Nematology,2002,V ol.4(8), 953-963

Thecellular structure of the female reproductive system within the Heteroderinae andMeloidogyninae (Nematoda)

1; 2 1 1 Wim BERT ¤, Gerrit KARSSEN , Rita VAN DRIESSCHE andEtienne G ERAERT 1 Departmentof Biology,Ghent University, Ledeganckstraat 35, 9000 Gent, Belgium 2 PlantProtection Service, Nematology Section, P .O.Box 9102, 6700 HC Wageningen,The Netherlands Received:18 March2002; revised: 5 August2002 Acceptedfor publication:5 August2002

Summary – Gonadsfrom living young females, representing 23 different species, were extracted to study the cellular structure of thefemale genital structure within the Meloidogyninae and Heteroderinae. All genera studied can be characterised by their cellular spermathecamorphology. Within Meloidogyne asphericalspermatheca is found with lobe-like protruding cells, most species having 16 to18 spermatheca cells with interlaced cell boundaries while M.microtyla and M.ichinohei havemore spermatheca cells with different cellboundaries. Heterodera and Globodera reveala comparablegonad structure. The spermatheca cells of Heterodera arecolumnar andarranged in a restrictednumber of rows, whereas in Globodera thespermatheca cells are squarish to rounded,depending on the species.The gonad morphology of Afenestratakoreana isclearlydifferent from what would be expectedbased on therelated genera Globodera and Heterodera.Theapparently simplest genital system was found in Meloidodera oridensis wherethe uterus has a limited numberof cells.In the other genera studied a largeand variable cell number was found. Keywords – Afenestrata ,femalegenital system, Globodera, Heterodera, Meloidodera , Meloidogyne ,morphology,SEM, variablecell number.

Analysisof the cellular structure of thefemale repro- anduterine sac. The terms tricolumella (Hirschmann & ductivesystem for Secernenteawas mainlyexecuted by Triantaphyllou,1968), quadricolumella (Wu, 1958) and Geraert (1972,1976, 1978) and Geraert et al. (1980a, polycolumella(Chizhov & Swiliam,1986) describe the b)who also pointed out the importance of the female spatialarrangements of theuteruscells. Diversity and evo- reproductivesystem in nematode systematics (Geraert, lutionof Heteroderinae and Meloidogyninae are of spe- 1981,1983). More isolated studies of the reproductive cialinterest because world-wide they include some of the systemof Tylenchidawere undertakenby severalauthors mostdestructive plant-parasitic nematodes. In their com- (Wu,1958, 1967; Seinhorst, 1968; Bert & Geraert,2000). prehensivestudy, based on comparativedetailed morphol- Chizhovand Swiliam (1986) and Chizhov and Berez- ogy,of thephylogeny of theHeteroderinae, Baldwin and ina(1988a, b) studied several nematode species, espe- Schouest(1990) commented on the cellular structure of ciallyincluding Heteroderinae and Meloidogyninae .The thefemale reproductive tract as being potentially useful Ž rst Meloidogyne spermathecadrawing was publishedby for interpretingphylogeny. However, they excluded ex- Nagakura(1930). Triantaphyllou and Hirschmann (1962) istinginformation from theirmatrix pending examination madea detailedillustration of the reproductive system ofmoreexcised reproductive tracts. More recently, Zunke of Heteroderaglycines andTriantaphyllou (1962, 1987) andEisenback (1998) remarked that additional investiga- focusedon some Meloidogyne species.The terminology tionsare needed, particularly on the Ž nestructure of the ofthe reproductive system used here is basedon Geraert reproductivesystem, to improve our understanding of the (1983)who in turn followed the interpretation of Chit- phylogeneticrelationships among the numerous members woodand Chitwood (1950). The genital system consists ofthisgroup. In this paper, we examinethe cellular struc- of an ovary ( gonad)and gonoduct. The oviduct is the tureof several species and genera within the Heteroderi- D constrictedregion between the ovary and spermatheca naeand Meloidogynina einorder to shed new light on oruterus. The rest of the gonoduct is called the uterus suchquestions.

* Correspondingauthor, e-mail: [email protected]

© KoninklijkeBrill NV ,Leiden,2002 953 Alsoavailable online - www.brill.nl W. Bert et al.

