Nematology, 2008, Vol. 10(3), 405-431

Trichodoridae (Nematoda: ) from the Tara National Park, Serbia, and proposal of pseudobursatus n. sp. ∗ Wilfrida DECRAEMER 1,2, , Milan RADIVOJEVIC´ 3 and Eduardo DE LA PEÑA 4,5 1 Royal Belgian Institute of Natural Sciences, Vautierstraat 29, 1000 Brussels, Belgium 2 Department of Biology, Nematology, Ghent University, Ledeganckstraat 35, 9000 Ghent, Belgium 3 Plant Protection Institute, Faculty of Agriculture, Nemnjina 6, 11080 Beograd, Serbia 4 Institute for Agricultural and Fisheries Research, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium 5 Department of Biology, Terrestrial Ecology Unit, Ghent University, Ledeganckstraat 35, 9000 Ghent, Belgium Received: 13 August 2007; revised: 12 November 2007 Accepted for publication: 12 November 2007

Summary – A survey for plant-root parasites in Tara National Park, Serbia, revealed the widespread occurrence of , present in nearly one-third of the samples. Several species appeared to belong to the Trichodorus aequalis species complex sensu Loof (1973). The taxonomic value of the observed variations in diagnostic features within this species complex is discussed. A new species Trichodorus pseudobursatus n. sp. is described. It is characterised by males with a nearly straight habitus, two ventromedian cervical papillae anterior to the secretory-excretory pore, medium-sized and mostly straight spicules with wider manubrium continuous with the blade; blade with a smooth narrower part with a few bristles at mid-spicule, followed by a wider striated posterior part devoid of bristles. Females are characterised by a large, well developed pear-shaped vagina with medium-sized, rounded triangular and well separated vaginal sclerotised pieces in lateral optical view. In addition to the diagnostic morphological features, differentiation of the new species from other Serbian morphotypes of the Trichodorus sparsus species complex was supported by canonical discriminant analyses (CDA). Additional information is provided for T. sparsus and three T. aff. sparsus morphotypes from Serbia. The discovery of the genus Trichodorus in Serbia represents a new geographical record. Keywords – canonical discriminant analysis, description, morphology, morphometrics, , Trichodorus aequalis species complex, Trichodorus sparsus.

Serbia occupies a central position within the Balkan They represent the first records of trichodorid species Peninsula, a world centre of biodiversity. Its nematofauna, from Serbia. Taxonomic analysis revealed the presence however, is poorly known and since Serbia is mainly an of Trichodorus species belonging to the Trichodorus agricultural country, focus lies on plant-parasitic nema- aequalis complex sensu Loof (1973). Identification to todes. From 2002 until the present time, Milan Radivo- species level is difficult. Populations appear rather similar, jevic´ carried out a survey of plant-root parasitic nema- although showing small differences in diagnostic morpho- todes in the Tara National Park, the most westward of logical features and measurements, the extremes of which all national parks in Serbia. It is very diverse in habitat suggest different species when compared with intra- and and extends over a surface of about 20 000 ha at 1000 m interspecific ranges known within the genus. Loof (1973) a.s.l. mean altitude. Tara National Park is recognised as a sanctuary for wildlife and accommodates numerous rare mentioned that species of the T. aequalis complex (at that and/or endemic species as it escaped quaternary glaciation time T. aequalis Allen, 1957, T. sparsus Szczygiel, 1968 and was located near the tertiary Panonian Sea (Miocene). and T. hooperi Loof, 1973) are more variable in morpho- Even today, the park area has a mild, humid, continental logical and morphometric features compared to other Tri- climate, supporting rich vegetation, primarily forests. chodorus species (for example in stylet and spicule length, So far, populations of trichodorid species have been variation in number of precloacal supplements, position of found in 14 (nearly one-third) of the localities sampled. ventromedian cervical papillae in relation to the secretory-

∗ Corresponding author, e-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2008 405 Also available online - www.brill.nl/nemy W. Decraemer et al. excretory pore and distance between them and the vagina Materials and methods shape in female). Until now, taxonomic papers hardly ever make an ex- The whole territory of Tara is situated within the UTM plicit statement of the species concept used, which means grid CP quadrant of 100 × 100 km; codes 66-86 refer that the hypothesis ‘a new species’ cannot be tested (Dod- to 10 × 10 km unit quadrants, while the letters A to Z son & Lee, 2006). Without knowing if the author(s) used, of the code refer to the 25 sample stations in the 2 × 2 for example, the biological, morphological or phyloge- km sampling units. Habitats sampled include forests, pre- netic species concept, citing the most commonly used dominantly mixtures of spruce, fir and beech, as well as ones, we have no precise idea of what the author meant meadows and transition zones. Single point samples were by ‘species’ and we cannot test the boundaries of the taken, using a spade, per 4 km2 unit, from all over the ter- designated species. Barely distinguishable morphological ritory, as a block of about 1 kg soil from up to 20 cm groups, such as recently separated sibling species, can depth. were extracted by sieving the super- show clear genetic divergence and reproductive isolation natant of soil mixed with water, followed by the Baermann yet the reverse also occurs. Another confounding problem funnel technique, hot-fixed with TAF (Courtney et al., in the use of morphological characters is that they can be 1955), then slowly transferred to glycerin prior to mount- affected by phenotypic plasticity in response to environ- ing on glass slides. Morphometrics were obtained using mental factors, as well as by morphological stasis. an Olympus BX50 compound microscope equipped with To obtain a better understanding of the taxonomic value image-capture software. Drawings and photographs were of the observed differences in diagnostic features within made with a Reichert Polyvar microscope using interfer- this group and to be able to understand what the author(s) ence contrast. Morphological data of females and males considered to be a species, the T. aequalis species group from all populations were analysed using a forward step- will be discussed, based upon the study of type specimens, wise canonical discriminant analysis (Genstat 8.0). An voucher material and Serbian populations. In a later stage, unidentified juvenile specimen of Trichodorus was found molecular data will be added. at Barski dol location 76N: mixed forest of mainly spruce Although species of the T. aequalis species complex and beech forest on limestone. are gonochoristic, mating experiments to test reproductive isolation between putative species are hardly realistic; OTHER MATERIAL STUDIED mixed populations are common and trichodorids are difficult to culture on agar plates. In Trichodoridae, the Trichodorus aequalis, specimens from Alaska, courtesy morphological species concept is currently the concept in E. Bernard. vogue. Consequently, species are defined on the basis of T. coomansi De Waele & Carbonell, 1983, holotype particular essential features, whereby there is no proof of male, slide RUG284, paratypes, slide RUG285, RUG288, gene flow but morphological distinctiveness is considered Collection, Ghent University, Ghent, Belgium. as a surrogate to lineage independence. Moreover, testing T. giennensis Decraemer, Roca, Castillo, Peña-Santiago the hypothesis of a new species using the morphological & Gomez-Barcina, 1993: paratype specimens, slides species concept is easier than testing new species based RIT397, RIT417 from the Nematode Collection of the on the phylogenetic species concept, where it is often Royal Belgian Institute of Natural Sciences, Brussels, difficult to determine the polarity of the characters, several Belgium. features being probably convergent. T. hooperi: paratypes, slides WT1627, WT1632, WT1635 This contribution presents an overview of the T. ae- from the Nematode Collection of the Agricultural Univer- qualis species complex based on data from the litera- sity of Wageningen, courtesy P. Loof. ture, type material and other specimens. A new species, T. orientalis De Waele & Hashim, 1984, holotype female, Trichodorus pseudobursatus sp. n., is described and il- slide RUG269-272, paratypes, slides RUG 269, RUG 273 lustrated. Additional support for its differentiation from from the Nematode Collection, Ghent University, Ghent, closely related species based on morphometric data is Belgium and voucher specimens from the Nematode substantiated by canonical discriminant analyses. Three Collection of the Royal Belgian Institute of Natural different morphotypes of T. aff. sparsus from Serbia are Sciences, Brussels, Belgium. described and additional data are given for Trichodorus T. parorientalis Decraemer & Kilian, 1992, paratypes, sparsus. slides RIT378, RIT 379 from the Nematode Collection of

