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. 2017 Jun;49(2):189–206. doi: 10.21307/jofnem-2017-064

Redefinition of Genus Malenchus Andrássy, 1968 (Tylenchomorpha: Tylenchidae) with Additional Data on Ecology

Xue Qing 1,✉, Wim Bert 1
PMCID: PMC5507140  PMID: 28706319

Abstract

Malenchus is the second specious genus in Tylenchidae. In the presented study, we examined 22 populations including 12 type/paratype species. Detailed morphology was recovered using light microscopy, scanning- and transmission- electron microscopy. All population and type slides were recorded as picture and video vouchers, which are available online. We have compared inter- or intraspecific variations and extracted taxonomically informative traits. Amended definitions of the Malenchus as well as the closely related Ottolenchus were given based on a combination of morphology and recent molecular data, and their phylogeny were analyzed in a context of Tylenchidae. Furthermore, we test different fungi and moss as a food resource of Malenchus and their feeding behavior is also discussed.

Keywords: Duosulcius, Filenchus, morphology, Ottolenchus, taxonomy, Tylenchomorpha, Zanenchus.

Tylenchidae is one of the most important soil inhabiting nematode groups (Andrássy, 1981), and species belonging to Tylenchidae may constitute up to 30% of the nematode individuals in a soil sample (Yeates and Bird, 1994; Ferris and Bongers, 2006). As early-branching Tylenchomorpha, they do not comprise economically important plant parasites and are characterized by ancestral characters, such as weak feeding apparatus, undifferentiated nonmuscular corpus, filiform tails, and four cell rows in uterus (Luc et al., 1987; Siddiqi, 2000; Bert et al., 2008). Knowledge of their food resources remains limited, albeit, given their numeric importance, this subject is important for tropic guild analysis or soil quality evaluation. Furthermore, their small body size and a lack of clearly homologous characters prevented us from deriving a consistent systematic framework. As a result, the delimitation of taxa in this group remains poorly documented and highly uncertain (Bongers and Bongers, 1998; Yeates, 2003; Ferris and Bongers, 2006).

In this study, we focus on the cosmopolitan genus Malenchus, which is the second most specious (after Filenchus) in Tylenchidae. Although several species have been proposed, morphology details have been often only poorly described. The only genus review was made more than 30 years ago based on a limited number of morphological details (Andrássy, 1981). Recently, molecular methods have revealed a phylogenetic position for the genus Malenchus (Yaghoubi et al., 2015; Qing et al., 2016, 2017), but the need for a review is growing. In this present study, we examined type or paratype of 12 species together with 10 populations worldwide. We do not intend to establish new nor to synonymize current taxon but rather to summarize morphological variations and analyze the results in a phylogenical context, as most of the taxonomically important characters are generally absent or incomplete in the original description (Qing et al., 2017).

Materials and Methods

All specimen examined in this study are listed in Table 1. Classification of Malenchus and Tylenchidae follows Geraert (2008). Geographic distributions were plotted using QGIS 2.82 based on original descriptions and other reports (Andrássy, 1981; Geraert and Raski, 1986; Gómez-Barcina et al., 1992; Geraert, 2008; Holovachov, 2014; Mundo-Ocampo et al., 2015; Panahandeh et al., 2015a; Panahandeh et al., 2015b; Yaghoubi et al., 2015; Qing et al., 2016). Measurements and drawings from slides were prepared manually with a drawing tube mounted on an Olympus BX51 DIC Microscope (Olympus Optical, Tokyo, Japan), equipped with an Nikon DS-FI2 camera (Nikon Corporation, Tokyo, Japan) for photography. All examined populations as well as type slides were recorded as a video clips mimicking a multifocal observation through a light microscope (LM) following the video capture and editing procedures (De Ley and Bert, 2002). The resulting virtual specimens are available at http://nematodes.myspecies.info. Extraction and examination of female reproductive system was based on the method of Geraert (1973) and Bert et al. (2008). Illustrations were prepared based on LM drawings and modified by Adobe Illustrator CS3 and Adobe Photoshop CS6.

Table 1.