Materialand methods cellularstructure. Because of theelongation of theuterus innearlyall Heteroderinae and Meloidogyninae ,onlythe Alistof thespecies examined (and their authorities) is partfollowing the spermatheca was illustratedin those presentedin T able1. Tostudythe cellular structure of the caseswhere the uterus was demonstrablyhomogeneous femalereproductive system, gonads were Žrst extruded. instructure. Fourto six young females of every population were put ina dropof water on the glass slide and then cut with Results aneye-knife, leading to expulsion of gut and gonad. After removalof mostof thenon-reproductive tissue, the Asummaryof theresults with emphasis on the diver- preparation,covered by a coverslip,was studiedat once gentcharacters is presentedin T able2. underthe light microscope. In order to have an ideaabout thevariability of thestudied structure, this procedure was repeateduntil at least 20 preparations could be observed MELOIDOGYNE GÖLDI, 1892 for eachpopulation. The dissection process must have Theoviduct-spermatheca region of allthe species stud- anin uence on the size and shape of the cells due iedis characteristicand typical of thegenus Meloidogyne . toa changingosmolarity. However, repeated dissections Theoviduct comprises two rows offourthick cells. The demonstratedthat such in uences are relatively constant spermathecais spherical and formed by avariablenumber withina species.When preparations are stained with oflobe-likecells. The uterus consists of threelong rows aceticorcein (2% aqueous solution of orcein in acetic of cells ( atricolumella).In M.incognita 3 65 to 3 95 D £ £ acid)or acidfuchsin (aqueous solution), the nuclei of the cellshave been counted. cellsare more clearly visible. This can be very helpful Thespermathecae of M. arenaria, M.ardenensis,M. for smallernematodes, but for membersof Heteroderinae artiellia, M.chitwoodi , M. exigua, M.graminicola , M. andMeloidogyninae no additionalinformation is obtained hapla, M.hispanica , M.incognita , M.javanica,M. from suchstaining. morocciensis and M.trifoliophila aresimilar, comprising Observationson the internal structures can best be 16to 18 lobe-like cells with interlaced cell boundaries madeon freshly dissected specimens, which usually plas- (Fig.1A). Thespermatheca of M. fallax is slightly molyseand decompose within 1 h.With the purpose of differentin that in 35% of the specimens four or Ž ve permanentlyrecording the morphology of the reproduc- spermathecacells are clustered together to form separate tivesystem, we havealso produced some short video clips lobes(Fig. 1B). Meloidogynemicrotyla hasa spermatheca thatmimic multifocal observation through the lightmicro- with18 to26cellsand thecellboundaries are only slightly scope.This was donewith the aid of aVideoCapture and interlaced(Fig. 1C). The spermatheca of M.ichinohei is Editing(VCE) method(De Ley& Bert,in press) andthe clearlydifferent from theremaining Meloidogyne species resultscan be viewedon http://www.vce.be.tf . andhas 18 to30cells forming irregular lobes (Fig. 1D). For scanningelectron microscopic (SEM) observation, Sperm was onlyobserved in the spermatheca of M. excisedreproductive systems from 50specimens were artiellia and M. fallax.SEMof the reproductive system transferredand Ž xedin freshly prepared 4% formalde- of M.incognita (Fig.2) conŽ rms thelight microscopic hyde(from paraformaldehyde)in phosphate buffered observations.The two rows offourlarge oviduct cells can saline(PBS) pH7 atroom temperature. The reproduc- beobserved as protruding cells. The membranes of the tivesystems were Žxedovernight and subsequently dehy- lasttwo oviduct cells seem to befusedin somespecimens dratedin 25, 50,75 and95% ethanolat 2-hourlyintervals, andappear more like spermatheca cells (Fig. 2B). The followedby anovernight dehydration in absoluteethanol lobe-likecells of thespermatheca are pronounced, but the andŽ nalremoval of theremaining water using silica gel. interlacedcell boundaries, as seen by light microscopy After critical-pointdrying using CO 2 (Wergin,1981) as (Fig.1), cannot be seenwith SEM. SEM alsofails to show thedrying liquid and sputtercoating with 20 nmgold,they theexternal cell boundaries of theuterus. were examinedwith a JEOL JSM-840at 15 kV. Thecellular structure of theovary is excludedfrom this GLOBODERA SKARBILOVICH , 1959 study,because it proved difŽ cult to observethe cellular ar- chitectureusing only lightmicroscopy. Furthermore, there Theoviduct is made up of two rows offour cells. isno reported evidence for anytaxonomic value of this Thespermatheca of an excised gonad forms thecorner