406 Nematology Trichodoridae from Serbia the Royal Belgian Institute of Natural Sciences, Brussels, recorded T. aequalis from the rhizosphere of Rhododen- Belgium. dron obtusum from Japan but the author could not con- T. paucisetosus Bernard, 1992, paratypes, courtesy firm if the species is native to Japan. He also described a E. Bernard. new species, T. tricaulatus, which he claimed belonged to T. sparsus: paratype specimens, slide WT1643, WT1645, the T. aequalis species group. A few years later, another WT 1647 and voucher specimens from Oosterbeek (slide three species were added to the T. aequalis species group: 6878), Terschelling (slide 5520 c49-50), Wieringermeer T. coomansi, associated with the rhizosphere of several (slide 5400), The Netherlands and from Bodenrod (slides plants (e.g., Poaceae, Ericaceae, Lobeliaceae) from Mount 4862, 7031), Hondsdorf (slides 7013, 7014), Königstädten Kenya, T. orientalis from Jordanian lowlands in associa- (slide 7032), Germany and Tekirdog (slide 6825), Turkey, tion with Phaseolus, Lycopersicon and grapevines, and T. from the Nematode Collection of the Agricultural Uni- parorientalis from roots of grasses in Eastern Transvaal, versity of Wageningen, courtesy T. Bongers and P. Loof, South Africa. More recently, T. paucisetosus from forests and voucher specimens from several localities in Belgium in Alaska was described as morphologically similar to T. from the Nematode Collection of the Royal Belgian Insti- sparsus, while T. variabilis recorded from the rhizosphere tute of Natural Sciences, Brussels, Belgium. of artichoke from Greece was described as closely resem- T. taylori De Waele, Mancini, Roca & Lamberti, 1982, bling T. giennensis and T. sparsus. paratype specimens, slides RUG695-699 from the Nema- Within Europe, of the five Trichodorus species be- tode Collection, Ghent University, Ghent, Belgium. longing to the T. aequalis species group, four show a T. tricaulatus Shishida, 1979, paratype specimens, slides restricted distribution: T. giennensis (Spain), T. hooperi RUG3528-3529 from the Nematode Collection, Ghent (England), T. taylori (Northern Italy) and T. variabilis University, Ghent, Belgium. (Greece), whilst T. sparsus appears widespread, especially T. variabilis Roca, 1998, paratype specimens from the in central Europe where it is very common. To date, T. Nematode Collection of the Instituto di Nematologia sparsus has been recorded from Austria, Belgium, Bul- Agraria, C.N.R. Bari, Italy, courtesy F. Roca. garia, France, Germany, Hungary, Italy, The Netherlands, Poland, Romania, Sweden, Switzerland, UK and Turkey (European part). Of all European records, T. sparsus oc- The Trichodorus aequalis species group curred most frequently in the Slovak Republic, i.e., 58% of all trichodorid records (Liskova & Sturhan, 1999). Tri- SPECIES DISTRIBUTION chodorus sparsus occurs in different types of soil from sandy to loamy clay, but is prevalent in light soils. In Slo- Loof (1973) considered the distribution of Trichodorus vakia, as in most records from other countries, the species species in western and central Europe to be well under- is mostly associated with forests of coniferous and decid- stood apart from the T. aequalis–group. Based upon the uous trees but also occurs in soils of riverbanks with trees study of type material he could differentiate the American and sporadically in arable soils, e.g., in association with species T. aequalis from the European species T. spar- potato, sugar beet, onions, and in the rhizosphere of straw- sus and he agreed with Wyss (1970) that T. aequalis re- berry and grapevine (Decraemer, 1995). ported from Germany belongs to T. sparsus. Loof (1973) added to this group a new species, T. hooperi from Eng- HOW TO CHARACTERISE THE T. AEQUALIS SPECIES land, currently considered to be endemic to England (Tay- GROUP? lor & Brown, 1997). Two records of T. hooperi from out- side the UK, respectively from Florida (Mead, 1986) and According to Loof (1973) the main diagnostic features from China (Ye, 1994), need confirmation. The record of of this species group (three species at that time) were T. aequalis from northern Italy (Mancini et al., 1979) sub- a three-layered cuticle, males with two ventromedian sequently appeared to refer to a mixture of a new species, cervical papillae (CP) anterior to the secretory-excretory T. taylori, and T. sparsus (De Waele et al., 1982). Tri- pore (S-E pore) and three precloacal supplements (SP) chodorus aequalis, recorded from Spain in Peña-Santiago with the posterior one (SP1) near the manubrium of the et al. (1987), was later described as T. giennensis.The retracted spicule, and females with two pairs of lateral record of T. sparsus from a forest nursery in Great Britain body pores (one postadvulval and one prevulval, though could possibly represent an introduction with imported the latter may be absent on one side or both sides), vagina plants and soil (Alphey & Boag, 1976). Shishida (1979) often barrel-shaped and sclerotised pieces rather small.

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As mentioned previously, 11 species have been at- ornamentations (bristles, striae), sperm structure (used tributed to the T. aequalis species group: T. aequalis, T. only to differentiate T. variabilis from T. giennensis), coomansi, T. giennensis, T. hooperi, T. orientalis, T. paror- terminal tail cuticle with or without thickening and degree ientalis, T. paucisetosus, T. sparsus, T. taylori, T. tricaula- of thickening and habitus of posterior body region (degree tus and T. variabilis. The deciding characters for assigning of ventral curvature and presence/absence of a dorso- a species to the T. aequalis species group differ accord- ventral flattening resulting in a bursa-like structure). ing to author. In the male, emphasis is put on one feature or a combination of the following features: i) the num- Female ber of ventromedian cervical papillae, i.e., two CP (except Number and position of lateral body pores in relation to T. tricaulatus with three); ii) the position of the ventro- vulva, vulva shape in ventral view (a pore, except for T. median cervical papillae, i.e., mostly anterior to the S-E hooperi which has a transverse slit), shape and length of pore (except in T. coomansi and T. variabilis where mostly vagina in relation to corresponding body diam. and size, in between CP1 and CP2 or also posterior to them in T. shape of vaginal sclerotised pieces and distance between variabilis); iii) number of precloacal supplements (three them in lateral optical section. in all species of the group, though exceptions of four or two occur); and iv) position of SP1 in relation to retracted spicule (used in all species but in most situated at level of EUROPEAN SPECIES OF THE T. AEQUALIS COMPLEX manubrium) or v) spicule shape (slightly ventrally arcuate Interspecific differentiation is often based on: i)small with widened manubrium and distal tip of lamina bent; differences of a few morphological characters, some of distal half of spicules striated or not and provided or not which appear rather variable; and ii) differences in mor- with delicate bristles). In female, emphasis is either on: i) number of lateral body pores, mostly two; or ii)small phometric data, the latter often with overlapping ranges, size of vaginal sclerotised pieces in lateral optical view. more so when dealing with more than one population per Based on this list of characters, we can already deduce species (Tables 1, 2). that T. taylori, with a different spicule shape with long Intraspecific differences are often subtle and may result sharply offset manubrium and large vaginal sclerotised from differences in condition of fixed specimens or be due pieces, and T. tricaulatus, with three CP, do not fit in the T. to morphological plasticity (variation within and between aequalis species group. However, several of these ‘diag- populations). For example, in male spicule shape the nostic’ features are present in about 50% of the species width of the manubrium and width of the blade may of the genus. For example, nearly 50% of Trichodorus appear slightly different between specimens/populations, species possess two CP (always present in 22 species and especially when dealing with differences in spicule length. occasionally so in five species) and in nearly all of these Also, the presence or absence of a slightly narrower part at species (except three) the S-E pore is located posterior about mid-spicule level is subtle since a minor narrowing to the ventromedian cervical papillae. Further, all of the can often be observed at the level of the posterior border of ‘two CP species’ possess three ventromedian precloacal the suspension muscles capsule in Trichodorus species in supplements and two-thirds of them have the posterior- general and may be due to the impact of contraction of the most precloacal supplement (SP1) located at the level of protractor muscles on the spicule. Presence or absence of the manubrium of the retracted spicules. ornamentations, such as bristles and/or transverse striae, may be biased by the condition of the fixed specimens, DIAGNOSTIC MORPHOLOGICAL FEATURES WITHIN e.g., T. variabilis spicules were described with very fine THE T. AEQUALIS SPECIES GROUP AND INTRASPECIFIC transverse striations at distal third of spicule but the VARIABILITY WITH FEATURES FROM LOOF (1973) IN paratype specimen examined appeared smooth even at × ITALICS 2000 magnification (Fig. 5K). Moreover, ‘bristles’ are in reality not bristles at all but the edges of scale- Male like structures that are more or less divergent from the Position of CP1 and CP2 in relation to S-E-pore and blade core (Rodriguez-M. & Bell, 1978; observations, to one another, number of SP and position of SP1 in first author). Further, the degree of curvature of the relation to retracted spicule, spicule shape, i.e., with or spicules may be influenced by fixation, contraction of the without a narrower blade portion and degree of spicule protractor or retractor muscles and degree of bending of curvature, presence and location or absence of spicule the posterior body region (see Loof, 1973).