Number of cuticle annules and width in different species or populations of the genus Malenchus.a

graphic file with name 189tbl1.jpg

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For scanning electron microscopy (SEM), specimens from DESS were gradually transfered to water, then dehydrated in a battery of ethanol solutions and dried by critical point dried with CO2. After mounting on stubs samples were coated with gold and observed with a JSM-840 EM (JEOL, Tokyo, Japan) at 12 kV. For transmission electron microscope (TEM), specimens were fixed, ultra-thin sections were cut and sections were stained as detailed by Qing et al. (2017). Sections were observed with a JEOL JEM 1010.

To test the feeding type, four fungal species (Flammulina velutipes, Lepista nuda, Botrytis cinerea, and Pleurotus sp.) were inoculated on potato dextrose agar medium with three repeats for each species and incubated at 25°C for 10 d until the mycelium covered the culture plates. Forty individuals of M. pachycephalus and M. acarayensis were transferred to each plate and nematodes were extracted by Baermann tray after 2 months. To test moss as a potential host, Eurhynchium sp. was isolated from soil habited by M. pachycephalus, rinsed five times with distill water to remove attached detritus and then carefully transplant to culture plates with 1% agar in tap water. Controls plates were made using 1% agar in tap water to compare with the two treatments. Forty individuals were transferred to each plate and directly checked in binocular every 3 d for 2 months.

Result and Discussion

Taxonomic overview

The genus Malenchus was established by Andrássy (1968) with M. machadoi (formerly Aglenchus machadoi Andrássy, 1963) as the type species. Later, several new genera have been erected and later synonymized with this genus, for details see Geraert (2008). Within the genus, three subgenera are valid: Malenchus, Neomalenchus, and Telomalenchus. Neomalenchus was initially established as a genus for species with indistinct median bulb in Malenchus (Siddiqi, 1979), but this genus was synonymized (Andrássy, 1981) in his comprehensive review of Malenchus and later considered as a subgenus (Siddiqi, 2000). Subgenus Telomalenchus was introduced to accommodate three species (M. williamsi Geraert and Raski, 1986; M. parthenogeneticus Geraert and Raski, 1986 and M. leiodermi Geraert and Raski, 1986) with straight amphideal aperture and less lateral incisures (four or six) (Siddiqi, 2000). Although Andrássy (2007) synonymized Telomalenchus with Fraglenchus, such an action was rejected by Geraert (2008). Currently, Malenchus contains 36 valid species and 3 nomina nuda (Geraert, 2008; Mundo-Ocampo et al., 2015; Qing et al., 2016).

Geographic distribution

Malenchus is a cosmopolitan genus and is reported from all continents except for Antarctica (Fig. 1). Among them, Malenchus bryophilus (Steiner, 1914) Andrássy 1981, and M. acarayensis Andrássy, 1968 are the most frequently encountered species, whereas 18 species are only reported once from their type location (M. angustus Talavera and Siddiqi, 1996; M. anthrisulcus (Sumenkova, 1988) Ebsary, 1991; M. fusiformis (Thorne and Malek, 1968) Siddiqi, 1979; M. gratiosus Andrássy 1981; M. holochmatus (Singh, 1971) Siddiqi, 1986; M. herrerai Mundo-Ocampo, Holovachov and Pereira, 2015; M. kausari Khan and Ahmad, 1991; M. macrodorus Geraert and Raski, 1986; M. nobilis Andrássy, 1981; M. pampinatus Andrássy, 1981; M. paramonovi Katalan-Gateva and Alexiev, 1985; M. parvus Brzeski, 1988; M. sexlineatus Qing, Sánchez-monge, Janssen, Couvreur and Bert, 2016; M. shaheenae Khan and Ahmad, 1991; M. solovjovae Brzeski, 1988; M. subtilis Lai and Khan, 1988; M. truncates Knobloch, 1976; M. parthenogeneticus Geraert and Raski, 1986; M. williamsi Geraert and Raski, 1986).

Fig. 1.

