halicephalobus gingivalis Turbatrix aceti) 86 X X 79 X

Clade C(Plectonchus hunti Panagrobelus stammeri) 100 X X 100 X(Clade C (Clade A Clade B)) (DPanagrolaimidae) 100 X X 100 XMonophyly of Strongyloidoidea (Steinernema X X X 51 X

carpocapsae (Rhabditophanes Strongyloides stercolaris))Monophyly of Panagrolaimoidea and Strongyloidoidea X X X except X X

(DInfraorder Panagrolaimomorpha1 SteinernemaRhabditomorpha1 (DRhabditina) as sister taxon to X X

Panagrolaimomorpha1

Panagrolaimomorpha1 as sister X Xtaxon to Cephalobomorpha1 Tylenchomorpha1

1De Ley and Blaxter (2002)

hunti Stock De Ley De Ley Mundo-Ocampo Baldwinamp Nadler 2002 and Panagrobelus stammeri Ruumlhm 1956(clade C) and to a basal position for this clade withinPanagrolaimidae In other respects our results are com-parable to the SSU rDNA sequence analysis of Feacutelix etal (2000) for example the position of Brevibucca withinRhabditida is uncertain variously assuming a positionat the base of either infraorder Cephalobomorpha Pana-grolaimomorpha or Rhabditomorpha (De Ley amp Blaxter2002) Steinernema carpocapsae (Weiser 1955) WoutsMraacutedaggercek Gerdin amp Bedding 1982 is another nematodewhose position is unclear both in our trees and thoseof Feacutelix et al (2000) but analysis with a more fo-cused dataset con rms its placement in a monophyleticsuperfamily Strongyloidoidea (Dorris et al 2002) Asthe purpose of our study was to explore phylogeneticaf nities between Baujardia gen n and its closest rel-atives we limited the number of sequences included inour dataset and it is therefore not surprising that resolu-tion of relationships among higher taxa remains unclearin our trees (eg aphelenchs are not found to be mono-phyletic)

BIOLOGY

We assume that the pitchers of Nepenthes mirabilis arethe natural habitat of Baujardia mirabilis gen n sp nconsidering that the species was found alive in severalpitchers These pitchers contained a relatively diversefauna dominated by mosquito larvae midges and thenew genus in addition to a few dipteran and coleopteranlarvae The most abundant prey insects in the pitcherswere ants and diverse ying hymenopterans Apparentlya diverse assemblage of organisms lives in associationwith the pitchers of this Nepenthes species This mayrepresent an ecologically complex community similarto the better-known association inhabiting Sarraceniapitchers (eg Kitching 2001 Greeney 2001) Possiblythe nematodes have a phoretic relationship with oneor more insect species Closely related genera are alsocapable of withstanding harsh environments Turbatrixaceti (the vinegar eelworm) Panagrellus redivivus Psilusiae and P nepenthicola inhabit vinegar bookbinderrsquospaste beer lters and pitcher plants respectively

Not much is known about the occurrence of nematodesin pitcher plants and they were not even mentioned in

Vol 5(3) 2003 415

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Fig 5 Neighbour joining tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupnumbers next to branches are percentage bootstrap support values from corresponding50 majority-ruleconsensus tree obtained withNJ analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within the Panagrolaimidae

a reference work of the Nepenthes-inhabiting fauna byThienemann (1933) More extensive study is needed toinvestigate the life cycle and biochemistry of this unusualnew genus Moreover pitcher plants and other epiphytesare clearly a promising source of surprising new nematodetaxa

Discussion

The nematofauna of pitcher plants in Indonesia wasstudied by Menzel (1922) who reported the genera Plec-tus Dorylaimus Rhabditis and Diplogaster He verybrie y described a new species Anguillula nepenthicolaMenzel 1922 which he considered as the only true inhab-itant of pitcher plants and not an accidental recovery An-guillula nepenthicola together with several other speciesincluding Panagrellus pycnus Thorne 1938 was laterplaced in the newly erected genus Turbator by Goodey

(1943) which later (Goodey 1945) became a junior syn-onym of Panagrellus

As P nepenthicola was described from the same habi-tat and similar geographical region as our new genus wehave brie y examined Menzelrsquos (1922) very limited de-scription to justify proposal of a new species for our ma-terial especially in the absence of original illustrations forP nepenthicola Both species are comparable in length(18-27 vs 23-29 mm for P nepenthicola) and in vulvaposition (average of 76 vs 77 for P nepenthicola) Therelative tail length is however clearly longer in our ne-matodes being on average 125 of the vulva-anus dis-tance vs only half the vulva-anus distance length in P ne-penthicola the male tail is on average twice the spiculalength vs three times the spicula length in P nepenthicolathe female c value is 62-89 vs 9-10 in P nepenthicolaand the male c value is 67-11 vs 12-15 in P nepenthicolaThe identity of P nepenthicolabecame somewhat clearerwith the redescription by Micoletzky and Menzel (1928)although only the male tail was illustrated The most ap-

416 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 6 First of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup numbers next to branches are bootstrap values from corresponding 50 majority-ruleconsensus tree obtained with MP analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within thePanagrolaimidae Broken line in the bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to theothers (Figs 5 8)

parent similarities of the new genus with the redescribedP nepenthicolaare the muscular vagina and long postvul-val sac (103-149 vs 78-150 for P nepenthicola) Howeverthe spicules of our new genus and species have more an-gular manubria and their tips are not bi d (an indistinctbi d tip can be observed on the original illustration of Pnepenthicola) Furthermore the male tail is more stronglycurved compared to P nepenthicola and the number andpositioning of the genital papillae is different The differ-ence in tail curvature however may be due to cold xa-tion of our nematodes

The genital system of the new genus is panagrolaimidand shows similarities with the morphology of the femalegonad of Panagrellus redivivus (according to results ob-tained after extraction of the genital system by Geraertet al 1980) As in P redivivus the exure of the gen-ital system is formed by the oviduct and a pair of dis-tinct oviduct cells are observed at the spermatheca side

However the two opposing and offset spermatheca-likepouches observed here represent a unique feature amongstdescribed nematodes These pouches are probably glan-dular rather than having a function in sperm storage assuggested by the unusually large size of the sperm cellsand their occurrence throughout the uterus of gravid fe-males Possibly each compressed cluster of sperm orig-inated from successive copulations as multiple matingsper female might be more frequent in a limited and liquidenvironment Alternatively the movement of eggs couldcause the break-up of single clusters of sperm

On morphological grounds alone it was not immedi-ately apparent in which family Baujardiagen n shouldbeplaced The molecular analysis however robustly placesthe new genus as a sister taxon of Panagrellus within thePanagrolaimidae As a matter of fact the morphologicalsimilarities with Panagrellus as discussed above are rel-atively obvious a posteriori but were not evident before

Vol 5(3) 2003 417

W Bert et al

Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

W Bert et al

Fig 4 Baujardia mirabilis gen n sp n SEM studies of male A Longitudinal section of neck region B Longitudinal section of stomaregion showing the subventral sectors C Longitudinal section of stoma region showing the dorsal sector black arrow D projection ofsubventral sector white arrows D basal lamina D Cloacal and tail region lateral view E Cloacal region ventral view F Lateral eld (at middle of body) (Scale bars A B C D E D 10 sup1m F D 1 sup1m)

410 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 Morphometric data of two populations of Baujardia mirabilis gen n sp n Measurements are in sup1m and presented as meansect standard deviation (range)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

n 19 17 12L 2138 2316 sect 238 103 1694 sect 175 103 2113 sect 380 18

(1837-2745) (1350-1944) (1512-2550)a 347 31 sect 35 113 343 sect 41 118 325 sect 45 14

(22-36) (27-43) (23-37)b 57 62 sect 05 87 52 sect 05 96 63 sect 12 19

(53-74) (41-59) (44-77)c 7 75 sect 06 84 87 sect 11 124 66 sect 05 72

(62-89) (67-11) (61-75)c0 105 101 sect 1 101 63 sect 07 108 106 sect 23 21

(73-117) (46-75) (65-135)Maximum body diam 616 76 sect 15 198 50 sect 77 155 643 sect 132 21

(54-107) (36-68) (48-84)Stoma length 20 20 sect 08 41 18 sect 1 55 19 sect 17 87

(18-22) (16-20) (17-22)Head diam (at amphid) 133 13 sect 10 78 12 sect 08 71 12 sect 07 6

(12-15) (9-13) (11-13)Stoma lengthhead diam 15 15 sect 01 69 15 sect 02 104 16 sect 02 97

(13-17) (13-2) (14-18)Seta length 41 28 sect 06 23 27 sect 05 172 26 sect 04 17

(17-42) (17-35) (2-3)Amphid width 67 6 sect 11 19 54 sect 11 195 55 sect 09 17

(4-85) (3-7) (4-7)Neck length 375 372 sect 16 44 326 sect 19 58 336 sect 287 85

(337-402) (285-352) (294-378)Corpus length 252 253 sect 163 64 216 sect 15 69 219 sect 19 87

(222-280) (186-237) (189-250)Procorpus length 145 135 sect 117 87 117 sect 86 73 118 sect 116 98

(117-153) (97-127) (108-132)Metacorpus length 107 115 sect 11 95 984 sect 81 82 114 sect 91 79

(97-132) (78-112) (103-123)Isthmus length 60 57 sect 41 71 512 sect 44 85 538 sect 51 95

(51-62) (40-57) (48-61)Bulb length 425 46 sect 34 73 43 sect 53 123 40 sect 38 94

(40-52) (35-52) (35-46)Corpusisthmus 42 44 sect 04 87 42 sect 04 86 41 sect 03 83

(37-52) (34-48) (36-46)Excretory pore position 232 224 sect 22 98 199 sect 273 137 220 sect 255 12

(180-260) (155-252) (186-252)Nerve ring position 270 269 sect 207 77 237 sect 152 64 255 sect 303 12

(235-312) (210-237) (216-294)EP (as Neck length) 62 59 sect 42 71 615 sect 71 116 66 sect 56 85

(53-66) (48-73) (57-73)NR (as Neck length) 72 722 sect 34 48 73 sect 35 48 758 sect 68 89

(66-80) (65-79) (66-86)Tail length 305 307 sect 18 6 195 sect 154 79 317 sect 481 15

(275-345) (162-225) (243-378)

Vol 5(3) 2003 411

W Bert et al

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

Anal body diam 291 31 sect 3 98 31 sect 27 85 31 sect 83 27(26-40) (258-36) (22-50)

Rectum length 50 49 sect 42 86 47 sect 6 13(42-56) (38-56)

RectumAnal body diam 17 16 sect 02 128 16 sect 03 17(12-21) (11-2)

TailRectum 6 63 sect 05 77(55-73)

Gonad length 1250 1370 sect 187 136 1049 sect 110 105 1251 sect 327 26(1012-1700) (775-1206) (768-1635)

Vulva position 1625 1768 sect 179 101 1620 sect 335 21(1375-2062) (1104-2070)

Vulva body diam 466 57 sect 178 31 46 sect 87 19(42-107) (33-59)

Vagina length 266 29 sect 32 112 26 sect 4 15(25-35) (21-34)

Vulva-Anus distance 207 240 sect 737 306 175 sect 565 322(120-439) (102-270)

Vulva-AnusTail 07 08 sect 02 286 06 sect 02 35(04-14) (03-08)

Postvulval sac length 121 126 sect 106 84 115 sect 154 13(103-149) (93-141)

Postvulval sac length 26 23 sect 05 202 25 sect03 12vulva body diam (12-3) (2-29)

Postvulval stalk length 29 34 sect 53 153 34 sect 64 19(26-44) (23-40)

Postvulval pouch length 91 91 sect 112 123 81 sect 11 14(75-120) (70-101)

Vaginal cell cluster length 20 17 sect 22 126 15 sect 26 18(15-21) (9-17)

Vaginal cell cluster diam 108 13 sect 2 161 9 sect 24 25(10-18) (6-12)

No of eggs in uterus 20 sect 10 491 20 sect 84 42(4-34) (8-31)

V 76 764 sect 21 27 763 sect 3 4(72-81) (72-81)

Gonad (as L) 585 59 sect 28 47 584 sect 55 95(55-64) (51-66)

Vagina (as Vulva 57 531 sect 141 265 59 sect 132 22body diam) (24-74) (40-76)

T 621 sect 45 73(52-74)

Spicule length 57 sect 38 67(50-62)

SpiculeAnal body diam 18 sect 02 118(15-21)

Gubernaculum length 24 sect 22 89(20-28)

412 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

GubernaculumSpicule 04 sect 0 89(04-05)

Spicate tail portion 972 sect 84 84(85-117)

Spicate tailTail length 05 sect 01 141(04-07)

source of specimens for DNA extraction males were not recovered

and stegostom in ratio 1 1 2 Cheilostom without dis-tinct sclerotisations Stegostom divided into two parts insectioned specimens observed with SEM Posterior partapparently with seven denticles or digitate projections ca2 sup1m long reclining against and posteriorly connectedto stoma wall Projections as observed with SEM in twospecimens and consistent with light microscope obser-vations on three females positioned as follows three indorsal sector and two in each subventral sector (the sec-ond projection in the right subventral sector was presum-ably sliced through in Fig 4B) Dorsal gland opening atbase of these projections Basal lamina visible betweengymnostom and stegostom (arrows in Fig 4C) Neck re-gion forming one fth to one seventh of body Pharynxdivided into a cylindrical corpus comprising on average68 of neck length a short isthmus and an oval bulbuswith transverse valves Cardia well developedNerve ringat transition of corpus-isthmus Excretory pore 15-80 sup1manterior to nerve ring wide but often dif cult to nd be-cause of its lack of sclerotisation Deirid not found In-testine with wide lumen demarcated by refractive liningmicrovilli visible anterior to rectum Rectum long withlarge sphincter cells at its beginningReproductivesystemmonodelphic prodelphicwith posteriorly re exed ovaryOviduct always with two distinct nuclei located at ex-ure between ovary and uterus Two opposing and offsetspermatheca-like pouches present at junction of oviductand uterus Each pouch with one distinct terminal cell itslining with a membranous appearance Granulated spermcells very large (15-34 sup1m long) and often compressedinto serial clusters erratically dispersed throughoututerusoften alternating with developing eggs Vulva a trans-verse cleft with overhanging anterior vulval lip locatedat 75 of body length Vagina with thickened walls andsurrounded by strong muscles Prominent postvulval sac

with anterior stalk and posterior pouchTransition vagina-postvulvalsac guarded by a cluster of four cells consistingof two pairs lying side by side Anus a broad curved slitin ventral view Tail elongate-conical with ne tip Irreg-ular pattern of lines substituting annulation over a shortdistance at middle of tail as seen by SEM No phasmidsor phasmid-likestructures observed with SEM or light mi-croscope