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Table 1. Comparison of male diagnostic features of species of the Trichodorus aequalis complex from Europe. Character T. pseudobursatus T. giennensis T. hooperi T. sparsus T. taylori T. variabilis n. sp. Spicule – Wider head, – Wider head, – Wider head, – Wider head, – Head long, – Head wide, shape blade slightly blade slightly blade often blade about wide and sharply gradually narrower at narrower at slightly narrower equally wide, offset from merging with an mid-spicule, mid-spicule at mid-spicule. posteriorly narrower equally wide tapered tapered slightly tapered calamus, wider blade tapering to posteriorly to posteriorly to to bifid tip. blade. a sharp distal bifid tip. bifid tip. end. – Spicule slightly – Spicule slightly – Spicule almost – Spicule mostly – Spicule slightly –Spicules curved to about curved straight largely straight curved ventrally curved straight and only but curved at curved anteriorly extremities or slightly ventrally curved Spicule or- Posterior half Posterior half Finely striated, Finely striated, Blade finely Distal third with namentation finely striated; a finely striated; no bristles bristles present transverse fine transverse few bristles at usually two pairs over large part of striated; no striae; no bristles mid-spicule level of bristles at blade (posterior bristles mid-spicule level 2/3rd) Tail end Cuticle clearly Variable Thickened Thickened More or less Drop-like cuticle thickened (variable degree, thickened thickened cuticle rarely absent) Precloacal With thin bursa No subventral Asymmetrical, Ventrally Asymmetrical No subventral region thickening bursa-like concave, thickening bursa-like Number SP 3 (exc. 2, 4) 3 (exc. 4, 5) 3 (exc. 4) 3 (exc. 2, 4) 3 3 Position At spicule head to Around spicule At spicule head At spicule head At spicule head At level spicule SP1 in along anterior head (just to opposite to opposite to opposite head relation to 1/5th spicule anterior to just anterior 1/5th anterior 1/5th anterior 1/5th spicule posterior) spicule spicule length spicule length Number CP 2 2 (exc. 1) 2 2 (exc. 1) 2 2 (exc. 1, 0)

Description of Serbian Trichodorus species and DESCRIPTION morphotypes Male Trichodorus pseudobursatus* n. sp. Body largely cylindrical and nearly straight. Body (Figs 1-4) cuticle usually slightly swollen, more so dorsally than ventrally (4-7 µm). Lip region rounded, marked by four slightly raised sets of double papillae composed of a MEASUREMENTS cephalic papilla and a subdorsal or subventral papilla of outer crown of labial papillae. Amphidial aperture a large See Tables 3-6. transverse slit located just posterior to lip region and fovea cup-shaped. Stoma small; strengthening rods (4-6 *Species name refers to the presence of a narrow, non-functional, µm) clearly visible. Pharyngostom with ventrally curved bursa in male. onchiostyle, with onchium ca as long as onchiophore

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Table 2. Comparison of female diagnostic features species of the Trichodorus aequalis species complex from Europe. Character T. pseudobursatus T. giennensis T. hooperi T. sparsus T. taylori T. variabilis n. sp. Vagina shape More or less Barrel (relaxed), Pear-shaped Barrel to Rhomboid to Barrel to pear-shaped cylindrical cylindrical pear-shaped cylindrical Shape Rounded Variable: small Round Broad rounded Large round Triangular vaginal triangular to oval triangular or triangular (oval) sclerotised rounded pieces Vaginal Medium-sized, Minute Medium-sized, Small (mean Medium-sized Small, sclerotised 3-4.5 µm; (variable); 5 µmapart 2.5 µm) but (3 µm); 3 µm 0.9-1.1 µm pieces 2.5-4 µmapart 2.3 µmapart variable apart apart Length 40-50% 40-50% ca 40% Variable, relaxed ca 50% 41% (37-46) vagina as % ca 40% of corresp. body diam. Number of 2 (1 postadvulvar, 1 (type)or 1-2 1 postadvulvar 2 (1 anterior, 1 1 postadvulvar 1 postadvulvar lateral body 1anteriorto postadvulvar) or pores vulva) anterior one absent on one or both sides Shape vulva Pore-like to wider ? Transverse slit Pore-like Round, not Pore-like round pore-like support. Slender mid-part of pharynx gradually widening gradually tapering and continuous with blade; blade posteriorly to a glandular bulb; only dorsal and both narrower at about mid-spicule (in retracted spicules at posterior ventrosublateral gland nuclei visible in TAF- level of posterior border of capsule of suspensor muscles) fixed specimens. Pharyngeal bulb offset except for a short and provided with a few bristles; posteriorly blade with (7 and 11 µm) anterior dorsal overlap of intestine over fine transverse striae except for distal end where tapering pharyngeal bulb in two specimens. Nerve ring in general slightly to a bifid tip. Gubernaculum fine, with thickened located close to base of pharyngostom, but varying from keel. Three ventromedian precloacal supplements (SP); level base pharyngostom to 11.5 µm posterior. Two in type population (85R) three out of ten specimens distinct ventromedian cervical papillae (CP) present; both with four SP and in population from 86G one specimen located anterior to S-E pore. Anteriormost CP1 may with only two precloacal supplements. Posterior two be situated opposite base of pharyngostom (rarely just SP papilliform, anteriormost precloacal supplement (two anterior to it), but mostly at a short distance (less than anteriormost supplements when four SP) smaller, not 10 µm) posterior. Distance between both ventromedian protruding. Copulatory muscles of medium development. cervical papillae (CP1 and CP2) ranging between 16 Posteriormost precloacal supplement (SP1) located at and 23 µm; posterior CP2 at level of S-E pore or level of retracted spicule or up to 14 µm(ca one-fifth of at short distance (up to 10 µm; exceptionally 18 µm spicule length) posterior to tip of manubrium. One pair of in one specimen). Lateral cervical pore on both sides, small subventral postcloacal papillae located just posterior posterior to nerve ring (rarely opposite) and between CP1 to cloacal opening and flanked by protruding and CP2 (rarely at level of CP1). Single testis reaching cuticular flap of a thin bursa. Bursa or caudal alae weakly far anteriorly to within 44 µm of pharyngo-intestinal developed but clearly visible, i.e., protruding beyond junction. Sperm cells with large sausage-shaped nucleus. ventral wall and extending from level SP1 to subterminal Spicule anteriorly and posteriorly ventrally curved with on tail. Pair of caudal pores present just posterior to mid-region more or less straight. Manubrium widened, postcloacal papillae. Tail very short, ventrally flattened

410 Nematology Trichodoridae from Serbia

Fig. 1. Trichodorus pseudobursatus n. sp. A: Female paratype, entire body; B: Anterior genital branch, paratype female; C: Male holotype, neck region; D: Male holotype, posterior body region; E-G: Male paratypes, tail region and copulatory apparatus; H, I: Female paratypes, vaginal region. (Scale bars = 20 µm.)

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Fig. 2. Trichodorus pseudobursatus n. sp., light micrographs. A: Male from Raˇca, entire body; B: Holotype male, posterior body region and female paratype reproductive system; C: Vaginal region in female from Orlovac; D: Moulting juvenile male showing male copulatory apparatus (specimen from Orlovac). (Scale bars = 20 µm.)

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Fig. 3. Canonical discriminant analysis for Trichodorus species isolated from the Tara National Park, Serbia, (A) for females and (B) males, carried out with four morphometrical characters (see Table 1). ()representsTrichodorus pseudobursatus n. sp.; (*) T. aff. sparsus morphotype 1; () T. aff. sparsus morphotype 2; (◦) T. aff. sparsus morphotype 3.

Fig. 4. Trichodorus pseudobursatus n. sp., juveniles from Orlovac. Scatter diagram plotting body length and length of genital primordium, with indication of different juvenile stages.