Fig. 1

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World distribution of Malenchus species. Species are labeled with different colors. 1. M. acarayensis. 2. M. Andrassyi Merny, 1970. 3. M. angustus. 4. M. anthrisulcus. 5. M. bryanti Knobloch, 1976. 6. M. bryophilus (Steiner, 1914) Andrássy, 1981. 7. M. exiguus (Massey, 1969) Andrássy, 1981. 8. M. fusiformis. 9. M. gratiosus. 10. M. holochmatus. 11. M. herrerai. 12. M. kausari. 13. M. labiatus Maqbool and Shahina, 1985. 14. M. laccocephalus Andrássy, 1981. 15. M. leiodermis Geraert and Raski, 1986. 16. M. machadoi (Andrássy, 1963) Andrássy, 1968. 17. M. macrodorus. 18. M. nanellus Siddiqi, 1979. 19. M. neosulcus Geraert and Raski, 1986. 20. M. nobilis. 21. M. novus Mukhina and Kazachenko, 1981. 22. M. ovalis (Siddiqi,1979) Andrássy, 1981. 23. M. pachycephalus Andrássy, 1981. 24. M. pampinatus. 25. M. paramonovi. 26. M. parthenogeneticus. 27. M. parvus. 28. M. platycephalus (Thorne and Malek, 1968) Andrássy, 1981. 29. M. pressulus (Kazachenko, 1975) Andrássy, 1981. 30. M. sexlineatus. 31. M. shaheenae. 32. M. solovjovae. 33. M. subtilis. 34. M. truncates. 35. M. undulates Andrássy, 1981 and 36. M. williamsi.

General morphology

The body size of Malenchus ranges from 250 µm to 900 µm, the largest species is M. novus, whereas M. sexlineatus, M. bryanti, and M. parvus are the three smallest species (Fig. 2). A ventrally arcuate habitus is the most common appearance, but a straight or “S” shape can also occasionally be found. Body behind vulva usually tapers markedly so that width at anus is about half of that at vulva, but an elongated-cylindrical shape similar to that of other genera in Tylenchidae is also possible (e.g., Malenchus sp. C163 nested within Malenchus clade [Qing et al., 2017] but with elongated-cylindrical shape behind vulva).

Fig. 2.

Fig. 2

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Body habitus and size in genus Malenchus. Size measured in µm and shown in longitudinal axis. A. M. novus from China. B, C. M. pachycephalus C116 from China. D. M. williamsi from Chile. E. M. solovjovae, from Poland. F, G. M. pachycephalus from Spain. H–J. M. pachycephalus C161 from Belgium. K, L. M. exiguus from China. M. M. undulates, from Philippines. N–P. M. acarayensis from Spain. Q, R. M. tantulus, from Malawi. S, T. M. nanellus, from Nigeria. U. M. parvus, from Mexico. V–X. M. sexlineatus, from Philippines. Female ventral views: A, B, D, G, H, L, T. Female lateral views: C, E, F, I, K, N, O, P, R, S, U, V, X. Male later view: J, M, Q, W.

Cuticle annulation

The cuticle in genus Malenchus is generally thick and folded between annuli (Figs. 3J–L; 4C–E; 5A) (Andrássy, 1981), in contrast to the typical finely striated Filenchus (Figs. 4B; 6C,F). The cuticle surface is smooth in most species but longitudinal striae can be observed occasionally under SEM (Fig. 7I,J). Annulations are prominent with a width of 0.76 to 2.38 µm, conspicuous even under low magnification. Although with some variations, the annulation number (especially from anterior to vulva/cloacal) and width shows different ranges interspecifically and is a taxonomically useful reference (see details in Table 1).

Fig. 3.

Fig. 3

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Selected anatomic structures in Malenchus pachycephalus (A–I, L, M), M. sexlineatus (J) and M. exiguus (K). A. Lateral view of amphideal fovea. B. Head region, arrow indicates ventral view of amphideal fovea. C. Spicule, after dissection. D. Gubernaculum, after dissection. E. Anterior part of intestine, arrow indicates brown granule. F. Female gonad after dissection. G. Ventral view of spicule. H. Ventral view of cloacal, arrow indicates distal end of spicule and gubernaculum. I. Lateral view of vulva region, arrow indicates swollen vagina. J. Folded cuticle of type 1. K. Folded cuticle of type 2. L. Folded cuticle of type 3. M. Ventral view of vulva, arrow indicates epiptygmata. ova = ovarium, ovi = oviduct, sp = spermatheca. Scale bar: A–D, J = 5 µm; E–I, K–M = 10 µm.

Fig. 4.

Fig. 4

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Diagrammatic example of cuticle layers in Malenchus (A) and the variation of the cuticle as observed based on LM observation (B–E). A. Illustration of ultrastructure in M. pachycephalus based on TEM, adapted from Qing et al. (2016). (1) epicuticle. (2) Cortical zone. (3) Median zone. (4) Basal zone. (a) Annuli width. (b) Groove depth. (c) Groove width. B–E. Schematically representation of the most common cuticle appearances in Tylenchidae. B. Filenchus type with indistinct annuli. C–E. cuticle types in Malenchus.