Male

General morphology similar to female Body 13-19 mm long arcuate ventrad and especially so in pos-terior region with tail usually curved into 15 coils Cu-ticle nely annulated 08 sup1m wide at midbody Lat-eral eld indistinct with several discontinuous lines ex-tending slightly posterior to cloacal opening 3 sup1m wideat midbody Neck region comprising 20-25 of bodylength Excretory pore from 80 sup1m anterior to nervering to 6 sup1m posterior to nerve ring Reproductive tractoccupying 50-75 of body length monorchic dextraltestis ventrally re exed for 36-120sup1m Vesicula seminaliscontaining large granulated spermatocytes Spicules mas-sive arcuate each with complex angular manubrium andwell developed velum Demarcation of shaft and velumstrongly sclerotised spicule tip not divided Gubernacu-lum with ventral triangular extension and prominent crurawhich may protrude from cloacal aperture Papilla for-mula 1 C 1 C 1=1 C 2 C 1 Seven precloacal papillae rst pair subventral on cuticular sockets at about twoanal body diam anterior to cloaca second pair lateralless than one anal body diam before cloaca third pairsubventral on cuticular sockets at level of precloacal lipand one medioventral papilla located on higher socket atsame level as second precloacal pair Four papilla pairson tail rst and fourth pair lateral situated respectively

Vol 5(3) 2003 413

W Bert et al

at ca one and two anal body diam posterior to cloacaSecond and third papilla pair close to each other subven-trally with second pair more pronounced Tail elongate-conical narrowing sharply at midpoint and with ne tailtip No phasmids observed with SEM or light micro-scope

TYPE LOCALITY

Pitchers of Nepenthes mirabilis (Lour) Druce in Tung-Kai Botanical Garden Trang Province Southern Thai-land

TYPE MATERIAL

Holotype female (slide MDNC 4021) two paratype fe-males (slides MDNC 4022 4023) and four paratype males(slides MDNC 4024 4025)deposited in the nematodecol-lection of the Zoology Museum Department of BiologyGhent University Belgium four paratype females andfour paratype males on slides with UCR-NC AccessionNo 30059-30063 in the nematode collection of the De-partment of Nematology University of California River-side USA three paratype females and two paratype maleson UCNC slide numbers 5234-5237 in the nematode col-lection of the Department of Nematology University ofCalifornia Davis USA two paratype females and twoparatype males in the nematode type collection of CABIBioscience Egham UK

DIAGNOSIS AND RELATIONSHIPS

Baujardia mirabilis gen n sp n differs from allknown nematodes in having two opposing and offsetspermatheca-like pouches at the junction of oviduct anduterus It also differs from nearly all known Rhabditidain having four cephalic setae instead of papillae in thepresence of six liplets and in the apparent absence ofphasmids

This new genus resembles Panagrellus Thorne 1938in the long body size in the elongate-conical tail and inthe monodelphic prodelphic female reproductive systemwith posteriorlyplaced vulva thickenedvaginalwalls andprominent postvulval sac However Baujardia gen ndiffers from Panagrellus in the characters mentionedabove in the comparatively longer stegostom and inthe more robust male spicules with complex angularmanubrium and undivided tip

The liplets cephalic setae and lack of phasmids arecharacters reminiscent of the genus Teratocephalus de

Man 1876 in the family Teratocephalidae but our newgenus differs from this family in numerous respects egthe amphidial apertures are located on the lips there is adistinct differentiationbetween the pharyngealcorpus andisthmus and the female has an elongate reproductive sys-tem Another taxon in which liplets and setae occur onthe lip region is the family Chambersiellidae Howevermembers of this family have distinct labial setae moreposteriorly located amphids a much narrower stegostomand the tail is usually equipped with a hook-like mucroOverall the mixture of characters resembling membersof different families precludes unequivocal placement ofthe new genus on morphological grounds alone In viewof the molecular analysis discussed below we concludethat it belongs in the infraorder Panagrolaimomorphasu-perfamily Panagrolaimoideaand family PanagrolaimidaeThe morphological diagnosis of Panagrolaimidae mustbe revised signi cantly to accommodate our new genusand the new information on some other panagrolaimsrecently described by Stock et al (2002) As a resultvery few unequivocal diagnostic characters currently re-main for Panagrolaimidae especially when compared tofamilies Cephalobidae Chambersiellidae or Brevibucci-dae (we consider the latter two to be incertae sedis ndash seeDe Ley amp Blaxter 2002) Molecular data are clearly es-sential as an additional source of diagnostic characters inthese nematodes

MOLECULAR PHYLOGENY

The nearly complete SSU sequences of the 22 in-cluded taxa consisted of 1491 (Rhabditophanes sp) to1611 (Panagrolaimus sp) bp with an intermediate valuefor Baujardia mirabilis gen n sp n (1587 bp) Thesecondary structure alignment comprised 1769 positionsof which 971 (55) were variable including 715 (40)parsimony-informative characters The A + T content ofBaujardia gen n was 53

Table 2 summarises the bootstrap support for andmonophyly of clades in the obtained trees NJ MP andML trees consistently support (100 bootstrap) a sistertaxon relationship between the new genus and Panagrel-lus redivivus (L 1767) Goodey 1945 (cf clade A Figs 5-8) Panagrolaimus sp has an uncertain position relativeto these two genera it is placed within the same clade inthe NJ and ML trees while the MP trees place it closerto the strongly supported sister taxa Halicephalobus gin-givalis (Stefanski 1954) Andraacutessy 1984 and Turbatrixaceti (Muumlller 1783) Peters 1927 (clade B) Consistentmaximal support was also given to sister taxa Plectonchus

414 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 2 Monophyly (X D yes X D no) and bootstrap support () for clades

Clades NJ MP1 MP2 MP majority ML(Fig 5) (Fig 6) (Fig 7) rule (Fig 6) (Fig 8)

Clade A(Panagrolaimus (Baujardia (Panagrellus redivivus 57 X X 52 X

AF036599 Panagrellus redivivus AF083007)))(Baujardia (Panagrellus redivivus AF036599 100 X X 100 X

Panagrellus redivivus AF083007))(Panagrolaimus sp (Halicephalobus gingivalis X X X 52 X

Turbatrix aceti))

Clade B(Halicephalobus gingivalis Turbatrix aceti) 86 X X 79 X

Clade C(Plectonchus hunti Panagrobelus stammeri) 100 X X 100 X(Clade C (Clade A Clade B)) (DPanagrolaimidae) 100 X X 100 XMonophyly of Strongyloidoidea (Steinernema X X X 51 X

carpocapsae (Rhabditophanes Strongyloides stercolaris))Monophyly of Panagrolaimoidea and Strongyloidoidea X X X except X X

(DInfraorder Panagrolaimomorpha1 SteinernemaRhabditomorpha1 (DRhabditina) as sister taxon to X X

Panagrolaimomorpha1

Panagrolaimomorpha1 as sister X Xtaxon to Cephalobomorpha1 Tylenchomorpha1

1De Ley and Blaxter (2002)

hunti Stock De Ley De Ley Mundo-Ocampo Baldwinamp Nadler 2002 and Panagrobelus stammeri Ruumlhm 1956(clade C) and to a basal position for this clade withinPanagrolaimidae In other respects our results are com-parable to the SSU rDNA sequence analysis of Feacutelix etal (2000) for example the position of Brevibucca withinRhabditida is uncertain variously assuming a positionat the base of either infraorder Cephalobomorpha Pana-grolaimomorpha or Rhabditomorpha (De Ley amp Blaxter2002) Steinernema carpocapsae (Weiser 1955) WoutsMraacutedaggercek Gerdin amp Bedding 1982 is another nematodewhose position is unclear both in our trees and thoseof Feacutelix et al (2000) but analysis with a more fo-cused dataset con rms its placement in a monophyleticsuperfamily Strongyloidoidea (Dorris et al 2002) Asthe purpose of our study was to explore phylogeneticaf nities between Baujardia gen n and its closest rel-atives we limited the number of sequences included inour dataset and it is therefore not surprising that resolu-tion of relationships among higher taxa remains unclearin our trees (eg aphelenchs are not found to be mono-phyletic)

BIOLOGY

We assume that the pitchers of Nepenthes mirabilis arethe natural habitat of Baujardia mirabilis gen n sp nconsidering that the species was found alive in severalpitchers These pitchers contained a relatively diversefauna dominated by mosquito larvae midges and thenew genus in addition to a few dipteran and coleopteranlarvae The most abundant prey insects in the pitcherswere ants and diverse ying hymenopterans Apparentlya diverse assemblage of organisms lives in associationwith the pitchers of this Nepenthes species This mayrepresent an ecologically complex community similarto the better-known association inhabiting Sarraceniapitchers (eg Kitching 2001 Greeney 2001) Possiblythe nematodes have a phoretic relationship with oneor more insect species Closely related genera are alsocapable of withstanding harsh environments Turbatrixaceti (the vinegar eelworm) Panagrellus redivivus Psilusiae and P nepenthicola inhabit vinegar bookbinderrsquospaste beer lters and pitcher plants respectively

Not much is known about the occurrence of nematodesin pitcher plants and they were not even mentioned in

Vol 5(3) 2003 415

W Bert et al

Fig 5 Neighbour joining tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupnumbers next to branches are percentage bootstrap support values from corresponding50 majority-ruleconsensus tree obtained withNJ analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within the Panagrolaimidae

a reference work of the Nepenthes-inhabiting fauna byThienemann (1933) More extensive study is needed toinvestigate the life cycle and biochemistry of this unusualnew genus Moreover pitcher plants and other epiphytesare clearly a promising source of surprising new nematodetaxa

Discussion

The nematofauna of pitcher plants in Indonesia wasstudied by Menzel (1922) who reported the genera Plec-tus Dorylaimus Rhabditis and Diplogaster He verybrie y described a new species Anguillula nepenthicolaMenzel 1922 which he considered as the only true inhab-itant of pitcher plants and not an accidental recovery An-guillula nepenthicola together with several other speciesincluding Panagrellus pycnus Thorne 1938 was laterplaced in the newly erected genus Turbator by Goodey

(1943) which later (Goodey 1945) became a junior syn-onym of Panagrellus

As P nepenthicola was described from the same habi-tat and similar geographical region as our new genus wehave brie y examined Menzelrsquos (1922) very limited de-scription to justify proposal of a new species for our ma-terial especially in the absence of original illustrations forP nepenthicola Both species are comparable in length(18-27 vs 23-29 mm for P nepenthicola) and in vulvaposition (average of 76 vs 77 for P nepenthicola) Therelative tail length is however clearly longer in our ne-matodes being on average 125 of the vulva-anus dis-tance vs only half the vulva-anus distance length in P ne-penthicola the male tail is on average twice the spiculalength vs three times the spicula length in P nepenthicolathe female c value is 62-89 vs 9-10 in P nepenthicolaand the male c value is 67-11 vs 12-15 in P nepenthicolaThe identity of P nepenthicolabecame somewhat clearerwith the redescription by Micoletzky and Menzel (1928)although only the male tail was illustrated The most ap-

416 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 6 First of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup numbers next to branches are bootstrap values from corresponding 50 majority-ruleconsensus tree obtained with MP analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within thePanagrolaimidae Broken line in the bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to theothers (Figs 5 8)

parent similarities of the new genus with the redescribedP nepenthicolaare the muscular vagina and long postvul-val sac (103-149 vs 78-150 for P nepenthicola) Howeverthe spicules of our new genus and species have more an-gular manubria and their tips are not bi d (an indistinctbi d tip can be observed on the original illustration of Pnepenthicola) Furthermore the male tail is more stronglycurved compared to P nepenthicola and the number andpositioning of the genital papillae is different The differ-ence in tail curvature however may be due to cold xa-tion of our nematodes

The genital system of the new genus is panagrolaimidand shows similarities with the morphology of the femalegonad of Panagrellus redivivus (according to results ob-tained after extraction of the genital system by Geraertet al 1980) As in P redivivus the exure of the gen-ital system is formed by the oviduct and a pair of dis-tinct oviduct cells are observed at the spermatheca side

However the two opposing and offset spermatheca-likepouches observed here represent a unique feature amongstdescribed nematodes These pouches are probably glan-dular rather than having a function in sperm storage assuggested by the unusually large size of the sperm cellsand their occurrence throughout the uterus of gravid fe-males Possibly each compressed cluster of sperm orig-inated from successive copulations as multiple matingsper female might be more frequent in a limited and liquidenvironment Alternatively the movement of eggs couldcause the break-up of single clusters of sperm

On morphological grounds alone it was not immedi-ately apparent in which family Baujardiagen n shouldbeplaced The molecular analysis however robustly placesthe new genus as a sister taxon of Panagrellus within thePanagrolaimidae As a matter of fact the morphologicalsimilarities with Panagrellus as discussed above are rel-atively obvious a posteriori but were not evident before

Vol 5(3) 2003 417

W Bert et al

Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 Morphometric data of two populations of Baujardia mirabilis gen n sp n Measurements are in sup1m and presented as meansect standard deviation (range)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

n 19 17 12L 2138 2316 sect 238 103 1694 sect 175 103 2113 sect 380 18

(1837-2745) (1350-1944) (1512-2550)a 347 31 sect 35 113 343 sect 41 118 325 sect 45 14

(22-36) (27-43) (23-37)b 57 62 sect 05 87 52 sect 05 96 63 sect 12 19

(53-74) (41-59) (44-77)c 7 75 sect 06 84 87 sect 11 124 66 sect 05 72

(62-89) (67-11) (61-75)c0 105 101 sect 1 101 63 sect 07 108 106 sect 23 21

(73-117) (46-75) (65-135)Maximum body diam 616 76 sect 15 198 50 sect 77 155 643 sect 132 21