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Table 3. Morphometric data for male Trichodorus pseudobursatus n. sp. from type locality and other localities. Measurements are in µm and in the form: mean ± s.d. (range). Parameter Ladjevac Racaˇ Orlovac Holotype Paratypes n–574 L 725 694 ± 48.8 (615-760) 743 ± 51.4 (685-810) 768 ± 18.5 (755-795) a 19.1 16.7 ± 1.8 1 (3.8-19.1) 18.3 ± 1.4 (16.25-20.53) 16.6 ± 0.59 (5.9-17.27) b4.64.6± 0.4 (3.9-5.0) 4.9 ± 0.3 (4.5-5.3) 4.4 ± 0.06 (4.4-4.5) c 76.3 60.3 ± 10.6 (48.2-76.3) 65.3 ± 6.2 (54.8-72.3) 60.6 ± 11.7 (50.3-72.2) T 66.7 58.6 ± 7.3 (46.6-65.9) 69.0 ± 3.3 (63.5-74.0) 65.0 ± 1.3 (65.4-66.5) Onchium 31 30 ± 1.4 (28-32) 28 ± 0.5 (28-29) 31 ± 1.9 (28-32) Onchiostyle 57 59 ± 2.1 (56-62) 54 ± 0.8 (53-55) 65 ± 1.63 (63-67) Pharynx length 158 152 ± 4.7 (147-158) 152 ± 6.8 (139-160) 173 ± 4.5 (167-178) Pharyngostom 68 70 ± 1.5 (68-72) 67 ± 2.2 (62-69) 78 ± 1.0 (77-79) Ant. to nerve ring 71 73 ± 1.89 (71-75) 68 ± 5.1 (60-76) 82 ± 4.04 (79-88) Anterior to CP1 89 79 ± 6.6 (70-89) 70 ± 4.5 (65-78) 77 ± 6.2 (72-86) Distance CP1-CP2 21.5 20 ± 2.7 (16-23) 19 ± 1.6 (17-21) 20 ± 2.9 (17-23) Dist. CP2-S-E pore 9 6.0 ± 2.4 (2.5-9) 10.2 ± 4.7 (6-19) 7.2 ± 1.04 (6-8.5) Ant. to lateral pore 90 87 ± 6.4 (79-96) 76 ± 3.9 (72-82) 87 ± 6.1 (80-94) Ant. to guide ring 27 26 ± 0.7 (25-27) 26 ± 1.5 (25-29) 27 ± 0.7 (27-28) Length pharyngeal bulb 38 42 ± 4.04 (37-48) 43 ± 3.7 (37-48) 44 ± 4.1 (40-49) Diam. pharyngeal bulb 19 19 ± 1.2 (18-21) 18 ± 1.4 (17-21) 21 ± 1.0 (20-22) Body diam. at cardia 35 36 ± 0.9 (35-37) 37 ± 2.3 (34-41) 41 ± 1.3 (40-43) Mid-body diam. 38 42 ± 4.8 (38-49) 41 ± 2.4 (38-44) 46 ± 2.6 (44-50) Anal body diam. 22 23 ± 1.2 (22-25) 23 ± 1.9 (20-25) 27 ± 1.2 (26-28) Distance cloacal opening to SP1 34 35 ± 2.4 (33-40) 36 ± 5.7 (30-47) 35 ± 3.9 (31-39) Distance SP1-SP2 47 46 ± 6.6 (38-55) 47 ± 3.2 (43-52) 47 ± 7.9 (38-57) Distance SP2-SP3 47 49 ± 6.8 (39-58) 53 ± 3.7 (48-58) 53 ± 6.1 (47-58) Distance SP3-SP4 – – 34 ± 13.6 (20-47)* – Distance SP1 spicule head** 3 0-6.5 0-14 3-7 Anterior end to testis 220 260 ± 63.7 (216-383) 218 ± 23.4 (195-253) 256 ± 5.4 (250-263) Spicule length 56 56 ± 3.2 (51-61) 54 ± 4.1 (48-59) 59 ± 1.7 (58-62) Gubernaculum 26 26 ± 2.1 (24-29) 24 ± 1.9 (21.5-27) 27 ± 1.5 (25-28) Tail length 9.5 12 ± 1.8 (9.5-14) 11 ± 0.8 (10-12.5) 13 ± 2.3 (11-15) Tail tip diam. 7.5 7.3 ± 0.3 (7-7.5) 7.9 ± 0.7 (7-8.5) 8.6 ± 1.0 (7.5-10) Number of CP 2 2 2 2 Number of SP 3 3 (2) 3 (*) 3

*(n = 3); ** at level of spicule head (‘0’) to posterior to it. and provided with caudal alae; terminal cuticle posterior well developed, more or less pear-shaped and surrounded to caudal pore clearly swollen. by well developed constrictor muscles. Vagina extending Female for ca 40-50% of corresponding body diam. Vaginal sclerotised pieces in lateral optical section medium-sized Body largely straight or slightly curved. Digestive (3.5 µm at largest mean diam.), more or less rounded system as in male. Reproductive system didelphic-amphi- delphic with reflexed ovaries. At tip of flexure, a narrow triangular to oval and tips clearly separated (ca 4 µm ovarial sac occasionally observed when only few oocytes apart in type population). Vulva shape pore-like to wider are present. Two large, finely granular, oviduct cells rounded in ventral view; may appear with star-like border. enveloping neighbouring part of uterus (= spermatheca); One pair of post-advulvar sublateral body pores present at sperm present in spermathecae. Uteri rather short. Vagina ca half corresponding body diam. from vulva level. One

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Table 4. Morphometric data for female Trichodorus pseudobursatus n. sp. from type locality and other localities. Measurements are in µm and in the form: mean ± s.d. (range). Parameter Ladjevac Orlovac Racaˇ Paratypes n523 L 725 ± 68.9 (550-650) 715; 870 686 ± 16.0 (675-705) a 16.2 ± 1.5 (14-18) 16.6; 16.1 16.0 ± 0.9 (15-16.6) b4.2± 0.4 (3.61-4.70) 4.2; 5.0 4.5 ± 0.3 (4.2-4.7) V 58.9 ± 3.0 (56-63) 56.4; 43.0 55.1 ± 1.9 (53.8-57.19) G1 35.9 ± 5.6 (27-41) 30.8; 34.5 36.5 ± 1.9 (33.9-37.4) G2 35.6 ± 4.2 (31-34) 33.4; 33.7 38.4 ± 1.1 (37.2-39.3) Onchium 30 ± 1.0 (29-31) 33 28 Onchiostyle 59 ± 1.6 (57-61) 68 55.3 ± 0.6 (55-56) Pharynx length 157 ± 3.7 (152-161) 172; 175 154 ± 7.5 (150-163) Pharyngostom 70 ± 2.2 (67-72) 78; 79 66 ± 2.1 (64-68) Ant. to nerve ring 74 ± 2.6 (71-77) 80; 84 74 ± 1.7 (73-76) Ant. to S-E pore 98 ± 7.7 (92-110) 107; 110 94 ± 4 (93-101) Ant. to guide ring 25 28; 27 26 ± 0.6 (26-27) Length pharyngeal bulb 46 ± 1.8 (45-49) 46; 49 42 ± 3.5 (39-44) Diam. pharyngeal bulb 18 ± 0.5 (18-19) 21; 25 18 ± 1.2 (17-19) Body diam. at cardia 37 ± 0.3 (36-38) 43; 46 38 ± 0.6 (38-39) Body diam. at vulva 40 ± 1.2 (39-42) 43; 54 43 ± 2.0 (41-45) Length ant. genital branch to flexure 170 ± 29.3 (143-217) 159; 210 175 ± 3.5 (173-179) Length anterior ovary 61 ± 6.5 (53-70) 61; 90 73 ± 14.4 (56-81) Distance anterior to vulva 382 ± 28.9 (349-416) 403; 374 378 ± 8.5 (369-386) Length posterior genital branch to flexure 156 ± 2.6 (153-159) 169; 205 171 ± 2.1 (169-173) Length post. ovary 73 ± 14.11 (64-97) 70; 88 93.33 ± 1.5 (92-95) Length vagina 18 ± 2.0 (16-21) 18; 19 18 ± 1.5 (16-19) Width vagina 16.4 ± 0.9 (15-17) 17; 20 14.3 ± 0.6 (14-15) Distance vulva to ant. lateral body pore 76 ± 35.8 (33-118) 118; 154 93 ± 3.5 (91-96) Distance vulva to post. lateral body pore 21.4 ± 1.2 (20-23) 22; 18 18.7 ± 2.5 (16-21) Vagina as % of mbd 43.3 ± 2.9 (40-48) 41.9; 34.2 43.0 ± 4.5 (39-48) pair of anterior sublateral body pores (rarely advulvar), based upon body length and degree of development of the mostly about 2-3 vulvar body diam. anterior to vulval reproductive system (Fig. 4). In moulting specimen from level. Anus subterminal; caudal pores subventral, just first to second developmental stage, cuticle clearly sepa- posterior to anus; tail minute. rated at both extremities but no break into cuticle at level of base onchiostyle had yet occurred. Second-stage juve- Juveniles nile with organs completely formed. No shed onchium ob- From the type population a single third-stage male served in intercuticular space in the head region. juvenile was recovered. Body 590 µm long, habitus similar to female. Onchiostyle = 53 µm, reserve onchium TYPE LOCALITY AND HABITAT = 19 µm. Genital primordium, 44 µm long, and spicular Ladjevac, location 86 G, soil between limestone rocks primordium present. in predominantly coniferous forest on limestone cliff. In the population from Orlovac, three different juve- nile stages (fourth, third, second) were observed and two OTHER LOCALITIES moulting specimens (one towards adult male and one from first- into second-stage). For measurements see Table 5. Raca,ˇ alt. 500 m a.s.l., location 86 L, mixed vegetation Different juvenile stages can be clearly differentiated including conifers, Crataegus sp., Rosa sp., Corylus

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Table 5. Morphometric data for juvenile stages of Trichodorus pseudobursatus n. sp. from Orlova, Serbia. All measurements in µm and in the form: mean ± s.d. (range).