954 Nematology Cellularstructure of the female reproductive system

Table 1. Listof speciesstudied, identiŽ cation method, examination method(s) and source. Species IdentiŽcation Examination Source method method Meloidogynearenaria (Neal, Light LM & Video GerritKarssen 1889)Chitwood, 1949 microscopy Capture and PlantProtection Service, (LM) & Editing(VCE) Wageningen,The Netherlands isozymes M.ardenensis Santos,1968 Idem LM Idem M.artiellia Franklin,1961 Idem LM & VCE Idem M.chitwoodi Golden,O’ Bannon, Idem LM & VCE Idem Santo& Finley,1980 M. exigua Göldi, 1892 Idem LM Idem M. fallax Karssen,1996 Idem LM & VCE Idem M.graminicola Golden & Idem LM Idem BirchŽeld, 1965 M. hapla Chitwood,1949, race A Idem LM & VCE Idem M.hispanica Hirschmann,1986 Idem LM & VCE Idem M.ichinohei Araki, 1992 Idem LM Idem M.incognita (Kofoid& White, Idem LM & TEM Idem 1919)Chitwood, 1949 M. javanica (Treub,1885) Idem LM & VCE Idem Chitwood,1949 M.morocciensis Rammah & Idem LM Idem Hirschmann,1990 M.microtyla Mulvey,T ownshend Idem LM & VCE Idem &Potter,1975 M.trifoliophila Bernard & Idem LM & VCE Idem Eisenback,1997

Globoderapallida (Stone,1973) Protein LM & VCE AgriculturalResearch Centre, Behrens,1975 electrophoresis DepartmentCrop Protection, Ghent,Belgium G.rostochiensis (Wollenweber, Idem LM Idem 1923)Skarbilovich, 1959 G. tabacum (Lownsbery& LM & Protein LM & VCE GerritKarssen Lownsbery,1954) Behrens, 1975 electrophoresis PlantProtection Service, Wageningen,The Netherlands Heteroderaavenae Wollenweber, LM LM Botanicalgarden 1924 GhentUniversity, Belgium H. Ž ci Kirjanova,1954 LM LM GerritKarssen PlantProtection Service, Wageningen,The Netherlands H.schachtii Schmidt,1871 LM LM Departmentof Genetics, GhentUniversity, Belgium Afenestratakoreana Vovlas, LM LM RenatoInserra Lamberti& Choo,1992 FDACS, Gainesville,FL, USA Meloidodera oridensis LM LM & VCE ManuelMundo-Ocampo Chitwood,Hannon & Esser,1956 Universityof California, Riverside,CA, USA

Vol.4(8), 2002 955 W. Bert et al.

Table 2. Summaryof theresults with emphasis on thedivergent characters. Meloidogyne spermatheca:spherical, M. arenaria, M.ardenensis,M. artiellia , formedby lobe-likecells M.chitwoodi , M. exigua, M.graminicola , M. hapla, uterus:long tricolumella M.hispanica , M.incognita , M. javanica, M.morocciensis , M.trifoliophila and M. fallax spermatheca:16 cells with interlaced cell boundaries

M. fallax spermathecacells often form separate lobes

M.microtyla spermatheca:18 to 26 cells,the cell boundaries areonly slightly interlaced

M.ichinohei spermatheca:18 to30cells forming irregular lobes, no interlacedcell boundaries

Globodera spermatheca:variously shaped G. pallida cells,forms corner of aright spermatheca:mostly 12 cells,two big round cells anglebetween oviduct and uterus adjacentto oviduct uterus:polycolumella G.rostochiensis spermatheca:mostly 14 equallysized cells

G. tabacum spermatheca:mostly 12 cells,four cells adjacent to oviductsquarish to round and followed by attened cells

Heterodera spermatheca:ten to 16 H. avenae and H. Ž ci columnarcells basically uterus:tricolumella arrangedin tworows, bend at junctionof thespermatheca H.schachtii and uterus uterus:additional rows of cells along the tricolumella uterus:tricolumella (with in somecases additional rows)

Afenestrata spermatheca:oval shaped, consists koreana of20to 26variably arrangedcells uterus:tricolumella

Meloidodera spermatheca:partly offset,  oridensis composedof 12 variably shapedsmall cells withunclear cell boundaries, uterusforms a 90degreeangle withoviduct uterus:short tricolumella

956 Nematology Cellularstructure of the female reproductive system

Fig. 1. Oviduct-spermathecaregion of Meloidogyne spp. A: Meloidogynehispanica ; B: M. fallax C: M.microtyla ; D: M.ichinohei . Endof ovary (ova.), oviduct (ovi.), spermatheca (sp.), beginning of uterus (ut.). (Scale bars 30 ¹m.) D