(54-107) (36-68) (48-84)Stoma length 20 20 sect 08 41 18 sect 1 55 19 sect 17 87

(18-22) (16-20) (17-22)Head diam (at amphid) 133 13 sect 10 78 12 sect 08 71 12 sect 07 6

(12-15) (9-13) (11-13)Stoma lengthhead diam 15 15 sect 01 69 15 sect 02 104 16 sect 02 97

(13-17) (13-2) (14-18)Seta length 41 28 sect 06 23 27 sect 05 172 26 sect 04 17

(17-42) (17-35) (2-3)Amphid width 67 6 sect 11 19 54 sect 11 195 55 sect 09 17

(4-85) (3-7) (4-7)Neck length 375 372 sect 16 44 326 sect 19 58 336 sect 287 85

(337-402) (285-352) (294-378)Corpus length 252 253 sect 163 64 216 sect 15 69 219 sect 19 87

(222-280) (186-237) (189-250)Procorpus length 145 135 sect 117 87 117 sect 86 73 118 sect 116 98

(117-153) (97-127) (108-132)Metacorpus length 107 115 sect 11 95 984 sect 81 82 114 sect 91 79

(97-132) (78-112) (103-123)Isthmus length 60 57 sect 41 71 512 sect 44 85 538 sect 51 95

(51-62) (40-57) (48-61)Bulb length 425 46 sect 34 73 43 sect 53 123 40 sect 38 94

(40-52) (35-52) (35-46)Corpusisthmus 42 44 sect 04 87 42 sect 04 86 41 sect 03 83

(37-52) (34-48) (36-46)Excretory pore position 232 224 sect 22 98 199 sect 273 137 220 sect 255 12

(180-260) (155-252) (186-252)Nerve ring position 270 269 sect 207 77 237 sect 152 64 255 sect 303 12

(235-312) (210-237) (216-294)EP (as Neck length) 62 59 sect 42 71 615 sect 71 116 66 sect 56 85

(53-66) (48-73) (57-73)NR (as Neck length) 72 722 sect 34 48 73 sect 35 48 758 sect 68 89

(66-80) (65-79) (66-86)Tail length 305 307 sect 18 6 195 sect 154 79 317 sect 481 15

(275-345) (162-225) (243-378)

Vol 5(3) 2003 411

W Bert et al

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

Anal body diam 291 31 sect 3 98 31 sect 27 85 31 sect 83 27(26-40) (258-36) (22-50)

Rectum length 50 49 sect 42 86 47 sect 6 13(42-56) (38-56)

RectumAnal body diam 17 16 sect 02 128 16 sect 03 17(12-21) (11-2)

TailRectum 6 63 sect 05 77(55-73)

Gonad length 1250 1370 sect 187 136 1049 sect 110 105 1251 sect 327 26(1012-1700) (775-1206) (768-1635)

Vulva position 1625 1768 sect 179 101 1620 sect 335 21(1375-2062) (1104-2070)

Vulva body diam 466 57 sect 178 31 46 sect 87 19(42-107) (33-59)

Vagina length 266 29 sect 32 112 26 sect 4 15(25-35) (21-34)

Vulva-Anus distance 207 240 sect 737 306 175 sect 565 322(120-439) (102-270)

Vulva-AnusTail 07 08 sect 02 286 06 sect 02 35(04-14) (03-08)

Postvulval sac length 121 126 sect 106 84 115 sect 154 13(103-149) (93-141)

Postvulval sac length 26 23 sect 05 202 25 sect03 12vulva body diam (12-3) (2-29)

Postvulval stalk length 29 34 sect 53 153 34 sect 64 19(26-44) (23-40)

Postvulval pouch length 91 91 sect 112 123 81 sect 11 14(75-120) (70-101)

Vaginal cell cluster length 20 17 sect 22 126 15 sect 26 18(15-21) (9-17)

Vaginal cell cluster diam 108 13 sect 2 161 9 sect 24 25(10-18) (6-12)

No of eggs in uterus 20 sect 10 491 20 sect 84 42(4-34) (8-31)

V 76 764 sect 21 27 763 sect 3 4(72-81) (72-81)

Gonad (as L) 585 59 sect 28 47 584 sect 55 95(55-64) (51-66)

Vagina (as Vulva 57 531 sect 141 265 59 sect 132 22body diam) (24-74) (40-76)

T 621 sect 45 73(52-74)

Spicule length 57 sect 38 67(50-62)

SpiculeAnal body diam 18 sect 02 118(15-21)

Gubernaculum length 24 sect 22 89(20-28)

412 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

GubernaculumSpicule 04 sect 0 89(04-05)

Spicate tail portion 972 sect 84 84(85-117)

Spicate tailTail length 05 sect 01 141(04-07)

source of specimens for DNA extraction males were not recovered

and stegostom in ratio 1 1 2 Cheilostom without dis-tinct sclerotisations Stegostom divided into two parts insectioned specimens observed with SEM Posterior partapparently with seven denticles or digitate projections ca2 sup1m long reclining against and posteriorly connectedto stoma wall Projections as observed with SEM in twospecimens and consistent with light microscope obser-vations on three females positioned as follows three indorsal sector and two in each subventral sector (the sec-ond projection in the right subventral sector was presum-ably sliced through in Fig 4B) Dorsal gland opening atbase of these projections Basal lamina visible betweengymnostom and stegostom (arrows in Fig 4C) Neck re-gion forming one fth to one seventh of body Pharynxdivided into a cylindrical corpus comprising on average68 of neck length a short isthmus and an oval bulbuswith transverse valves Cardia well developedNerve ringat transition of corpus-isthmus Excretory pore 15-80 sup1manterior to nerve ring wide but often dif cult to nd be-cause of its lack of sclerotisation Deirid not found In-testine with wide lumen demarcated by refractive liningmicrovilli visible anterior to rectum Rectum long withlarge sphincter cells at its beginningReproductivesystemmonodelphic prodelphicwith posteriorly re exed ovaryOviduct always with two distinct nuclei located at ex-ure between ovary and uterus Two opposing and offsetspermatheca-like pouches present at junction of oviductand uterus Each pouch with one distinct terminal cell itslining with a membranous appearance Granulated spermcells very large (15-34 sup1m long) and often compressedinto serial clusters erratically dispersed throughoututerusoften alternating with developing eggs Vulva a trans-verse cleft with overhanging anterior vulval lip locatedat 75 of body length Vagina with thickened walls andsurrounded by strong muscles Prominent postvulval sac

with anterior stalk and posterior pouchTransition vagina-postvulvalsac guarded by a cluster of four cells consistingof two pairs lying side by side Anus a broad curved slitin ventral view Tail elongate-conical with ne tip Irreg-ular pattern of lines substituting annulation over a shortdistance at middle of tail as seen by SEM No phasmidsor phasmid-likestructures observed with SEM or light mi-croscope

Male

General morphology similar to female Body 13-19 mm long arcuate ventrad and especially so in pos-terior region with tail usually curved into 15 coils Cu-ticle nely annulated 08 sup1m wide at midbody Lat-eral eld indistinct with several discontinuous lines ex-tending slightly posterior to cloacal opening 3 sup1m wideat midbody Neck region comprising 20-25 of bodylength Excretory pore from 80 sup1m anterior to nervering to 6 sup1m posterior to nerve ring Reproductive tractoccupying 50-75 of body length monorchic dextraltestis ventrally re exed for 36-120sup1m Vesicula seminaliscontaining large granulated spermatocytes Spicules mas-sive arcuate each with complex angular manubrium andwell developed velum Demarcation of shaft and velumstrongly sclerotised spicule tip not divided Gubernacu-lum with ventral triangular extension and prominent crurawhich may protrude from cloacal aperture Papilla for-mula 1 C 1 C 1=1 C 2 C 1 Seven precloacal papillae rst pair subventral on cuticular sockets at about twoanal body diam anterior to cloaca second pair lateralless than one anal body diam before cloaca third pairsubventral on cuticular sockets at level of precloacal lipand one medioventral papilla located on higher socket atsame level as second precloacal pair Four papilla pairson tail rst and fourth pair lateral situated respectively

Vol 5(3) 2003 413

W Bert et al

at ca one and two anal body diam posterior to cloacaSecond and third papilla pair close to each other subven-trally with second pair more pronounced Tail elongate-conical narrowing sharply at midpoint and with ne tailtip No phasmids observed with SEM or light micro-scope

TYPE LOCALITY

Pitchers of Nepenthes mirabilis (Lour) Druce in Tung-Kai Botanical Garden Trang Province Southern Thai-land

TYPE MATERIAL

Holotype female (slide MDNC 4021) two paratype fe-males (slides MDNC 4022 4023) and four paratype males(slides MDNC 4024 4025)deposited in the nematodecol-lection of the Zoology Museum Department of BiologyGhent University Belgium four paratype females andfour paratype males on slides with UCR-NC AccessionNo 30059-30063 in the nematode collection of the De-partment of Nematology University of California River-side USA three paratype females and two paratype maleson UCNC slide numbers 5234-5237 in the nematode col-lection of the Department of Nematology University ofCalifornia Davis USA two paratype females and twoparatype males in the nematode type collection of CABIBioscience Egham UK

DIAGNOSIS AND RELATIONSHIPS

Baujardia mirabilis gen n sp n differs from allknown nematodes in having two opposing and offsetspermatheca-like pouches at the junction of oviduct anduterus It also differs from nearly all known Rhabditidain having four cephalic setae instead of papillae in thepresence of six liplets and in the apparent absence ofphasmids

This new genus resembles Panagrellus Thorne 1938in the long body size in the elongate-conical tail and inthe monodelphic prodelphic female reproductive systemwith posteriorlyplaced vulva thickenedvaginalwalls andprominent postvulval sac However Baujardia gen ndiffers from Panagrellus in the characters mentionedabove in the comparatively longer stegostom and inthe more robust male spicules with complex angularmanubrium and undivided tip

The liplets cephalic setae and lack of phasmids arecharacters reminiscent of the genus Teratocephalus de

Man 1876 in the family Teratocephalidae but our newgenus differs from this family in numerous respects egthe amphidial apertures are located on the lips there is adistinct differentiationbetween the pharyngealcorpus andisthmus and the female has an elongate reproductive sys-tem Another taxon in which liplets and setae occur onthe lip region is the family Chambersiellidae Howevermembers of this family have distinct labial setae moreposteriorly located amphids a much narrower stegostomand the tail is usually equipped with a hook-like mucroOverall the mixture of characters resembling membersof different families precludes unequivocal placement ofthe new genus on morphological grounds alone In viewof the molecular analysis discussed below we concludethat it belongs in the infraorder Panagrolaimomorphasu-perfamily Panagrolaimoideaand family PanagrolaimidaeThe morphological diagnosis of Panagrolaimidae mustbe revised signi cantly to accommodate our new genusand the new information on some other panagrolaimsrecently described by Stock et al (2002) As a resultvery few unequivocal diagnostic characters currently re-main for Panagrolaimidae especially when compared tofamilies Cephalobidae Chambersiellidae or Brevibucci-dae (we consider the latter two to be incertae sedis ndash seeDe Ley amp Blaxter 2002) Molecular data are clearly es-sential as an additional source of diagnostic characters inthese nematodes

MOLECULAR PHYLOGENY

The nearly complete SSU sequences of the 22 in-cluded taxa consisted of 1491 (Rhabditophanes sp) to1611 (Panagrolaimus sp) bp with an intermediate valuefor Baujardia mirabilis gen n sp n (1587 bp) Thesecondary structure alignment comprised 1769 positionsof which 971 (55) were variable including 715 (40)parsimony-informative characters The A + T content ofBaujardia gen n was 53

Table 2 summarises the bootstrap support for andmonophyly of clades in the obtained trees NJ MP andML trees consistently support (100 bootstrap) a sistertaxon relationship between the new genus and Panagrel-lus redivivus (L 1767) Goodey 1945 (cf clade A Figs 5-8) Panagrolaimus sp has an uncertain position relativeto these two genera it is placed within the same clade inthe NJ and ML trees while the MP trees place it closerto the strongly supported sister taxa Halicephalobus gin-givalis (Stefanski 1954) Andraacutessy 1984 and Turbatrixaceti (Muumlller 1783) Peters 1927 (clade B) Consistentmaximal support was also given to sister taxa Plectonchus

414 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 2 Monophyly (X D yes X D no) and bootstrap support () for clades

Clades NJ MP1 MP2 MP majority ML(Fig 5) (Fig 6) (Fig 7) rule (Fig 6) (Fig 8)

Clade A(Panagrolaimus (Baujardia (Panagrellus redivivus 57 X X 52 X

AF036599 Panagrellus redivivus AF083007)))(Baujardia (Panagrellus redivivus AF036599 100 X X 100 X

Panagrellus redivivus AF083007))(Panagrolaimus sp (Halicephalobus gingivalis X X X 52 X

Turbatrix aceti))

Clade B(Halicephalobus gingivalis Turbatrix aceti) 86 X X 79 X

Clade C(Plectonchus hunti Panagrobelus stammeri) 100 X X 100 X(Clade C (Clade A Clade B)) (DPanagrolaimidae) 100 X X 100 XMonophyly of Strongyloidoidea (Steinernema X X X 51 X

carpocapsae (Rhabditophanes Strongyloides stercolaris))Monophyly of Panagrolaimoidea and Strongyloidoidea X X X except X X

(DInfraorder Panagrolaimomorpha1 SteinernemaRhabditomorpha1 (DRhabditina) as sister taxon to X X

Panagrolaimomorpha1

Panagrolaimomorpha1 as sister X Xtaxon to Cephalobomorpha1 Tylenchomorpha1

1De Ley and Blaxter (2002)

hunti Stock De Ley De Ley Mundo-Ocampo Baldwinamp Nadler 2002 and Panagrobelus stammeri Ruumlhm 1956(clade C) and to a basal position for this clade withinPanagrolaimidae In other respects our results are com-parable to the SSU rDNA sequence analysis of Feacutelix etal (2000) for example the position of Brevibucca withinRhabditida is uncertain variously assuming a positionat the base of either infraorder Cephalobomorpha Pana-grolaimomorpha or Rhabditomorpha (De Ley amp Blaxter2002) Steinernema carpocapsae (Weiser 1955) WoutsMraacutedaggercek Gerdin amp Bedding 1982 is another nematodewhose position is unclear both in our trees and thoseof Feacutelix et al (2000) but analysis with a more fo-cused dataset con rms its placement in a monophyleticsuperfamily Strongyloidoidea (Dorris et al 2002) Asthe purpose of our study was to explore phylogeneticaf nities between Baujardia gen n and its closest rel-atives we limited the number of sequences included inour dataset and it is therefore not surprising that resolu-tion of relationships among higher taxa remains unclearin our trees (eg aphelenchs are not found to be mono-phyletic)