Parameter J4 J3 J2 Moult J1-J2 n1431 L 615 519 ± 18 (450-585) 416 ± 53 (390-465) 240* Onchium 22 23.5 ± 0.7 (23-24) 20 ± 2.1 (18-22) 21.5 Onchiostyle 50 48 ± 0.4 (74-51) 45 ± 3.5 (43-48) 41.5 Pharynx length 144 133 ± 8 (130-139) 120 ± 5.7 (115-127) 120 Pharyngostom 65 57 ± 2.0 (55-60) 51 ± 5.3 (45-58) 49 Ant. to nerve ring 74 59 ± 0.7 (56-61) 53 ± 5.0 (50-57) 50 Ant. to S-E pore 97 89 ± 5.0 (84-96) 80 ± 6.4 (75-87) 70 Ant. to guide ring 21 18 ± 3.2 (15-21.5) 19 ± 5.0 (15-22) 18 Length pharyngeal bulb 47 39 ± 4.2 (36-42) 35 ± 2.1 (30-39) 44 Diam. pharyngeal bulb 19 15 ± 0.4 (13.5-15.5) 14 ± 0 (13-15) 13 Body diam. at cardia 38 31 ± 2.8 (29-33) 26 ± 2.5 (24.5-28) 24 Body diam. at mid genital primordium 43 32 ± 2.8 (30-35) 26 ± 2.5 (24.5-28) 24 Length genital primordium 174 34 ± 1.8 (26-38.5) 17.5 ± 12.4 (10-30) 10 *Length of J2.

Table 6. Standardised coefficients for canonical variates for DIAGNOSIS AND RELATIONSHIPS adult males and females of Trichodorus species from Serbia. Trichodorus pseudobursatus n. sp. is characterised by Females Males a nearly straight habitus in male, a medium-sized body Root 1 Root 2 Root 1 Root 2 length (615-810 µm in male; 550-870 µm in female) and rather long onchiostyle (53-67 µm in male; 55- Variation (%) 86.83 9.82 69.27 30.63 68 µm in female). Males possess a weakly developed Selected characters Vector loadings Vector Loadings V −0.1881 −0.1246 bursa, two ventromedian cervical papillae (CP) anterior to Body length 0.0032 0.0043 0.00422 0.01052 the secretory-excretory pore (EP), the anteriormost CP1 Sclerotisation −1.7242 1.8065 close to base of the pharyngostom, the posterior CP2 at Stylet length −0.0571 −0.0014 −0.05631 0.05371 a short distance from secretory-excretory pore. Spicules, Spicule length −0.29593 0.08099 medium-sized (48-62 µm), mid-region largely straight, Nerve ring 0.04134 0.05327 manubrium wider, continuous with blade; blade with a few bristles at slightly narrower portion at mid-spicule; posteriorly blade finely striated except at extremity, distal tip bifid. Three ventromedian precloacal supplements (SP) avelana, Rubus sp., on limestone and Orlovac, alt. 800 m (less common four or rarely two); posteriormost SP1 a.s.l., location 85 R, meadow bordered by Alnus trees at level of head retracted spicule or shortly posterior close to the Matijaševica river, on serpentine soil. to it. Females have a large, well developed, more or less pear-shaped vagina (40-50% of corresponding body diam.), vaginal sclerotised pieces in lateral optical section TYPE MATERIAL medium-sized (3-4.5 µm), rounded triangular with tip clearly separated (2.5-4 µm), one pair of postadvulvar Holotype male (slide RIT725), four male and three fe- sublateral body pores and one prevulval pair at 3-4 body male paratypes (slides RIT725-728) deposited in the Ne- diam. from vulva level. matode Collection of the Royal Belgian Institute of Nat- The code in the polytomous key of Decraemer and ural Sciences, Brussels; one male, three female and one Baujard (1998) is as follows: for male: A = 210 (or 212: juvenile paratype (slides UGMD104109-UGMD104110) mean, min max), B = 23, C = 23, D = 2, E = 0(1),F= deposited in the Nematode Collection of Ghent Univer- 34 (2), G = 11, H = 22, I = 13, J = 23, K = 33, L = 44, sity, Department of Biology. M = 467, N = 11, O = 1, P = 1 (2); for female: A = 120

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(or 112), B = 23, C = 1, D = 1, E = 100, F = 130, G = ical discriminant analysis revealed a clear separation of 1, H = 11, I = 22, J = 11, K = 450, L = 33, M = 11, N = T. pseudobursatus n. sp. from the three different morpho- 1, O = 11, P = 11, Q = 1, R = 11, S = 1. types of Trichodorus aff. sparsus (Fig. 3). This separation The new species belongs to the T. aequalis species was observed using data either from females or males. For complex, mainly characterised by the presence of two females, the morphometrical characters, vaginal scleroti- ventromedian cervical papillae anterior to the secretory- sation, vulva position (V) and onchiostyle length, allowed excretory pore in male. Within Europe, four species (T. most of the separation between the populations compared taylori no longer included, see above) belong to this (see Table 6), and provided more than 86% of the sepa- species group of which three have a restricted distribution: ration between the populations (Fig. 3A; Canonical vari- T. giennensis (Spain), T. hooperi (UK), and T. variabilis ate 1). In the case of males, two morphometrical traits, (Greece), whilst T. sparsus appears widespread, especially spicule and onchiostyle length, allowed the main separa- in central Europe. The new species differs from T. tion between T. pseudobursatus n. sp. and the three dif- giennensis male in spicule shape (head less marked, blade ferent morphotypes of T. aff. sparsus (Fig. 3B; Canonical stouter and largely straight vs head marked, blade slightly variate 1). narrower and curved in T. giennensis) and in female by smaller sclerotised pieces in T. giennensis vs medium- sized in the new species; from T. hooperi male by the Trichodorus sparsus Szczygiel, 1968 different spicules (slightly curved, posterior half striated (Figs 5A-I; 6) and with a few bristles at mid-spicule vs almost straight, finely striated and without bristles in T. hooperi) and in Within populations of T. sparsus, but mostly between females by the vaginal sclerotised pieces (medium-sized, populations, quite a lot of variation can be observed in round triangular and tips clearly separated vs smaller, morphological and morphometric characters (Loof, 1973; round and widely separated in T. hooperi) and the number Peneva, 1988). Table 7 gives an overview of the wide of lateral body pores in female (two vs one in T. hooperi); ranges of taxonomically important morphometrics for T. from T. variabilis in body length (L = 615-810 µmin sparsus. male, 550-870 µm in female vs 775-1100 µminmale The widely used morphological species concept seems and 850-1040 µm in female T. variabilis), spicule length to fail when dealing with the T. sparsus species group. (48-62 vs 44-51.5 µminT. variabilis) and differently The group probably consists of several recently separated shaped and ornamented spicules (mostly equally wide sibling species. blade, no bristles, different striation), different location of Paratype specimens of T. sparsus have been exam- S-E pore (posterior to cervical papillae vs mainly between ined (see Materials and methods), although several are in- the cervical papillae in T. variabilis) and in female by wardly in rather poor condition and/or have the body cu- the smaller vaginal sclerotised pieces and number of ticle largely detached or absent. However, diagnostic fea- lateral body pores (two vs one in T. variabilis); and tures such as the spicules in male and the vaginal region from T. sparsus s. str. in male by spicule shape and with sclerotised pieces in female are still well conserved. ornamentation (blade slightly narrower at mid-spicule Loof (1973) re-examined 24 males of the type population and curved, bristles restricted to posterior half vs blade of T. sparsus and provided additional information on the equally wide, largely straight and bristles present over variation of diagnostic features. For females, no new in- large part of blade in T. sparsus s. str.), straight habitus formation was added compared with the original descrip- vs J-shaped and presence vs absence of bursa, and in tion nor did the small, rather schematic, illustrations of the female by size and shape of vagina (mainly ca 50% of vaginal region provide more detail. The vagina of the pa- corresponding body diam., pear-shaped vs ca 40% of ratype specimens studied is more or less barrel-shaped, corresponding body diam., cylindrical in T. sparsus s. str.) 44-50% of the corresponding body diam. (body cuticle and sclerotised pieces (rounded triangular to oval vs broad not included) long; sclerotised pieces are small (1.5 µm rounded triangular in T. sparsus s. str.). largest dimension) and broadly rounded triangular to oval in shape, with tips 2 µm apart at vulva (Figs 5B; 6). Sim- CANONICAL DISCRIMINANT ANALYSIS ilar observations have been made in specimens from The Netherlands (Figs 5G, H; 6). Between populations, small Four different morphometrical characters of males and differences can be observed in shape of vaginal scleroti- females were used in the analyses (Table 6). The canon- sation and in length and shape of vagina. Female speci-

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Fig. 5. Trichodorus sparsus. A: Spicule, paratype male; B: Vaginal region, paratype female; C, D, F: Variation in shape of spicules, populations from The Netherlands; E: Spicule, population from Germany; G, H: Variation in vaginal region, populations from The Netherlands; I: Vaginal region, population from Germany. Trichodorus variabilis. J: Vaginal region, paratype female; K: Tail region and copulatory apparatus, paratype male. (Scale bar = 20 µm.)