Vol.4(8), 2002 957 W. Bert et al. ofa rightangle between oviduct and uterus. Thirty eightof 45 females from thethree studied G. pallida populationshave 12 cells comprising the spermatheca. Tworounded cells, larger than the other spermatheca cells,are located adjacent to the oviduct. Sperm was observedin the vicinity of these cells. The remaining cellsare  attenedto slightly rounded (Fig. 3B). The spermathecaof G.rostochiensis hasa slightlydifferent cellulararchitecture with 12 to 16 (mostly 14) cells of moreor less equal in size with a roundedto squarish shape.Sperm was alwaysobserved dispersed uniformly in thespermatheca (Fig. 3A). Thespermatheca of Globodera tabacum consistsof 12 cells, the Ž rst fourcells being squarishto round and followedby attenedcells arranged intwo rows. Thehighest concentration of sperm was foundin that part of the spermatheca closest to the oviduct.The uterus of the studied Globodera species consistsof twoor threeshort rows (mostlyfour cells long) atthe connection with the spermatheca, but increases to multiple(six to ten)rows (anirregular polycolumella).

HETERODERA SCHMIDT, 1871

Fig. 2. SEMoffemale reproductive system of Meloidogyne Heterodera species(Fig. 3C, D) exhibita reproductive incognita:endof ovary(ova.), oviduct (ovi.), spermatheca (sp.) systemsimilar to that of Globodera.Theoviduct consists andbeginning of uterus (ut.). (Scale bars: A, C 10 ¹m, B D D oftworows offourlarge oval cells. The spermatheca of 100 ¹m.) H. avenae, H. Ž ci and H.schachtii iscomposed of tento 16high columnar cells, 12 cells being observed in most MELOIDODERAFLORIDENSIS cases.The cells are basically arranged in tworows, butin As istypical within T ylenchida,the oviduct of M. themiddle of thespermathecathree cells are positioned at  oridensis consistsof two rows offour cells each. The thesame level. A characteristicbend occurs at the junc- spermathecais partly offset and composed of 12,variably tionof thespermatheca and uterus. The uterus of H. ave- shaped,small cells, the boundaries of whichare unclear . nae and H. Ž ci consistsof anelongatedtricolumella. Het- After excision,the uterus forms a90 ± anglewith the eroderaschachtii hasadditional rows ofcells along the oviduct.The tricolumella forming the uterus is short and tricolumellaand, closer to the vulva, up toŽ vecell rows onlysix to seven cells long. The eggs are deposited in occur.Twonuclei were observedin some of thesecells. amembranous,outstretched uterine sac following the tricolumella(Fig. 4B).

AFENESTRATAKOREANA Discussion Tworows offour cells make up the oviduct. The THE OVIDUCT spermathecais more or less oval shaped and consists of 20 to26 variablyarranged cells (Fig. 4A). Countingthe exact Themorphology of the oviduct has been described numberof cellswas difŽcult because cell boundaries were asa remarkablystable structure within the T ylenchida notalways clearly visible. The transition to the uterus (Geraert, 1983).Our expandedlight microscopic studies isvague and no clear bend occurs at the junction of donotdeviatefrom thisas, without exception, the oviduct spermathecaand uterus as in Globodera or Heterodera. comprisestwo rows offour cells. However, with SEM Theuterus includes an elongatedtricolumella. theoviduct of M.incognita didnot appear to be a

958 Nematology Cellularstructure of the female reproductive system

Fig. 3. Femalereproductive system of Heteroderinae. A: Globoderarostochiensis ; B: G. pallida C: Heteroderaavenae ; D: H. schachtii . Endof ovary (ova.), oviduct (ovi.), spermatheca (sp.), beginning of uterus (ut.) and uterus closer to the vulva (utv .).(Scalebars D 30 ¹m.) clear,morphologically discrete, unit. Other studies (TEM, THESPERMATHECA immuno-histochemical)are required in order to determine exactly,on the basis of function, the separate gonoduct Thespermatheca has been shown to have particular components. valueas ataxonomiccharacter. All studied genera can be characterisedby a combinationof spermathecamorphol- ogyand cell number .

Vol.4(8), 2002 959 W. Bert et al.