Fig. 5.

Fig. 5

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Cuticle ultrastructure of M. pachycephalus C161. A. Longitudinal section in female middle body. B, C, E, F. Unknown organisms present in annulation grooves. D. Cross section in female middle body. Scale bar: A = 2 μm; B, C, E, F = 0.5 μm; D = 5 μm.

Fig. 6.

Fig. 6

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Ultrastructure of cuticle and lateral region in Malenchus and Filenchus. A, C. F. discrepans. B, F. F. vulgaris. D, E. M. acarayensis. A, B, D. Cross section of female middle body. C, E, F. Longtitudinal section in female middle body. Scale bar: A, D, E = 2 μm; B = 4 μm; C, F = 1 μm.

Fig. 7.

Fig. 7

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SEM of female M. pachycephalus C116 (A–H) and M. nanellus (I, J). A. en face view. B. Vulva. C. Ventral view of tail. D. Lateral view of female head. E. Lateral view of vulva region. F. Lateral view of middle body showing smooth cuticle surface. G. Lateral view of tail. H. Lateral region of tail showing small ridges are stopped or interrupted. I. Lateral region appears slightly crenated due to the extension of the cuticle annulations until ridge beneath. J. Ventral view of anus showing cuticle surface with longitudinal striae. Scale bar: A, D, F, H, I, J = 1 µm; B, C, E, G = 5 µm.

The cuticle has been considered as an important generic character ever since this genus was proposed (Siddiqi, 1979; Andrássy, 1981; Geraert, 2008). However, a recent study shows that annulations can vary from prominent and folded to rather faint (Qing et al., 2017). These variations can be explained by different combinations of annuli width (a in Fig. 4A), groove height (b in Fig. 4A) and groove width (c in Fig. 4A) and therefore can be roughly clustered into three groups: (i) with indistinct folded part (Figs. 3J; 4C), annuli narrow and groove hardly visible in LM (a > 4c, usually annuli width less than 1.2 µm), represented by species M. sexlineatus, M. parthenogeneticus, M. leiodermis, and Malenchus sp. C163; (ii) with moderated folded cuticle annuli width (Figs. 3K; 4D), groove narrow but visible (4c < a < 2c, usually annuli width 1.2–1.6 µm), with species M. parvus, M. acarayensis, M. exiguus, M. nanellus, and M. ovalis; (iii) cuticle prominently folded (Figs. 3L; 4E), with spacious grooves and wide annuli (a < 2c, usually annuli wider than 1.6 µm); typical species include M. pachycephalus, M. solovjovae, M. pressulus, and M. novus.

Within each type, the groove appears with a narrow opening, forming a nearly enclosed space. In TEM, this groove lumen was embedded by unknown organisms which resemble conidia, zoospore, or hypha of fungus (Fig. 5B,C,E,F). Remarkably, we recovered 18S rDNA of the fungus Malassezia sp. from M. pachycephalus, the sequence similar to a fungus associated with Malenchus sp. in forest soil (Renker et al., 2003). Such fungal sequences have been obtained five times during our studies on Malenchus using “nematode specific” primers (Qing et al., 2017). Fungi from the genus Malassezia are opportunists, causing infection in humans and animals; they are commonly isolated from the skin and scalp of humans (Cunningham et al., 1990; Marcon and Powell, 1992; Hay and Midgley, 2010) and also from insects (Zhang et al., 2003). Although it has been reported from several species (Malenchus spp., Meloidogyne sp., Acrobeloides sp., and Cephalobus sp.) and assumed to be selectively associated with nematodes (Renker et al., 2003) as a vector (Karabörklü et al., 2015) or in random adherence (Adam et al., 2014), the relationship of Malassezia and nematodes remain unknown. In this study, the recovered unknown cuticula-associated organisms confirm the association of nematodes with another organism, and such an organism is likely to be Malassezia sp.