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Fig. 6. Light micrographs showing variation in vaginal sclerotised pieces and vagina in Trichodorus sparsus. A, B: Paratype specimens; C, D: Specimens from Oosterbeek; E, F: Specimens from Hondsdorf; G: Specimen from Königstadten; H: Specimen from Bodemrod. (Scale bar = 20 µm.)

Table 7. Trichodorus sparsus: current ranges of taxonomically important morphometric features. Measurements are in µm and in the form: minimum-maximum (range of mean values). Holotype Paratype females Paratype males Female ranges Male ranges L 910 790-1140 (920) 790-1000 (850) 530-1200 (719-1050) 560-1150 (685-1070) Onchiostyle 50 46-50 (48.3) 46-52 (48.6) 44-78 (47.8-72) 44-76 (43-67.5) Spicule – – 47-51 (49.5) – 43-76 (43-72) V 54.4 52-59 (54.6) – 56-62.5 (53.3-56) –

Vol. 10(3), 2008 419 W. Decraemer et al. mens from Hondsdorf (Germany), for example, have the 1994) but its occurrence in all adult specimens, e.g., in vagina more rhomboid in shape and the sclerotised pieces minor, is considered an additional diag- rounded triangular to rounded quadrangular in optical sec- nostic feature for the species (Decraemer, 1995). tion (Figs 5I; 6). Precise illustrations of the vaginal sclero- Males of T. aff. sparsus morphotype 1 also differ from tised pieces are often difficult to achieve because of their T. sparsus in spicule ornamentation, i.e., blade smooth and small size and because optical sections through the vagi- without bristles, and female by vaginal sclerotised pieces nal ring are not always sharply outlined and the pieces (= more variable, i.e., from slightly smaller to the same size sections) may appear asymmetrical. Consequently, illus- as in T. sparsus and shape broad triangular as in T. sparsus trations by different authors in the literature are difficult but often round to oval. to interpret. Canonical discriminant analyses based upon the same four morphometrical characters for males and females as previously described for T. pseudobursatus n. sp. did not Trichodorus aff. sparsus populations from Serbia clearly separate morphotypes 1 and 3 (Fig. 3).

Among the T. aff. sparsus populations from Serbia, MATERIAL AND LOCALITY three different morphotypes, T. aff. sparsus morphotype 1, T. aff. sparsus morphotype 2 and T. aff. sparsus mor- Trichodorus aff. sparsus morphotype 1 occurred in two photype 3 could be differentiated based upon a com- CP quadrants. Hajduckaˇ cesma,ˇ alt. 1000 m a.s.l., location bination of morphological features and morphometrics. 86B; meadow at the edge of a mixed forest of white pines, Taking into account that intraspecific variation is sub- spruce, beech and fir and Prorokov grob, alt. 700-800 m stantial in the T. sparsus (see previously), canonical dis- a.s.l., location 85S, black pine forest. criminant analyses were done to check the presence of three morphotypes. Canonical discriminant analyses were REMARKS based upon the same four morphometrical characters for males and females previously described for T. pseudobur- Based on the results from the canonical discriminant satus n. sp. The separation between the three different analyses and taking into account the variations known for morphotypes of T. sparsus was not straightforward for morphological features in T. aequalis species complex, both sexes (Fig. 3) except for morphotype 2. The Serbian including T. sparsus, the specimens of morphotype 1 specimens were compared with paratype specimens and are provisionally not described as a separate species. voucher specimens of T. sparsus from several European Additional information from DNA sequences is needed countries. to understand the taxonomic value of the differences observed. Trichodorus aff. sparsus morphotype 1 (Figs7,8,9A,B,E) Trichodorus aff. sparsus morphotype 2 (Figs 9C, D; 10) MEASUREMENTS MEASUREMENTS See Tables 8 and 9. See Tables 8 and 9. REMARKS REMARKS Trichodorus aff. sparsus morphotype 1 closely resem- ble T. sparsus s. str. in morphometrics for both sexes ex- Canonical discriminant analyses using four morphome- cept for the shorter vagina in females (mean value 28 and tric features for females and males, clearly separated T. 30% of corresponding body diam.). Trichodorus aff. spar- aff. sparsus morphotype 2 from the other Serbian Tri- sus morphotype 1 mainly differ from T. sparsus s. str. by chodorus species found (Fig. 3). Trichodorus aff. sparsus the presence of an inner onchium in all adult specimens, morphotype 2 specimens resemble T. sparsus s. str. males a feature never described from T. sparsus s. str. In Tricho- by the possession of two ventromedian cervical papillae doridae, an inner onchium may sometimes occur within anterior to the secretory-excretory pore, three precloacal some specimens of a population (Decraemer & Cheng, supplements with SP1 at level of spicule head, thickened

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Fig. 7. Trichodorus aff. sparsus morphotype 1, Prorokov grob specimens. A: Male neck region; B: Male posterior body region; C: Male tail region and copulatory apparatus; D: Female reproductive system; E: Vaginal region; F: Female neck region.

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Fig. 8. Trichodorus aff. sparsus morphotype 1, Hajduˇcka specimens. A, C: Male neck region; B: Male tail region and copulatory apparatus; D: Vaginal region; E: Female reproductive system. (Scale bars = 20 µm.)

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Fig. 9. Light micrographs of Trichodorus aff. sparsus morphotype 1. A: Female, onchiostyle region showing presence of inner onchium (arrow); B: Posterior body region in male from Prorokov grob; E: Vaginal region. Trichodorus aff. sparsus morphotype 2, specimens from Drundevo; C: Male copulatory apparatus and tail region; D: Vaginal region in female. (Scale bars = 20 µm.)

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Table 8. Morphometric data for male Trichodorus aff. sparsus morphotypes 1 and 2. All measurements in µm and in the form: mean ± s.d. (range). Parameter Morphotype 1 Morphotype 2 Prorokov grob Hajduckaˇ Drundevo n7114 L 809 ± 49.5 (730-855) 774 ± 61.6 (690-875) 560 ± 20.0 (550-590) a 21.5 ± 1.2 (19.9-22.8) 19.2 ± 0.8 (18.2-20.8) 16.0 ± 0.9 (15.3-17.2) b5.8± 0.5 (5.0-6.5) 5.0 ± 0.4 (4.5-5.5) 4.3 ± 0.03 (4.0-4.6) c 80.9 ± 11.0 (69.1-100.0) 77.2 ± 15.4 (44.1-95.3) 48.2 ± 10.5 (36.7-59.0) T64± 2.5 (59-67) 69 ± 3.4 (64-75) 64 ± 2.9 (61-68) Onchium 21 ± 1.7 (19-24) 29 ± 0.7 (28-30) 25 ± 0.5 (24-25) Onchiostyle 45 ± 1.2 (43-47) 57 ± 1.5 (54-57) 48 ± 1.4 (47-50) Pharynx length 140 ± 6.6 (132-151) 154 ± 5.5 (146-163) 131 ± 7.5 (120-139) Pharyngostom 57 ± 1.6 (55-59) 69 ± 8.9 (62-95) 60 ± 1.7 (57-61) Ant. to nerve ring 65 ± 6.2 (58-75) 71 ± 5.1 (63-80) 64 ± 2.6 (61-66) Anterior to CP1 69 ± 5.3 (59-76) 72 ± 6.8 (58-83) 66 ± 3.4 (63-71) Distance CP1-CP2 15 ± 2.2 (12-19) 18 ± 3.6 (10-22) 20 ± 1.5 (18.5-22) Dist. CP2-S-E pore 8 ± 2.1 (5.5-11.5) 11 ± 1.8 (9-15) 4.8 ± 2.5 (2.5-8) Ant. to lateral pore 77 ± 8.3 (66-89) 82 ± 8.1 (68-94) 69 ± 3.8 (66-73) Ant. to guide ring 21 (n = 4) 25 ± 0.9 (24-27) 25 ± 5.3 (21-31) Length pharyngeal bulb 39 ± 3.1 (36-45) 44 ± 2.8 (37-47) 38 ± 2.3 (35-39) Diam. pharyngeal bulb 15 ± 0.8 (15-17) 18 ± 0.8 (17-19) 17 ± 1.3 (15-18) Body diam. at cardia 33 ± 1.0 (32-34) 35 ± 2.1 (31-38) 33 ± 1.9 (32-36) Mid-body diam. 38 ± 1.4 (36-40) 40 ± 3.1 (36-45) 35 ± 2.0 (32-36) Anal body diam. 21 ± 1.4 (18-22) 21 ± 3.3 (16-28) 24 ± 3.6 (21-29) Distance cloacal opening to SP1 34 ± 2.2 (33-38) 34 ± 1.9 (30-37) 34 ± 1.7 (32-36) Distance SP1-SP2 37 ± 2.1 (35-41) 39 ± 3.1 (33-44) 37 ± 1.7 (34-38) Distance SP2-SP3 55 ± 5.0 (48-62) 57 ± 7.4 (38-70) 43 ± 4.6 (39-48) Anterior to testis 284 ± 30.8 (246-334) 225 ± 17.8 (199-250) 190 ± 8.8 (180-200) Spicule length 50 ± 1.9 (48-52) 48 ± 2.5 (43-51) 44 ± 1.7 (42-46) Gubernaculum 21 ± 0.4 (21-22) 23 ± 1.7 (21-27) 19 ± 3.8 (13.5-21) Tail length 10 ± 1.4 (8-11.5) 10 ± 2.7 (7.5-17) 12 ± 2.5 (10-15) Tail tip diam. 6.4 ± 0.7 (5.5-7.5) 5.6 ± 0.7 (4.5-6.5) 6.5 ± 0.41 (6-7) Number of CP 2 2 2 Number of SP 3 3 3 cuticle at tail end and similar onchiostyle length. They Vaginal sclerotised pieces appear slightly larger than in T. mainly differ from T. sparsus s. str. by their shorter body sparsus (2.5-3 µm), more or less oval-shaped, oblique in (560-590 µm) which is outside the known range for T. orientation; tips 2.2-3 µm apart. sparsus, and rather short spicules (42-46 µm), i.e., at the Since only a few specimens of male and female lower range recorded for T. sparsus (Table 7). Males of from a single population were collected and because morphotype 2 further differ by stouter spicules with short morphological differences are rather small, the specimens narrowed portion of blade at level of posterior border of are not described as a new species. A larger number of specimens are needed as well as additional data, e.g., from capsule of protractor muscles and provided with a few molecular sequences. bristles. Females of morphotype 2 possess a body length (475, 690 µm) also within the lower range for T. sparsus, MATERIAL AND LOCALITY the vagina more or less barrel-shaped, with or without nar- rower part at transition with uterus, and 30 or 40% of cor- Morphotype 2 found at a single locality: Drundevo, alt. responding body diam. long in the two specimens found. 300 m a.s.l., location 76U, mixed forest.