Fig. 4. Femalereproduction systems. A: Afenestratakoreana ; B: Meloidodera oridensis .Endof ovary (ova.), oviduct (ovi.), spermatheca(sp.), uterus (ut.) and uterine sac (uts.). (Scale bars 30 ¹m.) D

Meloidogyne 1965)Whitehead, 1965, an amphimictic species with ap- proximately34 cells, used Chizhov’ s (1981)results to Sincespermatheca morphology and the number of cells suggestthat obligate mitotic parthenogenetic species with variesamong species of Meloidogyne ,oneconsideration manyspermatheca cells probably evolved from amphim- iswhether this variability is convergent or if it directly icticspecies. These amphimictic species also have a large reects evolution within the genus. Conversely, these numberof spermathecacells, the additional cells presum- differencesin spermathecamay be convergentfunctional ablybeing required to accommodate many spermatozoa. adaptationslinked to particular modes of reproduction Our resultsdo notsupportthis view. Nevertheless, the hy- thatalso vary within the genus. According to Chizhov pothesisthat strictly amphimictic species have a larger (1981),obligatory mitotic parthenogenetic species, such numberof spermathecacells can be acknowledged given as M.incognita ,havea spermathecawith 24 smallcells. thefact that M.microtyla ,anamphimictic species, dis- Conversely,facultative meiotic parthenogenetic species, playsa largenumber of cells. However, in other nema- such as some M. hapla, havea spermathecawith only 16 todegenera, such as Pratylenchus ,acorrelationwith the largecells. This does not correspond with our results or numberof spermathecacells and the mode of reproduc- withresults from theliterature as describedbelow . tionhas never been found (Bert, unpubl.). The presence of Wefounda spermathecacomposed of approximately malesmay in uence spermatheca shape, the spermatheca 16cells in both obligate, as well as facultative, mei- cells of a M. fallax populationwith abundant males being oticparthenogenetic species. These results are congru- clusteredtogether and forming lobes compared with the entwith those of Geraert (1978),who found a maxi- morphologicallysimilar M.chitwoodi . mumof 16 spermatheca cells for M.incognita , M. are- Triantaphyllou(1985) inferred the phylogeny of root- naria and M.javanica. Onlya maximumof 18 large knotnematodes from cytogeneticdata and hypothesised cellscan be inferred from Triantaphyllou’s (1962)draw- thatstrictly amphimictic species are ancestral compared ingof the spermatheca of M. javanica.Triantaphyllou totheparthenogenetic ones. If thisis trueit wouldsuggest (1987),who studied M.spartinae (Rau& Fassuliotis, aroot-knotspermatheca with a highernumber of cellsis

960 Nematology Cellularstructure of the female reproductive system themore primitive condition relative to those with fewer diagnosis.Nevertheless, spermatheca morphology can cells. supplementother tools to morphologically separate the Ina recentcomprehensive phylogenetic analysis of twocyst nematodes. Meloidogyne SSU rDNA (TandiganDe Ley et al., in press),the amphimictic species M.microtyla is,however, Heterodera positionedin a cladetogether with M. hapla. Inthis study, The Heterodera reproductivestructures as describedin M.ichinohei ,aspecieswith a morphologicallydivergent theliterature are comparable with our results. Heterodera spermatheca(18 to 30 cells forming four lobes), was glycines Ichinohe,1952 was illustratedby Triantaphyllou invariablyplaced with maximal support as asistertaxon andHirschmann (1962), and H. avenae and H.schachtii toall other species of Meloidogyne . were describedby Chizhovand Swiliam (1986). It can be Althoughwe havedemonstrated intraspeciŽ c sper- concludedthat the genus Heterodera exhibitsa