BIOLOGY

We assume that the pitchers of Nepenthes mirabilis arethe natural habitat of Baujardia mirabilis gen n sp nconsidering that the species was found alive in severalpitchers These pitchers contained a relatively diversefauna dominated by mosquito larvae midges and thenew genus in addition to a few dipteran and coleopteranlarvae The most abundant prey insects in the pitcherswere ants and diverse ying hymenopterans Apparentlya diverse assemblage of organisms lives in associationwith the pitchers of this Nepenthes species This mayrepresent an ecologically complex community similarto the better-known association inhabiting Sarraceniapitchers (eg Kitching 2001 Greeney 2001) Possiblythe nematodes have a phoretic relationship with oneor more insect species Closely related genera are alsocapable of withstanding harsh environments Turbatrixaceti (the vinegar eelworm) Panagrellus redivivus Psilusiae and P nepenthicola inhabit vinegar bookbinderrsquospaste beer lters and pitcher plants respectively

Not much is known about the occurrence of nematodesin pitcher plants and they were not even mentioned in

Vol 5(3) 2003 415

W Bert et al

Fig 5 Neighbour joining tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupnumbers next to branches are percentage bootstrap support values from corresponding50 majority-ruleconsensus tree obtained withNJ analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within the Panagrolaimidae

a reference work of the Nepenthes-inhabiting fauna byThienemann (1933) More extensive study is needed toinvestigate the life cycle and biochemistry of this unusualnew genus Moreover pitcher plants and other epiphytesare clearly a promising source of surprising new nematodetaxa

Discussion

The nematofauna of pitcher plants in Indonesia wasstudied by Menzel (1922) who reported the genera Plec-tus Dorylaimus Rhabditis and Diplogaster He verybrie y described a new species Anguillula nepenthicolaMenzel 1922 which he considered as the only true inhab-itant of pitcher plants and not an accidental recovery An-guillula nepenthicola together with several other speciesincluding Panagrellus pycnus Thorne 1938 was laterplaced in the newly erected genus Turbator by Goodey

(1943) which later (Goodey 1945) became a junior syn-onym of Panagrellus

As P nepenthicola was described from the same habi-tat and similar geographical region as our new genus wehave brie y examined Menzelrsquos (1922) very limited de-scription to justify proposal of a new species for our ma-terial especially in the absence of original illustrations forP nepenthicola Both species are comparable in length(18-27 vs 23-29 mm for P nepenthicola) and in vulvaposition (average of 76 vs 77 for P nepenthicola) Therelative tail length is however clearly longer in our ne-matodes being on average 125 of the vulva-anus dis-tance vs only half the vulva-anus distance length in P ne-penthicola the male tail is on average twice the spiculalength vs three times the spicula length in P nepenthicolathe female c value is 62-89 vs 9-10 in P nepenthicolaand the male c value is 67-11 vs 12-15 in P nepenthicolaThe identity of P nepenthicolabecame somewhat clearerwith the redescription by Micoletzky and Menzel (1928)although only the male tail was illustrated The most ap-

416 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 6 First of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup numbers next to branches are bootstrap values from corresponding 50 majority-ruleconsensus tree obtained with MP analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within thePanagrolaimidae Broken line in the bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to theothers (Figs 5 8)

parent similarities of the new genus with the redescribedP nepenthicolaare the muscular vagina and long postvul-val sac (103-149 vs 78-150 for P nepenthicola) Howeverthe spicules of our new genus and species have more an-gular manubria and their tips are not bi d (an indistinctbi d tip can be observed on the original illustration of Pnepenthicola) Furthermore the male tail is more stronglycurved compared to P nepenthicola and the number andpositioning of the genital papillae is different The differ-ence in tail curvature however may be due to cold xa-tion of our nematodes

The genital system of the new genus is panagrolaimidand shows similarities with the morphology of the femalegonad of Panagrellus redivivus (according to results ob-tained after extraction of the genital system by Geraertet al 1980) As in P redivivus the exure of the gen-ital system is formed by the oviduct and a pair of dis-tinct oviduct cells are observed at the spermatheca side

However the two opposing and offset spermatheca-likepouches observed here represent a unique feature amongstdescribed nematodes These pouches are probably glan-dular rather than having a function in sperm storage assuggested by the unusually large size of the sperm cellsand their occurrence throughout the uterus of gravid fe-males Possibly each compressed cluster of sperm orig-inated from successive copulations as multiple matingsper female might be more frequent in a limited and liquidenvironment Alternatively the movement of eggs couldcause the break-up of single clusters of sperm

On morphological grounds alone it was not immedi-ately apparent in which family Baujardiagen n shouldbeplaced The molecular analysis however robustly placesthe new genus as a sister taxon of Panagrellus within thePanagrolaimidae As a matter of fact the morphologicalsimilarities with Panagrellus as discussed above are rel-atively obvious a posteriori but were not evident before

Vol 5(3) 2003 417

W Bert et al

Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

W Bert et al

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

Anal body diam 291 31 sect 3 98 31 sect 27 85 31 sect 83 27(26-40) (258-36) (22-50)

Rectum length 50 49 sect 42 86 47 sect 6 13(42-56) (38-56)

RectumAnal body diam 17 16 sect 02 128 16 sect 03 17(12-21) (11-2)

TailRectum 6 63 sect 05 77(55-73)

Gonad length 1250 1370 sect 187 136 1049 sect 110 105 1251 sect 327 26(1012-1700) (775-1206) (768-1635)

Vulva position 1625 1768 sect 179 101 1620 sect 335 21(1375-2062) (1104-2070)

Vulva body diam 466 57 sect 178 31 46 sect 87 19(42-107) (33-59)

Vagina length 266 29 sect 32 112 26 sect 4 15(25-35) (21-34)

Vulva-Anus distance 207 240 sect 737 306 175 sect 565 322(120-439) (102-270)

Vulva-AnusTail 07 08 sect 02 286 06 sect 02 35(04-14) (03-08)

Postvulval sac length 121 126 sect 106 84 115 sect 154 13(103-149) (93-141)

Postvulval sac length 26 23 sect 05 202 25 sect03 12vulva body diam (12-3) (2-29)

Postvulval stalk length 29 34 sect 53 153 34 sect 64 19(26-44) (23-40)

Postvulval pouch length 91 91 sect 112 123 81 sect 11 14(75-120) (70-101)

Vaginal cell cluster length 20 17 sect 22 126 15 sect 26 18(15-21) (9-17)

Vaginal cell cluster diam 108 13 sect 2 161 9 sect 24 25(10-18) (6-12)

No of eggs in uterus 20 sect 10 491 20 sect 84 42(4-34) (8-31)

V 76 764 sect 21 27 763 sect 3 4(72-81) (72-81)

Gonad (as L) 585 59 sect 28 47 584 sect 55 95(55-64) (51-66)

Vagina (as Vulva 57 531 sect 141 265 59 sect 132 22body diam) (24-74) (40-76)

T 621 sect 45 73(52-74)

Spicule length 57 sect 38 67(50-62)

SpiculeAnal body diam 18 sect 02 118(15-21)

Gubernaculum length 24 sect 22 89(20-28)

412 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

GubernaculumSpicule 04 sect 0 89(04-05)

Spicate tail portion 972 sect 84 84(85-117)

Spicate tailTail length 05 sect 01 141(04-07)

source of specimens for DNA extraction males were not recovered

and stegostom in ratio 1 1 2 Cheilostom without dis-tinct sclerotisations Stegostom divided into two parts insectioned specimens observed with SEM Posterior partapparently with seven denticles or digitate projections ca2 sup1m long reclining against and posteriorly connectedto stoma wall Projections as observed with SEM in twospecimens and consistent with light microscope obser-vations on three females positioned as follows three indorsal sector and two in each subventral sector (the sec-ond projection in the right subventral sector was presum-ably sliced through in Fig 4B) Dorsal gland opening atbase of these projections Basal lamina visible betweengymnostom and stegostom (arrows in Fig 4C) Neck re-gion forming one fth to one seventh of body Pharynxdivided into a cylindrical corpus comprising on average68 of neck length a short isthmus and an oval bulbuswith transverse valves Cardia well developedNerve ringat transition of corpus-isthmus Excretory pore 15-80 sup1manterior to nerve ring wide but often dif cult to nd be-cause of its lack of sclerotisation Deirid not found In-testine with wide lumen demarcated by refractive liningmicrovilli visible anterior to rectum Rectum long withlarge sphincter cells at its beginningReproductivesystemmonodelphic prodelphicwith posteriorly re exed ovaryOviduct always with two distinct nuclei located at ex-ure between ovary and uterus Two opposing and offsetspermatheca-like pouches present at junction of oviductand uterus Each pouch with one distinct terminal cell itslining with a membranous appearance Granulated spermcells very large (15-34 sup1m long) and often compressedinto serial clusters erratically dispersed throughoututerusoften alternating with developing eggs Vulva a trans-verse cleft with overhanging anterior vulval lip locatedat 75 of body length Vagina with thickened walls andsurrounded by strong muscles Prominent postvulval sac

with anterior stalk and posterior pouchTransition vagina-postvulvalsac guarded by a cluster of four cells consistingof two pairs lying side by side Anus a broad curved slitin ventral view Tail elongate-conical with ne tip Irreg-ular pattern of lines substituting annulation over a shortdistance at middle of tail as seen by SEM No phasmidsor phasmid-likestructures observed with SEM or light mi-croscope

Male

General morphology similar to female Body 13-19 mm long arcuate ventrad and especially so in pos-terior region with tail usually curved into 15 coils Cu-ticle nely annulated 08 sup1m wide at midbody Lat-eral eld indistinct with several discontinuous lines ex-tending slightly posterior to cloacal opening 3 sup1m wideat midbody Neck region comprising 20-25 of bodylength Excretory pore from 80 sup1m anterior to nervering to 6 sup1m posterior to nerve ring Reproductive tractoccupying 50-75 of body length monorchic dextraltestis ventrally re exed for 36-120sup1m Vesicula seminaliscontaining large granulated spermatocytes Spicules mas-sive arcuate each with complex angular manubrium andwell developed velum Demarcation of shaft and velumstrongly sclerotised spicule tip not divided Gubernacu-lum with ventral triangular extension and prominent crurawhich may protrude from cloacal aperture Papilla for-mula 1 C 1 C 1=1 C 2 C 1 Seven precloacal papillae rst pair subventral on cuticular sockets at about twoanal body diam anterior to cloaca second pair lateralless than one anal body diam before cloaca third pairsubventral on cuticular sockets at level of precloacal lipand one medioventral papilla located on higher socket atsame level as second precloacal pair Four papilla pairson tail rst and fourth pair lateral situated respectively

Vol 5(3) 2003 413

W Bert et al

at ca one and two anal body diam posterior to cloacaSecond and third papilla pair close to each other subven-trally with second pair more pronounced Tail elongate-conical narrowing sharply at midpoint and with ne tailtip No phasmids observed with SEM or light micro-scope

TYPE LOCALITY

Pitchers of Nepenthes mirabilis (Lour) Druce in Tung-Kai Botanical Garden Trang Province Southern Thai-land

TYPE MATERIAL

Holotype female (slide MDNC 4021) two paratype fe-males (slides MDNC 4022 4023) and four paratype males(slides MDNC 4024 4025)deposited in the nematodecol-lection of the Zoology Museum Department of BiologyGhent University Belgium four paratype females andfour paratype males on slides with UCR-NC AccessionNo 30059-30063 in the nematode collection of the De-partment of Nematology University of California River-side USA three paratype females and two paratype maleson UCNC slide numbers 5234-5237 in the nematode col-lection of the Department of Nematology University ofCalifornia Davis USA two paratype females and twoparatype males in the nematode type collection of CABIBioscience Egham UK

DIAGNOSIS AND RELATIONSHIPS

Baujardia mirabilis gen n sp n differs from allknown nematodes in having two opposing and offsetspermatheca-like pouches at the junction of oviduct anduterus It also differs from nearly all known Rhabditidain having four cephalic setae instead of papillae in thepresence of six liplets and in the apparent absence ofphasmids

This new genus resembles Panagrellus Thorne 1938in the long body size in the elongate-conical tail and inthe monodelphic prodelphic female reproductive systemwith posteriorlyplaced vulva thickenedvaginalwalls andprominent postvulval sac However Baujardia gen ndiffers from Panagrellus in the characters mentionedabove in the comparatively longer stegostom and inthe more robust male spicules with complex angularmanubrium and undivided tip

The liplets cephalic setae and lack of phasmids arecharacters reminiscent of the genus Teratocephalus de

Man 1876 in the family Teratocephalidae but our newgenus differs from this family in numerous respects egthe amphidial apertures are located on the lips there is adistinct differentiationbetween the pharyngealcorpus andisthmus and the female has an elongate reproductive sys-tem Another taxon in which liplets and setae occur onthe lip region is the family Chambersiellidae Howevermembers of this family have distinct labial setae moreposteriorly located amphids a much narrower stegostomand the tail is usually equipped with a hook-like mucroOverall the mixture of characters resembling membersof different families precludes unequivocal placement ofthe new genus on morphological grounds alone In viewof the molecular analysis discussed below we concludethat it belongs in the infraorder Panagrolaimomorphasu-perfamily Panagrolaimoideaand family PanagrolaimidaeThe morphological diagnosis of Panagrolaimidae mustbe revised signi cantly to accommodate our new genusand the new information on some other panagrolaimsrecently described by Stock et al (2002) As a resultvery few unequivocal diagnostic characters currently re-main for Panagrolaimidae especially when compared tofamilies Cephalobidae Chambersiellidae or Brevibucci-dae (we consider the latter two to be incertae sedis ndash seeDe Ley amp Blaxter 2002) Molecular data are clearly es-sential as an additional source of diagnostic characters inthese nematodes