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Table 9. Morphometric data for female Trichodorus aff. sparsus morphotypes 1 and 2. All measurements in µm and in the form: mean ± s.d. (range). Parameter Morphotype 1 Morphotype 2 Prorokov grob Hajduckaˇ Drundevo n8102 L 845 ± 81.2 (680-945) 740 ± 61.6 (600-820) 475; 690 a 21.1 ± 1.7 (17.4-23.0) 17.19 ± 1.47 (16.3-19.6) 12.50; 17.7 b5.7± 0.6 (4.9-6.7) 4.7 ± 0.23 (4.6-5.2) 3.8; 4.93 V 54.5 ± 1.1 (53-56) 55.0 ± 1.3 (53-56) 45; 53 G1 35 ± 2.8 (31.4-38.9) 37 ± 1.7 (35.8-42.0) 25.26; 31.74 G2 36 ± 5.7 (26.5-45.8) 35 ± 2.1 (34.0-39.0) 29.05; 31.16 Onchium 23 ± 2.4 (20-26) 30 ± 0.9 (28-31) 25; 26 Onchiostyle 47 ± 2.9 (44-53) 58 ± 2.0 (54-60) 49; 52 Pharynx length 149 ± 10.4 (131-165) 158 ± 5.4 (152-168) 125; 140 Pharyngostom 61 ± 3.5 (57-60) 70 ± 7.5 (65-91) 58; 66 Ant. to nerve ring 68 ± 5.9 (60-78) 71 ± 2.0 (67-74 ) 59; 72 Ant. to S-E pore 99 ± 7.3 (88-108) 99 ± 5.2 (87-106) 89; 93 Ant. to guide ring 21 ± 0.7 (20-21) 24 ± 0.9 (23-25) 27; – Length pharyngeal bulb 44 ± 4.9 (36-51) 44 ± 4.5 (39-52) 31; 33 Diam. pharyngeal bulb 18. ± 2.0 (15-22) 19 ± 1.5 (17-21) 16; 17 Body diam. at cardia 36 ± 2.5 (32-38) 36 ± 2.8 (33-41) 34; 39 Body diam. at vulva 40 ± 2.9 (37-45) 41 ± 3.8 (36-47) 38; 39 Length ant. genital branch to flexure 194 ± 20.5 (171-230) 184 ± 22.0 (139-213) 99; 144 Length anterior ovary 103 ± 19.1 (83-138) 83 ± 21.0 (59-125) 21; 75 Distance anterior to vulva 461 ± 43.9 (371-521) 409 ± 28.4 (346-446) 215; 364 Length posterior genital branch to flexure 200 ± 30.4 (143-238) 177 ± 21.9 (148-213) 101; 139 Length post. ovary 103 ± 22.5 (66-140) 87 ± 14.3 (62-107) 37; 76 Length vagina 11.7 ± 0.9 (10.5-13) 12.3 ± 2.1 (10-17) 15; 13 Diam. vagina 8.9 ± 0.8 (7.5-10) 8.4 ± 1.9 (7-13) 10; 12 Distance vulva to ant. lateral body pore 161 ± 41.3 (103-206) 172 ± 53.2 (102-262) 149; – Distance vulva to post. lateral body pore 34 ± 9.7 (27-58) 34 ± 6.1 (22-45) 26; 18 Vagina as % of mbd 28 ± 1.6 (25-30) 30 ± 4.9 (24-42) 30; 40

Trichodorus aff. sparsus morphotype 3 sparsus morphotype 1 but differ by the absence of a re- (Figs 11, 12) serve onchium in the adult and by the male spicules usu- ally with bristles vs spicules without bristles in T. aff. MEASUREMENTS sparsus morphotype 1.

See Tables 10 and 11. MATERIAL AND LOCALITIES

REMARKS Three populations were collected from locations within UTM grid CP76 and one population from a site within Specimens from the T. aff. sparsus morphotype 3 most UTM grid 75: Crveni potok, location 76H, mixed forest closely resemble T. sparsus s. str. in morphometrics and mostly beech, spruce and fir; Bukvina voda, alt. 1250 m in male features. They mainly differ in the female by a a.s.l., location 76D, meadow on limestone, at the edge very short vagina, i.e., less than one-third of correspond- of a forest with predominantly spruce; Mitrovac, location ing body diam. long, a feature not characteristic for the 76M, meadow with some trees (spruce, Sorbus aucuparia, genus (Decraemer, 1995), and by slightly narrower trian- Corylus avelana), soil on limestone and Ljuto polje, alt. gular to rounded triangular vaginal sclerotised pieces. T. 1200 m a.s.l., location 75X, meadow on limestone and aff. sparsus morphotype 3 also closely resemble T. aff. close to an old beech tree.

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Fig. 10. Trichodorus aff. sparsus morphotype 2 from Drundevo. A: Male neck region; B, D: Variation in spicule shape; C: Male tail region and copulatory apparatus; E: Vaginal region; F: Female reproductive system, posterior branch. (Scale bar = 20 µm.)

426 Nematology Trichodoridae from Serbia

Fig. 11. Trichodorus aff. sparsus morphotype 3 from Serbia. A, B: Male, neck region; C, D: Spicules; E: Posterior body region male; F, G: Vaginal region; H: Female reproductive system.

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Fig. 12. Light micrographs of Trichodorus aff. sparsus morphotype 3. A: Male neck region (specimen from Bukvina voda); B: Posterior body region of male from Bukvina voda; C: Vaginal region in female from Bukvina voda; D: Protruding spicule showing bristles (specimen from Mitrovac). (Scale bars = 20 µm.)