uniform mathecavariability within the genus Meloidogyne , the andcharacteristic spermatheca. spermathecais always spherical and formed by avariable numberof thick, lobe-like cells, thus making it distinc- Afenestratakoreana tivefrom anyother known nematode genus. Members of Inmost recent phylogenetic analyses, Afenestrata is the genus Meloidogyne arestrongly uniŽ ed by thisfeature suggestedas a sistertaxon of Heterodera (Wouts,1985; (Triantaphyllou,1987) andthisfeature can beadded to the Baldwin& Schouest,1990; Baldwin, 1992) and A. orien- longlist of reasons for notconsidering root-knot nema- talis Kazachenko,1989 is evenplaced within Heterodera todesand cyst nematodes as closelyrelated taxa (Baldwin, basedon ribosomal DNA sequences(Subbotin et al., 1992;Geraert, 1997). This is alsocongruent with molec- 2001).However, in this study the spermatheca morphol- ularŽ ndingsas Meloidogyne specieswere foundto bere- ogy of Afenestrata was shownto beclearly different from markablydivergent from eachother in 18Ssequence and that of Heterodera, whereas Heterodera and Globodera geneticallydistant from othergenera in Tylenchida(Sza- appearto havesimilar spermathecas. Recently, Inserra et lanski et al.,1997).Our resultsdo not allow us tospecu- al.(1999)found clear differences between A. koreana and lateabout the relationship with other genera. Pourjam et Heterodera incystmorphology, physiology and other fea- al. (2000)working with a Zygotylenchus populationfrom tures.For instance,the syncytium induced by A. koreana Iran,described a spermathecaapparently consisting of nu- differedfrom thatof Heterodera spp.because it lackedin- merouscells, i.e.,reminiscentof Meloidogyne. However, growthsof thecell walls. All these new data suggest that theirobservation was basedon Žxednematodes mounted theposition of the Afenestrata specieswithin the subfam- inglycerine. After dissectionof living specimens of Zy- ilyHeteroderinae is still far from resolved. gotylenchus itappeared that some sperm cells were re- markablyswollen (Bert, unpubl.) and they were probably Meloidodera oridensis interpretedas spermatheca wall cells by Pourjam et al. Meloidodera isgenerally recognised as expressing (2000). mostlyconserved characters for thesubfamily. A sper- mathecawith 12 cells and a shorttricolumella is remi- Globodera niscentof thatdescribed for someBelonolaimidae, Praty- Thedifferent spermatheca morphology of G. pallida lenchidaeand Rotylenchidae (Geraert, 1981;Bert & Ger- and G.rostochiensis isremarkable. The two potato cyst aert,2000). The morphology of the female reproductive nematodespecies are known to exhibit exceptionally system,based on outgroup comparisons, also suggests similarmorphologies and were, until1970, considered thatthis character is plesiomorphic. tobe pathotypes of thespecies Heteroderarostochiensis Wollenweber,1923. Both sibling species, however, were THE UTERUS shownto diverge at themolecular level (Folkertsma et al., 1994),protein proŽ le (Bakker & Bouwman-Smits,1988) Geraert (1986)pointed out the importance of the cel- andbehaviour (Mugnié ry, 1979; Den Nijs, 1992). lulararrangement of the uterus, in particular the tricol- Theseven populations in this study were investigated umella/quadricolumelladifference, for theclassiŽ cation todetermine whether the spermatheca morphology could oftheT ylenchida.Although a tricolumellais clearlythe beused as a differentiatingcharacter. Unfortunately, the basicscheme in Heteroderinae and Meloidogynina e,in inter-and intrapopulation variation of thespermatheca is some Globodera and Heterodera multiplerows ofcells toohigh to be practically useful on its own for species arefound which enlarge the uterus to a polycolumella.