MOLECULAR PHYLOGENY

The nearly complete SSU sequences of the 22 in-cluded taxa consisted of 1491 (Rhabditophanes sp) to1611 (Panagrolaimus sp) bp with an intermediate valuefor Baujardia mirabilis gen n sp n (1587 bp) Thesecondary structure alignment comprised 1769 positionsof which 971 (55) were variable including 715 (40)parsimony-informative characters The A + T content ofBaujardia gen n was 53

Table 2 summarises the bootstrap support for andmonophyly of clades in the obtained trees NJ MP andML trees consistently support (100 bootstrap) a sistertaxon relationship between the new genus and Panagrel-lus redivivus (L 1767) Goodey 1945 (cf clade A Figs 5-8) Panagrolaimus sp has an uncertain position relativeto these two genera it is placed within the same clade inthe NJ and ML trees while the MP trees place it closerto the strongly supported sister taxa Halicephalobus gin-givalis (Stefanski 1954) Andraacutessy 1984 and Turbatrixaceti (Muumlller 1783) Peters 1927 (clade B) Consistentmaximal support was also given to sister taxa Plectonchus

414 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 2 Monophyly (X D yes X D no) and bootstrap support () for clades

Clades NJ MP1 MP2 MP majority ML(Fig 5) (Fig 6) (Fig 7) rule (Fig 6) (Fig 8)

Clade A(Panagrolaimus (Baujardia (Panagrellus redivivus 57 X X 52 X

AF036599 Panagrellus redivivus AF083007)))(Baujardia (Panagrellus redivivus AF036599 100 X X 100 X

Panagrellus redivivus AF083007))(Panagrolaimus sp (Halicephalobus gingivalis X X X 52 X

Turbatrix aceti))

Clade B(Halicephalobus gingivalis Turbatrix aceti) 86 X X 79 X

Clade C(Plectonchus hunti Panagrobelus stammeri) 100 X X 100 X(Clade C (Clade A Clade B)) (DPanagrolaimidae) 100 X X 100 XMonophyly of Strongyloidoidea (Steinernema X X X 51 X

carpocapsae (Rhabditophanes Strongyloides stercolaris))Monophyly of Panagrolaimoidea and Strongyloidoidea X X X except X X

(DInfraorder Panagrolaimomorpha1 SteinernemaRhabditomorpha1 (DRhabditina) as sister taxon to X X

Panagrolaimomorpha1

Panagrolaimomorpha1 as sister X Xtaxon to Cephalobomorpha1 Tylenchomorpha1

1De Ley and Blaxter (2002)

hunti Stock De Ley De Ley Mundo-Ocampo Baldwinamp Nadler 2002 and Panagrobelus stammeri Ruumlhm 1956(clade C) and to a basal position for this clade withinPanagrolaimidae In other respects our results are com-parable to the SSU rDNA sequence analysis of Feacutelix etal (2000) for example the position of Brevibucca withinRhabditida is uncertain variously assuming a positionat the base of either infraorder Cephalobomorpha Pana-grolaimomorpha or Rhabditomorpha (De Ley amp Blaxter2002) Steinernema carpocapsae (Weiser 1955) WoutsMraacutedaggercek Gerdin amp Bedding 1982 is another nematodewhose position is unclear both in our trees and thoseof Feacutelix et al (2000) but analysis with a more fo-cused dataset con rms its placement in a monophyleticsuperfamily Strongyloidoidea (Dorris et al 2002) Asthe purpose of our study was to explore phylogeneticaf nities between Baujardia gen n and its closest rel-atives we limited the number of sequences included inour dataset and it is therefore not surprising that resolu-tion of relationships among higher taxa remains unclearin our trees (eg aphelenchs are not found to be mono-phyletic)

BIOLOGY

We assume that the pitchers of Nepenthes mirabilis arethe natural habitat of Baujardia mirabilis gen n sp nconsidering that the species was found alive in severalpitchers These pitchers contained a relatively diversefauna dominated by mosquito larvae midges and thenew genus in addition to a few dipteran and coleopteranlarvae The most abundant prey insects in the pitcherswere ants and diverse ying hymenopterans Apparentlya diverse assemblage of organisms lives in associationwith the pitchers of this Nepenthes species This mayrepresent an ecologically complex community similarto the better-known association inhabiting Sarraceniapitchers (eg Kitching 2001 Greeney 2001) Possiblythe nematodes have a phoretic relationship with oneor more insect species Closely related genera are alsocapable of withstanding harsh environments Turbatrixaceti (the vinegar eelworm) Panagrellus redivivus Psilusiae and P nepenthicola inhabit vinegar bookbinderrsquospaste beer lters and pitcher plants respectively

Not much is known about the occurrence of nematodesin pitcher plants and they were not even mentioned in

Vol 5(3) 2003 415

W Bert et al

Fig 5 Neighbour joining tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupnumbers next to branches are percentage bootstrap support values from corresponding50 majority-ruleconsensus tree obtained withNJ analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within the Panagrolaimidae

a reference work of the Nepenthes-inhabiting fauna byThienemann (1933) More extensive study is needed toinvestigate the life cycle and biochemistry of this unusualnew genus Moreover pitcher plants and other epiphytesare clearly a promising source of surprising new nematodetaxa

Discussion

The nematofauna of pitcher plants in Indonesia wasstudied by Menzel (1922) who reported the genera Plec-tus Dorylaimus Rhabditis and Diplogaster He verybrie y described a new species Anguillula nepenthicolaMenzel 1922 which he considered as the only true inhab-itant of pitcher plants and not an accidental recovery An-guillula nepenthicola together with several other speciesincluding Panagrellus pycnus Thorne 1938 was laterplaced in the newly erected genus Turbator by Goodey

(1943) which later (Goodey 1945) became a junior syn-onym of Panagrellus

As P nepenthicola was described from the same habi-tat and similar geographical region as our new genus wehave brie y examined Menzelrsquos (1922) very limited de-scription to justify proposal of a new species for our ma-terial especially in the absence of original illustrations forP nepenthicola Both species are comparable in length(18-27 vs 23-29 mm for P nepenthicola) and in vulvaposition (average of 76 vs 77 for P nepenthicola) Therelative tail length is however clearly longer in our ne-matodes being on average 125 of the vulva-anus dis-tance vs only half the vulva-anus distance length in P ne-penthicola the male tail is on average twice the spiculalength vs three times the spicula length in P nepenthicolathe female c value is 62-89 vs 9-10 in P nepenthicolaand the male c value is 67-11 vs 12-15 in P nepenthicolaThe identity of P nepenthicolabecame somewhat clearerwith the redescription by Micoletzky and Menzel (1928)although only the male tail was illustrated The most ap-

416 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 6 First of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup numbers next to branches are bootstrap values from corresponding 50 majority-ruleconsensus tree obtained with MP analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within thePanagrolaimidae Broken line in the bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to theothers (Figs 5 8)

parent similarities of the new genus with the redescribedP nepenthicolaare the muscular vagina and long postvul-val sac (103-149 vs 78-150 for P nepenthicola) Howeverthe spicules of our new genus and species have more an-gular manubria and their tips are not bi d (an indistinctbi d tip can be observed on the original illustration of Pnepenthicola) Furthermore the male tail is more stronglycurved compared to P nepenthicola and the number andpositioning of the genital papillae is different The differ-ence in tail curvature however may be due to cold xa-tion of our nematodes

The genital system of the new genus is panagrolaimidand shows similarities with the morphology of the femalegonad of Panagrellus redivivus (according to results ob-tained after extraction of the genital system by Geraertet al 1980) As in P redivivus the exure of the gen-ital system is formed by the oviduct and a pair of dis-tinct oviduct cells are observed at the spermatheca side

However the two opposing and offset spermatheca-likepouches observed here represent a unique feature amongstdescribed nematodes These pouches are probably glan-dular rather than having a function in sperm storage assuggested by the unusually large size of the sperm cellsand their occurrence throughout the uterus of gravid fe-males Possibly each compressed cluster of sperm orig-inated from successive copulations as multiple matingsper female might be more frequent in a limited and liquidenvironment Alternatively the movement of eggs couldcause the break-up of single clusters of sperm

On morphological grounds alone it was not immedi-ately apparent in which family Baujardiagen n shouldbeplaced The molecular analysis however robustly placesthe new genus as a sister taxon of Panagrellus within thePanagrolaimidae As a matter of fact the morphologicalsimilarities with Panagrellus as discussed above are rel-atively obvious a posteriori but were not evident before

Vol 5(3) 2003 417

W Bert et al

Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

Baujardia mirabilis gen n sp n from pitcher plants

Table 1 (Continued)

1999 Population (Type) 2002 Population

Holotype Female CV Male CV Female CVparatypes paratypes paratypes

GubernaculumSpicule 04 sect 0 89(04-05)

Spicate tail portion 972 sect 84 84(85-117)

Spicate tailTail length 05 sect 01 141(04-07)

source of specimens for DNA extraction males were not recovered

and stegostom in ratio 1 1 2 Cheilostom without dis-tinct sclerotisations Stegostom divided into two parts insectioned specimens observed with SEM Posterior partapparently with seven denticles or digitate projections ca2 sup1m long reclining against and posteriorly connectedto stoma wall Projections as observed with SEM in twospecimens and consistent with light microscope obser-vations on three females positioned as follows three indorsal sector and two in each subventral sector (the sec-ond projection in the right subventral sector was presum-ably sliced through in Fig 4B) Dorsal gland opening atbase of these projections Basal lamina visible betweengymnostom and stegostom (arrows in Fig 4C) Neck re-gion forming one fth to one seventh of body Pharynxdivided into a cylindrical corpus comprising on average68 of neck length a short isthmus and an oval bulbuswith transverse valves Cardia well developedNerve ringat transition of corpus-isthmus Excretory pore 15-80 sup1manterior to nerve ring wide but often dif cult to nd be-cause of its lack of sclerotisation Deirid not found In-testine with wide lumen demarcated by refractive liningmicrovilli visible anterior to rectum Rectum long withlarge sphincter cells at its beginningReproductivesystemmonodelphic prodelphicwith posteriorly re exed ovaryOviduct always with two distinct nuclei located at ex-ure between ovary and uterus Two opposing and offsetspermatheca-like pouches present at junction of oviductand uterus Each pouch with one distinct terminal cell itslining with a membranous appearance Granulated spermcells very large (15-34 sup1m long) and often compressedinto serial clusters erratically dispersed throughoututerusoften alternating with developing eggs Vulva a trans-verse cleft with overhanging anterior vulval lip locatedat 75 of body length Vagina with thickened walls andsurrounded by strong muscles Prominent postvulval sac

with anterior stalk and posterior pouchTransition vagina-postvulvalsac guarded by a cluster of four cells consistingof two pairs lying side by side Anus a broad curved slitin ventral view Tail elongate-conical with ne tip Irreg-ular pattern of lines substituting annulation over a shortdistance at middle of tail as seen by SEM No phasmidsor phasmid-likestructures observed with SEM or light mi-croscope

Male

General morphology similar to female Body 13-19 mm long arcuate ventrad and especially so in pos-terior region with tail usually curved into 15 coils Cu-ticle nely annulated 08 sup1m wide at midbody Lat-eral eld indistinct with several discontinuous lines ex-tending slightly posterior to cloacal opening 3 sup1m wideat midbody Neck region comprising 20-25 of bodylength Excretory pore from 80 sup1m anterior to nervering to 6 sup1m posterior to nerve ring Reproductive tractoccupying 50-75 of body length monorchic dextraltestis ventrally re exed for 36-120sup1m Vesicula seminaliscontaining large granulated spermatocytes Spicules mas-sive arcuate each with complex angular manubrium andwell developed velum Demarcation of shaft and velumstrongly sclerotised spicule tip not divided Gubernacu-lum with ventral triangular extension and prominent crurawhich may protrude from cloacal aperture Papilla for-mula 1 C 1 C 1=1 C 2 C 1 Seven precloacal papillae rst pair subventral on cuticular sockets at about twoanal body diam anterior to cloaca second pair lateralless than one anal body diam before cloaca third pairsubventral on cuticular sockets at level of precloacal lipand one medioventral papilla located on higher socket atsame level as second precloacal pair Four papilla pairson tail rst and fourth pair lateral situated respectively

Vol 5(3) 2003 413

W Bert et al

at ca one and two anal body diam posterior to cloacaSecond and third papilla pair close to each other subven-trally with second pair more pronounced Tail elongate-conical narrowing sharply at midpoint and with ne tailtip No phasmids observed with SEM or light micro-scope

TYPE LOCALITY

Pitchers of Nepenthes mirabilis (Lour) Druce in Tung-Kai Botanical Garden Trang Province Southern Thai-land

TYPE MATERIAL

Holotype female (slide MDNC 4021) two paratype fe-males (slides MDNC 4022 4023) and four paratype males(slides MDNC 4024 4025)deposited in the nematodecol-lection of the Zoology Museum Department of BiologyGhent University Belgium four paratype females andfour paratype males on slides with UCR-NC AccessionNo 30059-30063 in the nematode collection of the De-partment of Nematology University of California River-side USA three paratype females and two paratype maleson UCNC slide numbers 5234-5237 in the nematode col-lection of the Department of Nematology University ofCalifornia Davis USA two paratype females and twoparatype males in the nematode type collection of CABIBioscience Egham UK

DIAGNOSIS AND RELATIONSHIPS

Baujardia mirabilis gen n sp n differs from allknown nematodes in having two opposing and offsetspermatheca-like pouches at the junction of oviduct anduterus It also differs from nearly all known Rhabditidain having four cephalic setae instead of papillae in thepresence of six liplets and in the apparent absence ofphasmids

This new genus resembles Panagrellus Thorne 1938in the long body size in the elongate-conical tail and inthe monodelphic prodelphic female reproductive systemwith posteriorlyplaced vulva thickenedvaginalwalls andprominent postvulval sac However Baujardia gen ndiffers from Panagrellus in the characters mentionedabove in the comparatively longer stegostom and inthe more robust male spicules with complex angularmanubrium and undivided tip