428 Nematology Trichodoridae from Serbia

Table 10. Morphometric data for male Trichodorus aff. sparsus morphotype 3, from several localities in Serbia. All measurements in µm and in the form: mean ± s.d. (range). Parameter Mitrovac 75M Ljuto 75X Bukvina 76D Crveni 76H n6534 L 958 ± 39 (890-990) 775 ± 93.9 (640-895) 926 ± 33.3 (890-955) 938 ± 139 (825-1115) a 24.1 ± 0.9 (23.0-25.5) 19.6 ± 3.8 (15.7-24.7) 23.4 ± 1.8 (21.7-25.3) 24.2 ± 3 (21.8-28.6) b6.1± 0.3 (5.6-6.4) 4.8 ± 0.7 (3.9-5.6) 6.3 ± 0.3 (6.0-6.5) 5.8 ± 1.05 (4.6-7.0) c 76.6 ± 8.4 (66-77.6) 54.3 ± 5.1 (49.2-62.3) 72.0 ± 7.8 (66.8-80.9) 69.0 ± 14.7 (48.5-83) T 64.6 ± 4.4 (57.1-69.3) 69.7 ± 2.0 (66.4-71.4) 70.8 ± 1.9 (68.7-72.2) 66.4 ± 4.0 (62.2-71.7) Onchium 23 ± 1.9 (20-25) 27 ± 4.3 (24-33) 24 ± 1.0 (23-25) 27 ± 0.8 (26-28) Onchiostyle 50 ± 2.9 (45-52) 52.5 ± 2.4 (51-56) 50 ± 1.5 (49-52) 56 ± 1.3 (55-58) Pharynx length 157 ± 2.8 (154-160) 161 ± 6.2 (151-167.2) 147 ± 2 (145-149) 162 ± 12.7 (152-181) Pharyngostom 61 ± 1.7 (59-63) 61 ± 1.9 (59-64) 60 ± 1.7 (59-62) 68 ± 2.6 (65-71) Ant. to nerve ring 69 ± 3.0 (65-74) 68 ± 8.2 (61-81) 67 ± 2.1 (65-69) 70 ± 3.1 (65-72) Anterior to CP1 81 ± 2.8 (77-83) 85 ± 7.8 (76-95) 76 ± 6.5 (69-82) 78 ± 8.6 (68-89) Distance CP1-CP2 18 ± 2.3 (15-22) 17 ± 2.5 (13-18.5) 17 ± 4.9 (14-23) 18 ± 2.3 (15-20) Dist. CP2-S-E pore 5 ± 2.5 (1-7.5) 9 ± 3.8 (4.5-13) 5 ± 3.5 (2-8.5) 5 ± 1.7 (3.8-7.5) Ant. to lateral pore 59 ± 2.4 (83-88) 89 ± 8.5 (79-102) 84 ± 6.8 (76-89) 87 ± 10.5 (73-99) Ant. to guide ring 21 ± 0.8 (20-22) 22 ± 1.5 (20-24) 20 ± 3.5 (17-22) 24 ± 1 (23-25) Length pharyngeal bulb 47 ± 3.7 (43-52) 49 ± 6.6 (42-59) 45 ± 3.1 (42-48) 50 ± 7.5 (43-58) Diam. pharyngeal bulb 18 ± 1.9 (16-21) 20 ± 1.5 (17-21) 18 ± 1.5 (16-19) 18 ± 1 (18-20) Body diam. at cardia 33 ± 1.4 (32-36) 39 ± 3.9 (35-45) 33 ± 0.6 (33-34) 35 ± 3.3 (31-39) Mid-body diam. 40 ± 2.2 (38-43) 41 ± 7.0 (32-49) 40 ± 2.3 (37-41) 39 ± 2.5 (36-42) Anal body diam. 22 ± 2.6 (18-25) 24 ± 1.5 (22-26) 21 ± 2.1 (19-23) 24 ± 4.1 (19-28) Distance cloacal opening to SP1 34 ± 1.6 (31-35) 36 ± 2.6 (33-39) 33 ± 1 (32-34) 35 ± 4.1 (33-36) Distance SP1-SP2 42 ± 3.7 (37-45) 46 ± 9.1 (38.5-61) 41 ± 2.1 (39-43) 40 ± 3.8 (35-44) Distance SP2-SP3 51 ± 2.9 (47-54) 48 ± 2.6 (46-52) 49 ± 5.5 (44-55) 49 ± 6.4 (44-58) Anterior to testis 325 ± 33.6 (285-372) 222 ± 42.8 (170-285) 258 ± 18 (244-279) 298 ± 28.2 (260-328) Spicule length 49 ± 31.6 (48-52) 51 ± 2.1 (48-53) 48 ± 0.3 (48-48.5) 51 ± 1.9 (48-52) Gubernaculum 21 ± 1.7 (18-23) 23 ± 1.9 (22-26) 22 ± 1 (21-23) 21 ± 2.2 (18-23) Tail length 13 ± 1.7 (10-15) 14 ± 1.4 (13-16) 13 ± 1.7 (11-14) 14 ± 2.9 (10-17) Tail tip diam. 7 ± 1.5 (5-9) 7 ± 0.9 (6-8) 7 ± 0.8 (6-7.5) 8 ± 1.0 (7-9) NumberofCP2222 NumberofSP3333

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Table 11. Morphometric data for female Trichodorus aff. sparsus morphotype 3 from type several localities in Serbia. All measurements in µm and in the form: mean ± s.d. (range). Parameter Mitrovac 75M Ljuto 75X Bukvina 76D Crveni 76H n 5 963 L 919 ± 49 (870-980) 803 ± 58 (705-855) 843 ± 42 (795-910) 911 ± 120 (795-1035) a 22.7 ± 2.8 (19.3-25.7) 20.4 ± 1.4 (17.7-22.2) 22.1 ± 1.0 (21.5-23.3) 22.0 ± 1.0 (20.9-23.0) b5.8± 0.33 (5.5-6.3) 5.2 ± 0.4 (4.7-6.1) 5.3 ± 0.2 (5.4-5.5) 5.8 ± 0.7 (5.0-6.4) V 53.3 ± 1.1 (51.8-54.4) 54.4 ± 1.6 (51.6-56.9) 55.9 ± 3.3 (54.5-62.5) 53.9 ± 1.0 (53.2-55.0) G1 53.3 ± 1.1 (51.8-54.4) 33.5 ± 4.5 (26.4-40.9) 33.4 ± 2.7 (29.9-37.2) 33.0 ± 1.4 (32.1-34.6) G2 33.4 ± 8.1 (23.0-35.5) 34.7 ± 4.5 (26.7-41.8) 26.5 ± 3.6 (25.0-31.2) 34.2 ± 7.3 (25.8-39.6) Onchium 24 ± 1.4 (21-25) 23 ± 1.6 (21- 25) 25 ± 1.5 (22-26) 27 ± 1.5 (26-29) Onchiostyle 52 ± 2.6 (48-55) 48 ± 2.7 (44-52) 50 ± 3.2 (46-55) 57 Pharynx length 159 ± 8.0 (149-168) 154 ± 9.5 (147-175) 159 ± 8 (148-171) 156 ± 7 (149-162) Pharyngostom 66 ± 2.6 (63-69) 59 ± 3.0 (56-63) 60 ± 3.9 (55-66) 68± 1.2 (67-69) Ant. to nerve ring 70 ± 5.9 (65-80) 67 ± 4.3 (61-72) 69 ± 4.6 (62-74) 68 ± 4.6 (64-73) Ant. to S-E pore 99 ± 3.1 (95-103) 103.5 ± 7.6 (90-118) 100 ± 4.9 (96-109) 96 ± 4.2 (91-99) Ant. to guide ring 23 ± 1.2 (21-24) 21 ± 1.0 (19-22) 21 ± 1.6 (19-24) 25 ± 0.7 (24-25) Length pharyngeal bulb 51 ± 5.0 (45-58) 54 ± 5.7 (45-63) 48 ± 6.0 (44-57) 49 ± 2.5 (47-52) Diam. pharyngeal bulb 19 ± 1.6 (16-20) 18 ± 0.9 (17-20) 19 ± 1.4 (17-21) 19.8 ± 1.4 (19-22) Body diam. at cardia 36 ± 2.6 (33-39) 36 ± 2.2 (34-41) 34 ± 1.8 (33-37) 36 ± 2.5 (33-38) Body diam. at vulva 40 ± 3.3 (38-45) 40 ± 3.2 (36-46) 38 ± 0.8 (37-39) 41 ± 3.5 (38-45) Length ant. genital branch 196 ± 13.4 (181-218) 189 ± 19 (165-221) 200 ± 21.5 (178-231) 217 ± 28 (185-239) to flexure Length anterior ovary 76 ± 19.9 (55-109) 77 ± 17.22 (59-103) 81 ± 14.8 (60-98) 84 ± 13.0 (70-96) Distance anterior to vulva 490 ± 34 (451-533) 437 ± 37 (369-460) 470 ± 43 (433-544) 491 ± 63 (425-551) Length posterior genital 178 ± 41 (125-228) 191 ± 22 (153-220) 194 ± 25 (171-228) 206 ± 24 (186-233) branch to flexure Length post. ovary 109.5 ± 30.03 (75-146) 88 ± 19.3 (58-125) 85 ± 15.8 (63-104) 100 ± 30 (66-125) Length vagina 12 ± 3.3 (10-18) 12 ± 1.3 (11-15) 11 ± 0.6 (10-12) 11 ± 0.6 (11-12) Diam. vagina 9.6 ± 0.6 (9-10) 10.4 ± 0.7 (10-11) 10.8 ± 0.6 (10-11.5) 11.2 ± 0.3 (11-11.5) Distance vulva to ant. 143 ± 15.6 (123-160) 119 ± 52.4 (62-200) 131 ± 29 (100-178) 162 ± 2.1 (160-163) lateral body pore Distance vulva to post. 22 ± 10.1 (6.5-34) 33 ± 5.4 (25-42) 40 ± 5.9 (32-48) 32 ± 8.3 (23-39) lateral body pore Vagina as % of mbd 26.9 ± 3.2 (22.2-29.0) 29.5 ± 3.3 (24.2-35.1) 29 ± 1.8 (26.3-31.1 ) 27.6 ± 3.6 (24.4-31.6)

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