Cuticle ultrastructure

The ultrastructure in the cuticle was conventionally divided into four layers (Decraemer et al., 2003): (i) epicuticle, (ii) cortical zone, (iii) median zone, (iv) basal zone (including basal lamina) and all of these layers are present in Malenchus (Fig. 4A). The epicuticle and cortical and median zones generally resemble those of other Tylenchomorpha, whereas the radial striae in the basal zone are reduced in M. pachycephalus and M. acarayensis (Qing et al., 2017). Although the cuticle ultrastructure shows intergeneric variation within Tylenchomorpha (Johnson et al., 1970; Mounport et al., 1991, 1993b, 1997; Valette et al., 1997), a radially striated layer in the basal zone was considered to be always present (Decraemer et al., 2003; Geraert, 2006). Although several structural cuticular elements are homoplasious within Nematoda, at less inclusive taxonomic levels (e.g., on a family or genus level) the cuticle appears to be a more reliable phylogenetic marker (Decraemer et al., 2003). Thus, the divergent cuticle structure supports Malenchus as an evolutionarily divergent lineage within Tylenchomorpha and this character therefore important to define the genus.

Head region

The head of genus Malenchus is generally elevated, dorso-ventrally compressed (Andrássy, 1981) but more continuous in some species such as M. exiguus, M. parthenogeneticus, and M. williamsi) (Figs. 8,9). Stylet usually delicate, comparable to Filenchus, but can be robust in some species (e.g., M. macrodorus, M. novus, M. pachycephalus, and M. solovjovae). Cone part of stylet always heavier sclerotized but distinctly shorter (1/3–1/2 vs shaft) and thinner than shaft (Fig. 3B). Basal knobs flattened, directed backwards, forming a triangle-like base in stylet.

Fig. 8.

Fig. 8

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Anterior part of different species in genus Malenchus. All specimens are from examined type/paratypes, except for M. exiguus from Chinese population. More picture and video vouchers see http://nematodes.myspecies.info. A. M. exiguus. B. M. pachycephalus. C. M. parvus. D. M. leiodermis. E. M. nanellus. F. M. ovalis. G. M. solovjovae. H. M. tantulus. I. M. williamsi. J. M. acarayensis. K. M. macrodorus. L. M. novus. M. M. malawiensis. N. M. sexlineatus O. M. parthenogeneticus. Scale bar = 10 µm.

Fig. 9.

Fig. 9

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Illustration of anterior part of five Malenchus species showing general head shape, stylet and start position of lateral lines. A. M. acarayensis. B. M. exiguus, C. M. pachycephalus. D. M. nanellus. E. leiodermis. F. M. labiatus. Adapt from Andrássy (1981), Geraert and Raski (1986), and Maqbool and Shahina (1985). Scale bar = 10 µm.

Amphideal fovea is usually invisible in Tylenchidae but is conspicuous spindle shaped (=large inner sacks) in Malenchus (Fig. 3A,B), a trait that corresponds to molecularly defined lineages and thus potentially useful in Malenchus delimitation (Qing et al., 2017). The amphideal fovea is wrapped in cuticular outgrowths, which form the finer clefts (Gómez-Barcina et al., 1992) resulting in either an S shaped (Andrássy, 1981) or straight (Geraert and Raski, 1986) amphideal aperture. Although the aperture shape can change during development by the modification of the two outgrowths, it never switches from S shaped to straight (Qing et al., 2016). The most common S-shaped aperture varies among species and can be roughly divided into two groups: (i) aperture starts with large round to oval-shaped hole, sharply narrowing to a slit and ending at head base, represented by M. macrodorus, M. nanellus, M. pachycephalus, M. solovjovae, and M. sexalineatus; (ii) the aperture slit is equally wide throughout its length, represented by M. acarayensis. Interestingly, the S-shape aperture is also present in some Filenchus species, which have only two lateral field incisures such as F. normanjonesi, F. facultativus, and F. helenae (Raski and Geraert, 1986b), but not in F. fungivorous (Bert et al., 2010), and never in Filenchus with four incisures. This is in line with the molecularly-based observation that Filenchus species with two incisures are more closed related to Malenchus than Filenchus species with four incisures (Atighi et al., 2013; Qing et al., 2017).