The liplets cephalic setae and lack of phasmids arecharacters reminiscent of the genus Teratocephalus de

Man 1876 in the family Teratocephalidae but our newgenus differs from this family in numerous respects egthe amphidial apertures are located on the lips there is adistinct differentiationbetween the pharyngealcorpus andisthmus and the female has an elongate reproductive sys-tem Another taxon in which liplets and setae occur onthe lip region is the family Chambersiellidae Howevermembers of this family have distinct labial setae moreposteriorly located amphids a much narrower stegostomand the tail is usually equipped with a hook-like mucroOverall the mixture of characters resembling membersof different families precludes unequivocal placement ofthe new genus on morphological grounds alone In viewof the molecular analysis discussed below we concludethat it belongs in the infraorder Panagrolaimomorphasu-perfamily Panagrolaimoideaand family PanagrolaimidaeThe morphological diagnosis of Panagrolaimidae mustbe revised signi cantly to accommodate our new genusand the new information on some other panagrolaimsrecently described by Stock et al (2002) As a resultvery few unequivocal diagnostic characters currently re-main for Panagrolaimidae especially when compared tofamilies Cephalobidae Chambersiellidae or Brevibucci-dae (we consider the latter two to be incertae sedis ndash seeDe Ley amp Blaxter 2002) Molecular data are clearly es-sential as an additional source of diagnostic characters inthese nematodes

MOLECULAR PHYLOGENY

The nearly complete SSU sequences of the 22 in-cluded taxa consisted of 1491 (Rhabditophanes sp) to1611 (Panagrolaimus sp) bp with an intermediate valuefor Baujardia mirabilis gen n sp n (1587 bp) Thesecondary structure alignment comprised 1769 positionsof which 971 (55) were variable including 715 (40)parsimony-informative characters The A + T content ofBaujardia gen n was 53

Table 2 summarises the bootstrap support for andmonophyly of clades in the obtained trees NJ MP andML trees consistently support (100 bootstrap) a sistertaxon relationship between the new genus and Panagrel-lus redivivus (L 1767) Goodey 1945 (cf clade A Figs 5-8) Panagrolaimus sp has an uncertain position relativeto these two genera it is placed within the same clade inthe NJ and ML trees while the MP trees place it closerto the strongly supported sister taxa Halicephalobus gin-givalis (Stefanski 1954) Andraacutessy 1984 and Turbatrixaceti (Muumlller 1783) Peters 1927 (clade B) Consistentmaximal support was also given to sister taxa Plectonchus

414 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 2 Monophyly (X D yes X D no) and bootstrap support () for clades

Clades NJ MP1 MP2 MP majority ML(Fig 5) (Fig 6) (Fig 7) rule (Fig 6) (Fig 8)

Clade A(Panagrolaimus (Baujardia (Panagrellus redivivus 57 X X 52 X

AF036599 Panagrellus redivivus AF083007)))(Baujardia (Panagrellus redivivus AF036599 100 X X 100 X

Panagrellus redivivus AF083007))(Panagrolaimus sp (Halicephalobus gingivalis X X X 52 X

Turbatrix aceti))

Clade B(Halicephalobus gingivalis Turbatrix aceti) 86 X X 79 X

Clade C(Plectonchus hunti Panagrobelus stammeri) 100 X X 100 X(Clade C (Clade A Clade B)) (DPanagrolaimidae) 100 X X 100 XMonophyly of Strongyloidoidea (Steinernema X X X 51 X

carpocapsae (Rhabditophanes Strongyloides stercolaris))Monophyly of Panagrolaimoidea and Strongyloidoidea X X X except X X

(DInfraorder Panagrolaimomorpha1 SteinernemaRhabditomorpha1 (DRhabditina) as sister taxon to X X

Panagrolaimomorpha1

Panagrolaimomorpha1 as sister X Xtaxon to Cephalobomorpha1 Tylenchomorpha1

1De Ley and Blaxter (2002)

hunti Stock De Ley De Ley Mundo-Ocampo Baldwinamp Nadler 2002 and Panagrobelus stammeri Ruumlhm 1956(clade C) and to a basal position for this clade withinPanagrolaimidae In other respects our results are com-parable to the SSU rDNA sequence analysis of Feacutelix etal (2000) for example the position of Brevibucca withinRhabditida is uncertain variously assuming a positionat the base of either infraorder Cephalobomorpha Pana-grolaimomorpha or Rhabditomorpha (De Ley amp Blaxter2002) Steinernema carpocapsae (Weiser 1955) WoutsMraacutedaggercek Gerdin amp Bedding 1982 is another nematodewhose position is unclear both in our trees and thoseof Feacutelix et al (2000) but analysis with a more fo-cused dataset con rms its placement in a monophyleticsuperfamily Strongyloidoidea (Dorris et al 2002) Asthe purpose of our study was to explore phylogeneticaf nities between Baujardia gen n and its closest rel-atives we limited the number of sequences included inour dataset and it is therefore not surprising that resolu-tion of relationships among higher taxa remains unclearin our trees (eg aphelenchs are not found to be mono-phyletic)

BIOLOGY

We assume that the pitchers of Nepenthes mirabilis arethe natural habitat of Baujardia mirabilis gen n sp nconsidering that the species was found alive in severalpitchers These pitchers contained a relatively diversefauna dominated by mosquito larvae midges and thenew genus in addition to a few dipteran and coleopteranlarvae The most abundant prey insects in the pitcherswere ants and diverse ying hymenopterans Apparentlya diverse assemblage of organisms lives in associationwith the pitchers of this Nepenthes species This mayrepresent an ecologically complex community similarto the better-known association inhabiting Sarraceniapitchers (eg Kitching 2001 Greeney 2001) Possiblythe nematodes have a phoretic relationship with oneor more insect species Closely related genera are alsocapable of withstanding harsh environments Turbatrixaceti (the vinegar eelworm) Panagrellus redivivus Psilusiae and P nepenthicola inhabit vinegar bookbinderrsquospaste beer lters and pitcher plants respectively

Not much is known about the occurrence of nematodesin pitcher plants and they were not even mentioned in

Vol 5(3) 2003 415

W Bert et al

Fig 5 Neighbour joining tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupnumbers next to branches are percentage bootstrap support values from corresponding50 majority-ruleconsensus tree obtained withNJ analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within the Panagrolaimidae

a reference work of the Nepenthes-inhabiting fauna byThienemann (1933) More extensive study is needed toinvestigate the life cycle and biochemistry of this unusualnew genus Moreover pitcher plants and other epiphytesare clearly a promising source of surprising new nematodetaxa

Discussion

The nematofauna of pitcher plants in Indonesia wasstudied by Menzel (1922) who reported the genera Plec-tus Dorylaimus Rhabditis and Diplogaster He verybrie y described a new species Anguillula nepenthicolaMenzel 1922 which he considered as the only true inhab-itant of pitcher plants and not an accidental recovery An-guillula nepenthicola together with several other speciesincluding Panagrellus pycnus Thorne 1938 was laterplaced in the newly erected genus Turbator by Goodey

(1943) which later (Goodey 1945) became a junior syn-onym of Panagrellus

As P nepenthicola was described from the same habi-tat and similar geographical region as our new genus wehave brie y examined Menzelrsquos (1922) very limited de-scription to justify proposal of a new species for our ma-terial especially in the absence of original illustrations forP nepenthicola Both species are comparable in length(18-27 vs 23-29 mm for P nepenthicola) and in vulvaposition (average of 76 vs 77 for P nepenthicola) Therelative tail length is however clearly longer in our ne-matodes being on average 125 of the vulva-anus dis-tance vs only half the vulva-anus distance length in P ne-penthicola the male tail is on average twice the spiculalength vs three times the spicula length in P nepenthicolathe female c value is 62-89 vs 9-10 in P nepenthicolaand the male c value is 67-11 vs 12-15 in P nepenthicolaThe identity of P nepenthicolabecame somewhat clearerwith the redescription by Micoletzky and Menzel (1928)although only the male tail was illustrated The most ap-

416 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 6 First of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup numbers next to branches are bootstrap values from corresponding 50 majority-ruleconsensus tree obtained with MP analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within thePanagrolaimidae Broken line in the bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to theothers (Figs 5 8)

parent similarities of the new genus with the redescribedP nepenthicolaare the muscular vagina and long postvul-val sac (103-149 vs 78-150 for P nepenthicola) Howeverthe spicules of our new genus and species have more an-gular manubria and their tips are not bi d (an indistinctbi d tip can be observed on the original illustration of Pnepenthicola) Furthermore the male tail is more stronglycurved compared to P nepenthicola and the number andpositioning of the genital papillae is different The differ-ence in tail curvature however may be due to cold xa-tion of our nematodes

The genital system of the new genus is panagrolaimidand shows similarities with the morphology of the femalegonad of Panagrellus redivivus (according to results ob-tained after extraction of the genital system by Geraertet al 1980) As in P redivivus the exure of the gen-ital system is formed by the oviduct and a pair of dis-tinct oviduct cells are observed at the spermatheca side

However the two opposing and offset spermatheca-likepouches observed here represent a unique feature amongstdescribed nematodes These pouches are probably glan-dular rather than having a function in sperm storage assuggested by the unusually large size of the sperm cellsand their occurrence throughout the uterus of gravid fe-males Possibly each compressed cluster of sperm orig-inated from successive copulations as multiple matingsper female might be more frequent in a limited and liquidenvironment Alternatively the movement of eggs couldcause the break-up of single clusters of sperm

On morphological grounds alone it was not immedi-ately apparent in which family Baujardiagen n shouldbeplaced The molecular analysis however robustly placesthe new genus as a sister taxon of Panagrellus within thePanagrolaimidae As a matter of fact the morphologicalsimilarities with Panagrellus as discussed above are rel-atively obvious a posteriori but were not evident before

Vol 5(3) 2003 417

W Bert et al

Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

W Bert et al

at ca one and two anal body diam posterior to cloacaSecond and third papilla pair close to each other subven-trally with second pair more pronounced Tail elongate-conical narrowing sharply at midpoint and with ne tailtip No phasmids observed with SEM or light micro-scope

TYPE LOCALITY

Pitchers of Nepenthes mirabilis (Lour) Druce in Tung-Kai Botanical Garden Trang Province Southern Thai-land

TYPE MATERIAL

Holotype female (slide MDNC 4021) two paratype fe-males (slides MDNC 4022 4023) and four paratype males(slides MDNC 4024 4025)deposited in the nematodecol-lection of the Zoology Museum Department of BiologyGhent University Belgium four paratype females andfour paratype males on slides with UCR-NC AccessionNo 30059-30063 in the nematode collection of the De-partment of Nematology University of California River-side USA three paratype females and two paratype maleson UCNC slide numbers 5234-5237 in the nematode col-lection of the Department of Nematology University ofCalifornia Davis USA two paratype females and twoparatype males in the nematode type collection of CABIBioscience Egham UK

DIAGNOSIS AND RELATIONSHIPS

Baujardia mirabilis gen n sp n differs from allknown nematodes in having two opposing and offsetspermatheca-like pouches at the junction of oviduct anduterus It also differs from nearly all known Rhabditidain having four cephalic setae instead of papillae in thepresence of six liplets and in the apparent absence ofphasmids

This new genus resembles Panagrellus Thorne 1938in the long body size in the elongate-conical tail and inthe monodelphic prodelphic female reproductive systemwith posteriorlyplaced vulva thickenedvaginalwalls andprominent postvulval sac However Baujardia gen ndiffers from Panagrellus in the characters mentionedabove in the comparatively longer stegostom and inthe more robust male spicules with complex angularmanubrium and undivided tip

The liplets cephalic setae and lack of phasmids arecharacters reminiscent of the genus Teratocephalus de

Man 1876 in the family Teratocephalidae but our newgenus differs from this family in numerous respects egthe amphidial apertures are located on the lips there is adistinct differentiationbetween the pharyngealcorpus andisthmus and the female has an elongate reproductive sys-tem Another taxon in which liplets and setae occur onthe lip region is the family Chambersiellidae Howevermembers of this family have distinct labial setae moreposteriorly located amphids a much narrower stegostomand the tail is usually equipped with a hook-like mucroOverall the mixture of characters resembling membersof different families precludes unequivocal placement ofthe new genus on morphological grounds alone In viewof the molecular analysis discussed below we concludethat it belongs in the infraorder Panagrolaimomorphasu-perfamily Panagrolaimoideaand family PanagrolaimidaeThe morphological diagnosis of Panagrolaimidae mustbe revised signi cantly to accommodate our new genusand the new information on some other panagrolaimsrecently described by Stock et al (2002) As a resultvery few unequivocal diagnostic characters currently re-main for Panagrolaimidae especially when compared tofamilies Cephalobidae Chambersiellidae or Brevibucci-dae (we consider the latter two to be incertae sedis ndash seeDe Ley amp Blaxter 2002) Molecular data are clearly es-sential as an additional source of diagnostic characters inthese nematodes

MOLECULAR PHYLOGENY

The nearly complete SSU sequences of the 22 in-cluded taxa consisted of 1491 (Rhabditophanes sp) to1611 (Panagrolaimus sp) bp with an intermediate valuefor Baujardia mirabilis gen n sp n (1587 bp) Thesecondary structure alignment comprised 1769 positionsof which 971 (55) were variable including 715 (40)parsimony-informative characters The A + T content ofBaujardia gen n was 53

Table 2 summarises the bootstrap support for andmonophyly of clades in the obtained trees NJ MP andML trees consistently support (100 bootstrap) a sistertaxon relationship between the new genus and Panagrel-lus redivivus (L 1767) Goodey 1945 (cf clade A Figs 5-8) Panagrolaimus sp has an uncertain position relativeto these two genera it is placed within the same clade inthe NJ and ML trees while the MP trees place it closerto the strongly supported sister taxa Halicephalobus gin-givalis (Stefanski 1954) Andraacutessy 1984 and Turbatrixaceti (Muumlller 1783) Peters 1927 (clade B) Consistentmaximal support was also given to sister taxa Plectonchus

414 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Table 2 Monophyly (X D yes X D no) and bootstrap support () for clades

Clades NJ MP1 MP2 MP majority ML(Fig 5) (Fig 6) (Fig 7) rule (Fig 6) (Fig 8)