Vol.4(8), 2002 961 W. Bert et al.

Atricolumellais found without exception in the fami- ofthis study. The SEM adviceof Gaë tan Borgonie is liesDolichodoridae, Belonolaimidae, Pratylenchidae and stronglyappreciated! Rotylenchidaeand in the subfamilies Heteroderinae and Meloidogyninae(Geraert, 1981;Chizhov & Berezina, 1988b;Bert, unpubl.). These taxa were collectivelygiven References asubordinalrank by Chizhov and Berezina (1988b) and thetricolumella was usedas one of thesynapomorphies, BAKKER, J. & BOUWMAN-SMIT S,L.(1988).Contrasting rates anaction supported by Siddiqi(2000). ofprotein and morphological evolution in cyst nematode Phytopathology Accordingto the literature and research on about 70 species. 78,900-904. BALDWIN,J.G.(1992).Evolution of cyst and noncyst-forming speciesof Tylenchidanot belonging to theHeteroderinae Heteroderinae. AnnualReview of Phytopathology 30, 271- andMeloidogyninae (Bert, unpubl.), the female genital 290. apparatusis composed of a smalland nearly constant BALDWIN, J.G. & SCHOUEST JR,L.P. (1990).Comparative numberof cells.Geraert (1981),based on observationson detailedmorphology of the Heteroderinae Filip’ ev & Schuur- severalnematode orders, but excluding Heteroderinae and mansStekhoven, 1941, sensu Luc et al. (1998):phylogenetic Meloidogyninae,concluded that the genital system had an systematicsand revised classiŽ cation. SystematicP arasitol- invariablenumber of cells. However, the Heteroderinae ogy 15, 81-106. andMeloidogyninae (except Meloidodera withconserved BERT, W. & GERAERT,E.(2000).Nematode species of the characters)have a largeand variable number of cellsin the orderT ylenchida,new to the Belgian nematofauna with uterus.Furthermore, in some uterus cells of Heterodera additionalmorphological data. BelgianJournal of Zoology schachtii morethan one nucleus was found,giving the 130, 47-57. CHITWOOD, B.G. & CHITWOOD,M.B.(1950). Anintroduc- impressionof anongoingcell division. These differences tionto nematology .Baltimore,MD, USA, MonumentalPress, from otherNematoda may be determinedby differencesin 213 pp. functionalrequirements such as the mode of reproduction CHIZHOV,V.N.(1981).[Some peculiarities of the structure associatedwith a sedentaryway of life. ofthe female sexual system in some species of Meloidogy- Thenumber of uterus cells in Meloidodera is limited nidaeand Heteroderidae]. Byulleten’Vsesoyuznogo Instituta andinvariable and the eggs are found in a membranous Gel’mintologii im. K.I. Skryabina 31, 66-73. structurewhich is not formed by separate cells. This CHIZHOV, V.N. & BEREZINA,N.V.(1988a).[Structure and canbe regarded as a primitiveway to accommodate the evolutionof the genital system in female nematodes of the largeegg numbers characteristic of theextremely fecund orderT ylenchida.1. Primary monodelphic species]. Zoologi- Heteroderinaeand Meloidogyninae . cheskyZhurnal 67,331-339. Evolutionof theuterus from onewith a smallnumber CHIZHOV, V.N. & BEREZINA,N.V.(1988b).[Structure and evolutionof the genital system in female nematodes of the ofcells to one with a largenumber must have arisen at orderT ylenchida.2. Primarily didelphic species]. Zoologi- leasttwice; once in the genus Meloidogyne and once in cheskyZhurnal 67,485-490. theHeteroderinae. CHIZHOV, V.N. & SWILIA M,M.(1986).[Structural peculiari- Thecellular enlargement of the uterus differs among tiesof thefemale reproductive system in somesedentary ne- thegenera studied. SpeciŽ cally, Meloidogyne , Afenestrata matodespecies of the order T ylenchida]. ZoologicheskyZhur- and Heteroderaavenae (Bidera)havea regularelongated nal 65,1788-1798. tricolumella, Heteroderaschachtii hasan elongated tri- DE LEY, P. & BERT,W.Video capture and editing as a tool columellawhich enlarges to avariablepolycolumella to- forthe storage, distribution and illustration of morphological wards thevulva while Globodera hasan elongated poly- charactersof nematodes. Journalof Nematology , in press. columellaalmost immediately proximal to the spermath- DEN NIJS,L.J.M.F. (1992).Do Globoderarostochiensis and G. pallida eca. interbreedin seminatural conditions? An examination ofthe next generation. Fundamentaland Applied Nematology 15,545-550. Acknowledgements FOLKERTSMA, R.T., ROUPPEVAN DER VOORT, J.N.A.M., VAN GENT-PELZER, M.P.E., DE GROOT, K.E., VAN DEN BOS, W.J., SCHOTS, A., BAKKER, J. & GOMMERS, F.J. Theauthorsthank Manuel Mundo-Ocampo and Renato (1994).Inter- and intraspeciŽ c variationbetween populations Inserra for theirenthusiastic supply of specimens and of Globoderarostochiensis and G. pallida revealedby Ran- theirvaluable comments. Paul and Irma De Leyare domAmpliŽ ed PolymorphicDNA. Phytopathology 84, 807- acknowledgedfor theirsplendid aid in many aspects 811.