Clade A(Panagrolaimus (Baujardia (Panagrellus redivivus 57 X X 52 X

AF036599 Panagrellus redivivus AF083007)))(Baujardia (Panagrellus redivivus AF036599 100 X X 100 X

Panagrellus redivivus AF083007))(Panagrolaimus sp (Halicephalobus gingivalis X X X 52 X

Turbatrix aceti))

Clade B(Halicephalobus gingivalis Turbatrix aceti) 86 X X 79 X

Clade C(Plectonchus hunti Panagrobelus stammeri) 100 X X 100 X(Clade C (Clade A Clade B)) (DPanagrolaimidae) 100 X X 100 XMonophyly of Strongyloidoidea (Steinernema X X X 51 X

carpocapsae (Rhabditophanes Strongyloides stercolaris))Monophyly of Panagrolaimoidea and Strongyloidoidea X X X except X X

(DInfraorder Panagrolaimomorpha1 SteinernemaRhabditomorpha1 (DRhabditina) as sister taxon to X X

Panagrolaimomorpha1

Panagrolaimomorpha1 as sister X Xtaxon to Cephalobomorpha1 Tylenchomorpha1

1De Ley and Blaxter (2002)

hunti Stock De Ley De Ley Mundo-Ocampo Baldwinamp Nadler 2002 and Panagrobelus stammeri Ruumlhm 1956(clade C) and to a basal position for this clade withinPanagrolaimidae In other respects our results are com-parable to the SSU rDNA sequence analysis of Feacutelix etal (2000) for example the position of Brevibucca withinRhabditida is uncertain variously assuming a positionat the base of either infraorder Cephalobomorpha Pana-grolaimomorpha or Rhabditomorpha (De Ley amp Blaxter2002) Steinernema carpocapsae (Weiser 1955) WoutsMraacutedaggercek Gerdin amp Bedding 1982 is another nematodewhose position is unclear both in our trees and thoseof Feacutelix et al (2000) but analysis with a more fo-cused dataset con rms its placement in a monophyleticsuperfamily Strongyloidoidea (Dorris et al 2002) Asthe purpose of our study was to explore phylogeneticaf nities between Baujardia gen n and its closest rel-atives we limited the number of sequences included inour dataset and it is therefore not surprising that resolu-tion of relationships among higher taxa remains unclearin our trees (eg aphelenchs are not found to be mono-phyletic)

BIOLOGY

We assume that the pitchers of Nepenthes mirabilis arethe natural habitat of Baujardia mirabilis gen n sp nconsidering that the species was found alive in severalpitchers These pitchers contained a relatively diversefauna dominated by mosquito larvae midges and thenew genus in addition to a few dipteran and coleopteranlarvae The most abundant prey insects in the pitcherswere ants and diverse ying hymenopterans Apparentlya diverse assemblage of organisms lives in associationwith the pitchers of this Nepenthes species This mayrepresent an ecologically complex community similarto the better-known association inhabiting Sarraceniapitchers (eg Kitching 2001 Greeney 2001) Possiblythe nematodes have a phoretic relationship with oneor more insect species Closely related genera are alsocapable of withstanding harsh environments Turbatrixaceti (the vinegar eelworm) Panagrellus redivivus Psilusiae and P nepenthicola inhabit vinegar bookbinderrsquospaste beer lters and pitcher plants respectively

Not much is known about the occurrence of nematodesin pitcher plants and they were not even mentioned in

Vol 5(3) 2003 415

W Bert et al

Fig 5 Neighbour joining tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupnumbers next to branches are percentage bootstrap support values from corresponding50 majority-ruleconsensus tree obtained withNJ analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within the Panagrolaimidae

a reference work of the Nepenthes-inhabiting fauna byThienemann (1933) More extensive study is needed toinvestigate the life cycle and biochemistry of this unusualnew genus Moreover pitcher plants and other epiphytesare clearly a promising source of surprising new nematodetaxa

Discussion

The nematofauna of pitcher plants in Indonesia wasstudied by Menzel (1922) who reported the genera Plec-tus Dorylaimus Rhabditis and Diplogaster He verybrie y described a new species Anguillula nepenthicolaMenzel 1922 which he considered as the only true inhab-itant of pitcher plants and not an accidental recovery An-guillula nepenthicola together with several other speciesincluding Panagrellus pycnus Thorne 1938 was laterplaced in the newly erected genus Turbator by Goodey

(1943) which later (Goodey 1945) became a junior syn-onym of Panagrellus

As P nepenthicola was described from the same habi-tat and similar geographical region as our new genus wehave brie y examined Menzelrsquos (1922) very limited de-scription to justify proposal of a new species for our ma-terial especially in the absence of original illustrations forP nepenthicola Both species are comparable in length(18-27 vs 23-29 mm for P nepenthicola) and in vulvaposition (average of 76 vs 77 for P nepenthicola) Therelative tail length is however clearly longer in our ne-matodes being on average 125 of the vulva-anus dis-tance vs only half the vulva-anus distance length in P ne-penthicola the male tail is on average twice the spiculalength vs three times the spicula length in P nepenthicolathe female c value is 62-89 vs 9-10 in P nepenthicolaand the male c value is 67-11 vs 12-15 in P nepenthicolaThe identity of P nepenthicolabecame somewhat clearerwith the redescription by Micoletzky and Menzel (1928)although only the male tail was illustrated The most ap-

416 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 6 First of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup numbers next to branches are bootstrap values from corresponding 50 majority-ruleconsensus tree obtained with MP analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within thePanagrolaimidae Broken line in the bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to theothers (Figs 5 8)

parent similarities of the new genus with the redescribedP nepenthicolaare the muscular vagina and long postvul-val sac (103-149 vs 78-150 for P nepenthicola) Howeverthe spicules of our new genus and species have more an-gular manubria and their tips are not bi d (an indistinctbi d tip can be observed on the original illustration of Pnepenthicola) Furthermore the male tail is more stronglycurved compared to P nepenthicola and the number andpositioning of the genital papillae is different The differ-ence in tail curvature however may be due to cold xa-tion of our nematodes

The genital system of the new genus is panagrolaimidand shows similarities with the morphology of the femalegonad of Panagrellus redivivus (according to results ob-tained after extraction of the genital system by Geraertet al 1980) As in P redivivus the exure of the gen-ital system is formed by the oviduct and a pair of dis-tinct oviduct cells are observed at the spermatheca side

However the two opposing and offset spermatheca-likepouches observed here represent a unique feature amongstdescribed nematodes These pouches are probably glan-dular rather than having a function in sperm storage assuggested by the unusually large size of the sperm cellsand their occurrence throughout the uterus of gravid fe-males Possibly each compressed cluster of sperm orig-inated from successive copulations as multiple matingsper female might be more frequent in a limited and liquidenvironment Alternatively the movement of eggs couldcause the break-up of single clusters of sperm

On morphological grounds alone it was not immedi-ately apparent in which family Baujardiagen n shouldbeplaced The molecular analysis however robustly placesthe new genus as a sister taxon of Panagrellus within thePanagrolaimidae As a matter of fact the morphologicalsimilarities with Panagrellus as discussed above are rel-atively obvious a posteriori but were not evident before

Vol 5(3) 2003 417

W Bert et al

Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

Baujardia mirabilis gen n sp n from pitcher plants

Table 2 Monophyly (X D yes X D no) and bootstrap support () for clades

Clades NJ MP1 MP2 MP majority ML(Fig 5) (Fig 6) (Fig 7) rule (Fig 6) (Fig 8)

Clade A(Panagrolaimus (Baujardia (Panagrellus redivivus 57 X X 52 X

AF036599 Panagrellus redivivus AF083007)))(Baujardia (Panagrellus redivivus AF036599 100 X X 100 X

Panagrellus redivivus AF083007))(Panagrolaimus sp (Halicephalobus gingivalis X X X 52 X

Turbatrix aceti))

Clade B(Halicephalobus gingivalis Turbatrix aceti) 86 X X 79 X

Clade C(Plectonchus hunti Panagrobelus stammeri) 100 X X 100 X(Clade C (Clade A Clade B)) (DPanagrolaimidae) 100 X X 100 XMonophyly of Strongyloidoidea (Steinernema X X X 51 X

carpocapsae (Rhabditophanes Strongyloides stercolaris))Monophyly of Panagrolaimoidea and Strongyloidoidea X X X except X X

(DInfraorder Panagrolaimomorpha1 SteinernemaRhabditomorpha1 (DRhabditina) as sister taxon to X X

Panagrolaimomorpha1

Panagrolaimomorpha1 as sister X Xtaxon to Cephalobomorpha1 Tylenchomorpha1

1De Ley and Blaxter (2002)

hunti Stock De Ley De Ley Mundo-Ocampo Baldwinamp Nadler 2002 and Panagrobelus stammeri Ruumlhm 1956(clade C) and to a basal position for this clade withinPanagrolaimidae In other respects our results are com-parable to the SSU rDNA sequence analysis of Feacutelix etal (2000) for example the position of Brevibucca withinRhabditida is uncertain variously assuming a positionat the base of either infraorder Cephalobomorpha Pana-grolaimomorpha or Rhabditomorpha (De Ley amp Blaxter2002) Steinernema carpocapsae (Weiser 1955) WoutsMraacutedaggercek Gerdin amp Bedding 1982 is another nematodewhose position is unclear both in our trees and thoseof Feacutelix et al (2000) but analysis with a more fo-cused dataset con rms its placement in a monophyleticsuperfamily Strongyloidoidea (Dorris et al 2002) Asthe purpose of our study was to explore phylogeneticaf nities between Baujardia gen n and its closest rel-atives we limited the number of sequences included inour dataset and it is therefore not surprising that resolu-tion of relationships among higher taxa remains unclearin our trees (eg aphelenchs are not found to be mono-phyletic)

BIOLOGY

We assume that the pitchers of Nepenthes mirabilis arethe natural habitat of Baujardia mirabilis gen n sp nconsidering that the species was found alive in severalpitchers These pitchers contained a relatively diversefauna dominated by mosquito larvae midges and thenew genus in addition to a few dipteran and coleopteranlarvae The most abundant prey insects in the pitcherswere ants and diverse ying hymenopterans Apparentlya diverse assemblage of organisms lives in associationwith the pitchers of this Nepenthes species This mayrepresent an ecologically complex community similarto the better-known association inhabiting Sarraceniapitchers (eg Kitching 2001 Greeney 2001) Possiblythe nematodes have a phoretic relationship with oneor more insect species Closely related genera are alsocapable of withstanding harsh environments Turbatrixaceti (the vinegar eelworm) Panagrellus redivivus Psilusiae and P nepenthicola inhabit vinegar bookbinderrsquospaste beer lters and pitcher plants respectively

Not much is known about the occurrence of nematodesin pitcher plants and they were not even mentioned in

Vol 5(3) 2003 415

W Bert et al

Fig 5 Neighbour joining tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupnumbers next to branches are percentage bootstrap support values from corresponding50 majority-ruleconsensus tree obtained withNJ analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within the Panagrolaimidae

a reference work of the Nepenthes-inhabiting fauna byThienemann (1933) More extensive study is needed toinvestigate the life cycle and biochemistry of this unusualnew genus Moreover pitcher plants and other epiphytesare clearly a promising source of surprising new nematodetaxa

Discussion

The nematofauna of pitcher plants in Indonesia wasstudied by Menzel (1922) who reported the genera Plec-tus Dorylaimus Rhabditis and Diplogaster He verybrie y described a new species Anguillula nepenthicolaMenzel 1922 which he considered as the only true inhab-itant of pitcher plants and not an accidental recovery An-guillula nepenthicola together with several other speciesincluding Panagrellus pycnus Thorne 1938 was laterplaced in the newly erected genus Turbator by Goodey

(1943) which later (Goodey 1945) became a junior syn-onym of Panagrellus

As P nepenthicola was described from the same habi-tat and similar geographical region as our new genus wehave brie y examined Menzelrsquos (1922) very limited de-scription to justify proposal of a new species for our ma-terial especially in the absence of original illustrations forP nepenthicola Both species are comparable in length(18-27 vs 23-29 mm for P nepenthicola) and in vulvaposition (average of 76 vs 77 for P nepenthicola) Therelative tail length is however clearly longer in our ne-matodes being on average 125 of the vulva-anus dis-tance vs only half the vulva-anus distance length in P ne-penthicola the male tail is on average twice the spiculalength vs three times the spicula length in P nepenthicolathe female c value is 62-89 vs 9-10 in P nepenthicolaand the male c value is 67-11 vs 12-15 in P nepenthicolaThe identity of P nepenthicolabecame somewhat clearerwith the redescription by Micoletzky and Menzel (1928)although only the male tail was illustrated The most ap-

416 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 6 First of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup numbers next to branches are bootstrap values from corresponding 50 majority-ruleconsensus tree obtained with MP analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within thePanagrolaimidae Broken line in the bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to theothers (Figs 5 8)

parent similarities of the new genus with the redescribedP nepenthicolaare the muscular vagina and long postvul-val sac (103-149 vs 78-150 for P nepenthicola) Howeverthe spicules of our new genus and species have more an-gular manubria and their tips are not bi d (an indistinctbi d tip can be observed on the original illustration of Pnepenthicola) Furthermore the male tail is more stronglycurved compared to P nepenthicola and the number andpositioning of the genital papillae is different The differ-ence in tail curvature however may be due to cold xa-tion of our nematodes

The genital system of the new genus is panagrolaimidand shows similarities with the morphology of the femalegonad of Panagrellus redivivus (according to results ob-tained after extraction of the genital system by Geraertet al 1980) As in P redivivus the exure of the gen-ital system is formed by the oviduct and a pair of dis-tinct oviduct cells are observed at the spermatheca side

However the two opposing and offset spermatheca-likepouches observed here represent a unique feature amongstdescribed nematodes These pouches are probably glan-dular rather than having a function in sperm storage assuggested by the unusually large size of the sperm cellsand their occurrence throughout the uterus of gravid fe-males Possibly each compressed cluster of sperm orig-inated from successive copulations as multiple matingsper female might be more frequent in a limited and liquidenvironment Alternatively the movement of eggs couldcause the break-up of single clusters of sperm