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GERAERT,E.(1972).A comparativestudy of the structure of SIDDIQI,M.R.(2000). Tylenchidaparasites of plants and thefemale gonads in plant-parasitic T ylenchida(Nematoda). insects.2nd Edition. Wallingford,UK, CABI Publishing,864 Annalesde la Socié té Royale Zoologique de Belgique 102, pp. 171-198. SUBBOTIN, S.A., VIERSTRAETE, A., DE LEY, P., ROWE, J., GERAERT,E.(1976).The female reproductive system in De- WAEYENBERGE, L., MOENS, M. & VANFLETEREN, J.R. ladenus and Hexatylus witha redeŽnition of the oviduct in (2001).Phylogenetic relationships within the cyst-forming theT ylenchida(Nematoda). Nematologica 22,437-445. nematodes(Nematoda, Heteroderidae) based on analyses GERAERT, E. (1978). Enkelenieuwe inzichten in demorfome- ofsequences from the ITS regionsof ribosomal DNA. trieen inde morfologie van de nematoden .GhentUniversity, MolecularPhylogenetics and Evolution 21, 1-16. Belgium,Thesis, 259 pp. SZALANSKI, A.L., ADAMS, B.J. & POWERS,T.O.(1997). GERAERT,E.(1981).The female reproductive system in nema- Molecularsystematics of T ylenchidausing 18S ribosomal todesystematics. Annalesde laSocié té Royale Zoologique de DNA. Journalof Nematology 29,607-608. [Abstr.] Belgique 110, 73-86. TANDIGAN DE LEY, I., DE LEY, P., VIERSTRAETE, A., GERAERT,E.(1983).The use of the female reproductive KARSSEN, G., MOENS, M. & VANFLETEREN,J.Phyloge- systemin nematode systematics. In: Stone, A.R., Platt, H.M. neticanalysis of Meloidogyne SSU rDNA. Journalof Nema- &Khalil,L.F .(Eds). Conceptsin nematode systematics. tology, in press. London,UK, AcademicPress, pp. 73-84. TRIANTAPHYLLOU ,A.C.(1962).Oogenesis in the root-knot GERAERT,E.(1986).The use of thefemale reproductive system nematode Meloidogynejavanica . Nematologica 7, 105-113. intheclassiŽ cation of theT ylenchida(Nematoda). Revue de TRIANTAPHYLLOU ,A.C.(1985).Cytogenetics, cytotaxonomy Nématologie 9,296.[Abstr.] andphylogeny of root-knot nematodes. In: Sasser, J.N. & GERAERT,E.(1997).Comparison of the head patterns in the CarterC.C. (Eds). Anadvanced treatise on Meloidogyne; Tylenchoidea(Nematoda). Nematologica 43,283-294. VolumeI: Biologyand control .Raleigh,NC, USA, North GERAERT, E., GROOTAERT, P. & DECRAEMER,W.(1980a). CarolinaState University Graphics, pp. 113-126. Structureof the female reproductive system in some Dory- TRIANTAPHYLLOU ,A.C.(1987).Cytogenetic status of laimidaand Enoplida (Nematoda). Nematologica 26, 255- Meloidogyne (Hypsoperine ) spartinae inrelation to other 271. Meloidogyne species. Journalof Nematology 19, 1-7. GERAERT, E., SUDHAUS, W. & GROOTAERT,P. (1980b).The TRIANTAPHYLLOU , A.C. & HIRSCHMANN,H.(1962).Ooge- structureof thefemale genital apparatus in the order Rhabdi- nesisand mode of reproductionin the soybean cyst nematode, tida(Nematoda). Annalesde laSocié té Royale Zoologique de Heteroderaglycines . Nematologica 7, 235-241. Belgique 109,91-108. WERGIN,W.P. (1981).Scanning electron microscopic tech- HIRSCHMANN, H. & TRIANTAPHYLLOU ,A.C.(1968).Mode niquesand application for use in nematology.In: Zuckerman, ofreproductionand development of thereproductive system B.M.& Rohde,R.A. (Eds). Plantparasitic nematodes. V ol- of Helicotylenchusdihystera . Nematologica 13,558-574. ume III.New York,NY ,USA, AcademicPress, pp. 175-204. INSERRA, R.N., VOVLAS, N. & LEHMAN,P.S. (1999).Para- sitismof Žshpolebamboo roots by Afenestratakoreana . Ne- WOUTS,W.M.(1985). Phylogenetic classiŽ cation of the family matropica 29,105-111. Heteroderidae(Nematoda: T ylenchida). SystematicP arasitol- ogy 7, 295-328. MUGNIÉRY,D.(1979).Hybridation entre Globoderaros- tochiensis (Wollenweber)et G. pallida (Stone). Revue de Né- WU,L.Y.(1958).Morphology of Ditylenchusdestructor matologie 2, 153-159. Thorne,1945 (Nematoda: T ylenchidae),from a purecul- NAGAKURA,K.(1930).Ueber den Bau die Lebensgeschichte ture,with special reference to reproductive systems and der Heteroderaradicicola (Greef)Mü ller. JapaneseJournal esophagealglands. CanadianJournal of Zoology 36, 569- of Zoology 78, 95-160. 576. POURJAM, E., GERAERT, E. & ALIZ ADEH,A.(2000).Some WU,L.Y.(1967).Differences of spermatheca cells in the pratylenchidsfrom Iran (Nematoda: T ylenchina). Nematology genera Ditylenchus Filipjev,1936 (T ylenchidae:Nematoda). 2, 855-869. CanadianJournal of Zoology 45, 27-30. SEINHORST,J.W.(1968). Three new Pratylenchus specieswith ZUNKE, U. & EISENBACK,J.D.(1998).Morphology and adiscussionof the structure of the cephalic framework and of ultrastructure.In: Sharma, S.B. (Ed.). Thecyst nematodes . thespermatheca in this genus. Nematologica 14,497-510. London,UK, Chapman& Hall,pp. 31-52.

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