On morphological grounds alone it was not immedi-ately apparent in which family Baujardiagen n shouldbeplaced The molecular analysis however robustly placesthe new genus as a sister taxon of Panagrellus within thePanagrolaimidae As a matter of fact the morphologicalsimilarities with Panagrellus as discussed above are rel-atively obvious a posteriori but were not evident before

Vol 5(3) 2003 417

W Bert et al

Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

W Bert et al

Fig 5 Neighbour joining tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupnumbers next to branches are percentage bootstrap support values from corresponding50 majority-ruleconsensus tree obtained withNJ analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within the Panagrolaimidae

a reference work of the Nepenthes-inhabiting fauna byThienemann (1933) More extensive study is needed toinvestigate the life cycle and biochemistry of this unusualnew genus Moreover pitcher plants and other epiphytesare clearly a promising source of surprising new nematodetaxa

Discussion

The nematofauna of pitcher plants in Indonesia wasstudied by Menzel (1922) who reported the genera Plec-tus Dorylaimus Rhabditis and Diplogaster He verybrie y described a new species Anguillula nepenthicolaMenzel 1922 which he considered as the only true inhab-itant of pitcher plants and not an accidental recovery An-guillula nepenthicola together with several other speciesincluding Panagrellus pycnus Thorne 1938 was laterplaced in the newly erected genus Turbator by Goodey

(1943) which later (Goodey 1945) became a junior syn-onym of Panagrellus

As P nepenthicola was described from the same habi-tat and similar geographical region as our new genus wehave brie y examined Menzelrsquos (1922) very limited de-scription to justify proposal of a new species for our ma-terial especially in the absence of original illustrations forP nepenthicola Both species are comparable in length(18-27 vs 23-29 mm for P nepenthicola) and in vulvaposition (average of 76 vs 77 for P nepenthicola) Therelative tail length is however clearly longer in our ne-matodes being on average 125 of the vulva-anus dis-tance vs only half the vulva-anus distance length in P ne-penthicola the male tail is on average twice the spiculalength vs three times the spicula length in P nepenthicolathe female c value is 62-89 vs 9-10 in P nepenthicolaand the male c value is 67-11 vs 12-15 in P nepenthicolaThe identity of P nepenthicolabecame somewhat clearerwith the redescription by Micoletzky and Menzel (1928)although only the male tail was illustrated The most ap-

416 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 6 First of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup numbers next to branches are bootstrap values from corresponding 50 majority-ruleconsensus tree obtained with MP analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within thePanagrolaimidae Broken line in the bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to theothers (Figs 5 8)

parent similarities of the new genus with the redescribedP nepenthicolaare the muscular vagina and long postvul-val sac (103-149 vs 78-150 for P nepenthicola) Howeverthe spicules of our new genus and species have more an-gular manubria and their tips are not bi d (an indistinctbi d tip can be observed on the original illustration of Pnepenthicola) Furthermore the male tail is more stronglycurved compared to P nepenthicola and the number andpositioning of the genital papillae is different The differ-ence in tail curvature however may be due to cold xa-tion of our nematodes

The genital system of the new genus is panagrolaimidand shows similarities with the morphology of the femalegonad of Panagrellus redivivus (according to results ob-tained after extraction of the genital system by Geraertet al 1980) As in P redivivus the exure of the gen-ital system is formed by the oviduct and a pair of dis-tinct oviduct cells are observed at the spermatheca side

However the two opposing and offset spermatheca-likepouches observed here represent a unique feature amongstdescribed nematodes These pouches are probably glan-dular rather than having a function in sperm storage assuggested by the unusually large size of the sperm cellsand their occurrence throughout the uterus of gravid fe-males Possibly each compressed cluster of sperm orig-inated from successive copulations as multiple matingsper female might be more frequent in a limited and liquidenvironment Alternatively the movement of eggs couldcause the break-up of single clusters of sperm

On morphological grounds alone it was not immedi-ately apparent in which family Baujardiagen n shouldbeplaced The molecular analysis however robustly placesthe new genus as a sister taxon of Panagrellus within thePanagrolaimidae As a matter of fact the morphologicalsimilarities with Panagrellus as discussed above are rel-atively obvious a posteriori but were not evident before

Vol 5(3) 2003 417

W Bert et al

Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

Baujardia mirabilis gen n sp n from pitcher plants

Fig 6 First of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup numbers next to branches are bootstrap values from corresponding 50 majority-ruleconsensus tree obtained with MP analysis of 3000 bootstrap replicates A B and C indicate the three obtained clades within thePanagrolaimidae Broken line in the bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to theothers (Figs 5 8)

parent similarities of the new genus with the redescribedP nepenthicolaare the muscular vagina and long postvul-val sac (103-149 vs 78-150 for P nepenthicola) Howeverthe spicules of our new genus and species have more an-gular manubria and their tips are not bi d (an indistinctbi d tip can be observed on the original illustration of Pnepenthicola) Furthermore the male tail is more stronglycurved compared to P nepenthicola and the number andpositioning of the genital papillae is different The differ-ence in tail curvature however may be due to cold xa-tion of our nematodes

The genital system of the new genus is panagrolaimidand shows similarities with the morphology of the femalegonad of Panagrellus redivivus (according to results ob-tained after extraction of the genital system by Geraertet al 1980) As in P redivivus the exure of the gen-ital system is formed by the oviduct and a pair of dis-tinct oviduct cells are observed at the spermatheca side

However the two opposing and offset spermatheca-likepouches observed here represent a unique feature amongstdescribed nematodes These pouches are probably glan-dular rather than having a function in sperm storage assuggested by the unusually large size of the sperm cellsand their occurrence throughout the uterus of gravid fe-males Possibly each compressed cluster of sperm orig-inated from successive copulations as multiple matingsper female might be more frequent in a limited and liquidenvironment Alternatively the movement of eggs couldcause the break-up of single clusters of sperm

On morphological grounds alone it was not immedi-ately apparent in which family Baujardiagen n shouldbeplaced The molecular analysis however robustly placesthe new genus as a sister taxon of Panagrellus within thePanagrolaimidae As a matter of fact the morphologicalsimilarities with Panagrellus as discussed above are rel-atively obvious a posteriori but were not evident before

Vol 5(3) 2003 417

W Bert et al

Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

W Bert et al

Fig 7 Second of two best trees obtained with Maximum Parsimony analysis based on secondary structure alignment of small subunitrDNA Plectus was designated as outgroup A B and C indicate the three obtained clades within the Panagrolaimidae Broken line inthe bracket for clade A denotes paraphyletic position of Panagrolaimus in this tree compared to the others (Figs 5 8)

the molecular analysis Morphological characters such asspermatheca structure stoma organisation the presenceof liplets setae and amphids are generally consideredto be appropriate to characterise a family However theplacement by molecular analysis of this morphologicallydeviant new genus illustrates the inconsistency of thesecharacters within the family Panagrolaimidae As a con-sequence a morphological rede nition of this family isrequired

Panagrolaimidae Thorne 1937

EMENDED DIAGNOSIS

Lip region without probolae or mbriate processesLabial sensilla papilliform cephalic sensilla papilliformor rarely setiform Cuticula nely annulated (annules usu-ally lt1 sup1m wide) Stoma fairly wide anteriorly stegos-tom usually tapering comprising between 50 and 66 ofstoma length never with distinct subdivisionsof its liningPharynx with cylindrical procorpus metacorpus cylin-

drical or with valveless median bulb terminal bulb val-vate Female reproductive system monodelphic prodel-phic Spermatheca usually absent or axial rarely offsetas in CephalobidaeUterus-oviduct junctionexceptionallywith two offset spermatheca-like pouches Postvulval sacpresent or absent Female tail conical with pointed tipMales without bursa with one unpaired papilla and veto seven pairs of genital papillae arrangement variableSpicules often with wide velum gubernaculumoften withposteriorly expanded outline

LIST OF GENERA

AnguilluloidesRuumlhm 1956Brevistoma Mukhina 1981HalicephalobusTimm 1956D Micronema Koumlrner 1954D PhytorhabditisLordello amp De Oliveira 1963PanagrellusThorne 1938D Anguillula de Man 1910 pro parteD NeocephalobusSteiner 1936 pro parteD Turbator Goodey 1943

418 Nematology

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

Baujardia mirabilis gen n sp n from pitcher plants

Fig 8 Maximum Likelihood tree based on secondary structure alignment of small subunit rDNA Plectus was designated as outgroupA B and C indicate the three obtained clades within the PanagrolaimidaeLineages of taxa are based on proposed classication schemeby De Ley and Blaxter (2002)

D TylorhabdusSukul 1971PanagrobelusThorne 1939PanagrobeliumAndraacutessy 1984Panagrolaimus Fuchs 1930Plectonchus Fuchs 1930ProcephalobusSteiner 1934Tricephalobus Steiner 1936D PseudorhabditisKreis 1929D Trilabiatus Goodey 1963Turbatrix Peters 1927

Acknowledgements

The authors thank P Pholpunthin S Chittapun and SKitichate of the Department of BiologyPrince of SongklaUniversityHat Yai Thailand for organising eld trips andfor providing the identi cation of the Nepenthes speciesWe thank J Van eteren and A Vierstraete for the DNAsequencing and alignment The support of G Borgonie inseveral aspects of this study is greatly appreciatedWe arealso grateful to M Yoder L Mundo and S Esfahani forassistance with the production of virtual specimens

References

DE LEY IT DE LEY P BALDWIN JG MUNDO-OCAMPO M amp NADLER SA (1999) Three new speciesof Nothacrobeles (Nemata Cephalobidae) from the MojaveDesert California Journal of Nematology 31 482-497

DE LEY P amp BERT W (2002) Video capture and editing asa tool for the storage distribution and illustration of morpho-logical characters of nematodes Journal of Nematology 34296-302

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon Taylor amp Francis pp 1-30

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DE RIJK P amp DE WACHTER R (1993) DCSE v254an interactive tool for sequence alignment and secondarystructure research Computer Applications in the Biosciences9 735-740 [software]

DORRIS M VINEY M amp BLAXTER M (2002) Molecularphylogenetic analysis of the genus Strongyloides and related

Vol 5(3) 2003 419

W Bert et al

nematodes International Journal for Parasitology 32 1507-1517

FELIX MA DE LEY P SOMMER RJ FRISSE LNADLER S THOMAS K VANFLETEREN J amp STERN-BERG PW (2000) Evolution of vulva development in theCephalobina (Nematoda) Developmental Biology 221 68-86

GERAERT E SUDHAUS W amp GROOTAERT P (1980) Thestructure of the female genital apparatus in the order Rhabdi-tida (Nematoda) Annales de la Socieacuteteacute Royale Zoologique deBelgique 109 91-108

GOODEY T (1943) On the systematic relationships of thevinegar eelworm Turbatrix aceti and its congeners with adescription of a new species Journal of Helminthology 211-9

GOODEY T (1945) A note on the subfamily Turbatricinae andthe genus Turbator Goodey 1943 Journal of Helminthology21 69-70

GREENEY HF (2001) The insects of plant-held waters areview and bibliography Journal of Tropical Ecology 17241-260

KISHINO H amp HASEGAWA M (1989)Evaluation of the max-imum likelihood estimate of the evolutionary tree topologiesfrom DNA sequence data and the branching order of theHominoidea Journal of Molecular Evolution 29 170-179

KITCHING RL (2001) Food webs in phytotelmata ldquoBottom-uprdquo and ldquotop-downrdquo explanations for community structureAnnual Review of Entomology 46 729-760

LOCKHART PJ STEEL MA HENDY MD amp PENNYD (1994) Recovering evolutionary trees under a morerealistic model of sequence evolutionMolecular Biology andEvolution 11 605-612

MENZEL R (1922) Beitraumlge zur Kenntnis der Mikrofauna vonNiederlaumlndisch Ost-Indien II Uumlber den tierischen Inhalt derKannen von Nepenthes melamphora Reinw mit besondererberuumlcksichtigung der Nematoden Treubia 3 116-122

MICOLETZKY H amp MENZEL R (1922) Beitraumlge zur Ken-ntnis der mikrofauna von Niederlaumlndisch Ost-Indien VII

Anguillula nepenthicola Menzel aus kannen von Nepenthesgymnaphora Nees bei Tjibodas Treubia 10 285-290

PAGE RDM (1996)TreeView an application to display phy-logenetic trees on personal computers Computer Applica-tions in the Biological Sciences 12 357-358 [software]

POSADA D amp CRANDALL KA (1998) MODELTESTtesting the model of DNA substitution Bioinformatics 14817-818

SEINHORST JW (1959) A rapid method for the transfer ofnematodes from xative to anhydrousglycerinNematologica4 67-69

STOCK SP DE LEY P DE LEY I MUNDO-OCAMPO M BALDWIN JG amp NADLER SA (2002) Panagrobelusstammeri Ruumlhm 1956 and Plectonchus hunti n sp implica-tions of new morphological observations for characterisationof these genera (Nematoda Panagrolaimoidea) Nematology4 403-419

SWOFFORD DL (2002) PAUP Phylogenetic Analysis UsingParsimony (and other methods) version 40 SunderlandMassachusetts USA Sinauer Associates [software]

TANDINGAN DE LEY I DE LEY P VIERSTRAETE AKARSSEN G MOENS M amp VANFLETEREN J (2002)Phylogenetic analyses of Meloidogyne SSU rDNA Journalof Nematology 34 319-327

THIENEMANN A (1933) Die Tierwelt der Nepenthes-KannenArchiv fuumlr Hydrobiologie 11 1-54

THORNE G (1938) Notes on free-livingand plant-parasiticne-matodes IV (1) Panagrellus pycnus n g n sp (Cephalo-bidae Panagrolaiminae) Proceedings of the Helminthologi-cal Society of Washington 5 64-65

VAN DE PEER Y ROBBRECHT E DE HOOG S CAERSA DE RIJK P amp DE WACHTER R (1998) Database onthe structure of small subunit ribosomal RNA Nucleic AcidResearch 271 179-183

WERGIN WP (1981) Scanning electron microscopic tech-niques and application for use in nematology In ZuckermanBM amp Rohde RA (Eds) Plant parasitic nematodes Vol-ume III New York Academic Press pp 175-204

420 Nematology