Lateral region

The lateral region is prominent, two incisures delimit a single narrow but elevated ridge (=protruding band, by Geraert and Raski [1986]; Geraert [2008]). In LM, it appears as a plain ridge but in SEM or TEM several small ridges can be discerned (Figs. 5D; 6D; 7F,H,I). This feature is different from genus Filenchus (Fig. 6A,B) as well as other known species in Tylenchomorpha (Baldwin and Hirschmann, 1975; Mounport et al., 1991, 1993a, 1993b, 1997; Valette et al., 1997). The number of these small ridges is an interspecific variable, ranging from 3 to 14 based on SEM (Geraert and Raski, 1986; Brzeski, 1988; Gómez-Barcina et al., 1992; Mundo-Ocampo et al., 2015; Qing et al., 2016). However, even based on SEM the actual number can be underestimated, as small ridges can be present below the elevated ridge of the lateral region and these are hard to observe based on a single SEM image plane (Figs. 7I,10B). Therefore, a cross section is crucial to determine the correct number of small ridges, which can be up to 22 based on TEM (Figs. 5D,6D) (Qing et al., 2017).

Fig. 10.

Fig. 10

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Illustration of lateral region in genus Malenchus. A–C. Longitudinal lateral ridge narrow at the base, forming overlap with transversal crenation at two image plane and appears as crenated margin. A, B. Cross section of lateral ridge. C. Lateral view of lateral ridge. D–F. Transversal crenation cannot reach bottom of lateral ridge, no overlap from lateral view and appears as smooth margin. D, E. Cross section of lateral ridge. F. Lateral view of lateral ridge. G, H. Anterior start of lateral ridge. I–K. Lateral ridge with small ridges stopped or interrupted. L, M. Posterior end of lateral ridge.

The boundary of lateral lines sometimes appears to be a crenated margin, based on LM (Knobloch, 1976; Siddiqi, 1979; Andrássy, 1981; Geraert and Raski, 1986; Siddiqi, 2000). However, unlike other species, this crenated lateral field appears to correspond with the width of the ridge base, and if narrow then the crenation can extend below the lateral ridge that appears as a crenated margin from a lateral view (the longitudinal lateral ridge overlap with transversal crenation in two focus planes, see Fig. 10A–C), while if the base is wide there is no overlap and the margin of the lateral field is smooth (transversal crenation cannot reach to bottom of longitudinal ridge, see Fig. 10D–F).

The beginning of the lateral field range from few annuli after the head to the median bulb level (Figs. 9,11) and ends at 1/4 to 1/3 of the tail. Interestingly, at least two start patterns have been observed (Fig. 10G,H), and the number of small ridges can be reduced at the anterior- or posterior-most part (Fig. 10I–M); they are clearly dissimilar to Cephalenchus (Mizukubo and Minagawa, 1985; Raski and Geraert, 1986a), which start from single ridge then hierarchically split three times to form five small ridges (six incisures). The start position of the lateral field has been used in species diagnosis and is indeed, consistent intraspecifically and varies interspecifically, based on our observations of 22 examined populations over 18 species. However, interpopulation differences have also been observed, for example the lateral field of M. nanellus starts at knob level (Troccoli and Geraert, 1995), the mid-region of procorpus (Siddiqi, 1979; Andrássy, 1981; Siddiqui and Khan, 1983; Geraert, 2008) or even at the base of the procorpus (Geraert and Raski, 1986). If this is a matter of real variation or the presence of cryptic species (the examined paratype start at mid-region of the procorpus, other different reports may be cryptic species) remains to be investigated, but based on our data the starting position of the lateral ridge is a consistent character and taxonomically informative. This also concurs with the key to species provided by Andrássy (1981) and Geraert (2008).

Fig. 11.

Fig. 11

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The relative origin positions of lateral lines in genus Malenchus. A. Anterior of stylet. B. Mid-region of stylet. C. Level of knob. D. Anterior of procorpus. E. Mid-region of procorpus. F. Base of procorpus. G. Median bulb region. 1. M. acarayensis. 2. M. andrassyi. 3. M. angustus. 4. M. bryanti. 5. M. bryophilus. 6. M. exiguus. 7. M. gratiosus. 8. M. herrerai. 9. M. kausari. 10. M. labiatus. 11. M. laccocephalus. 12. M. machadoi. 13. M. macrodorus. 14. M. nanellus. 15. M. neosulcus. 16. M. nobilis. 17. M. novus. 18. M. ovalis. 19. M. pachycephalus. 20. M. pampinatus. 21. M. parvus. 22. M. pressulus. 23. M. sexlineatus. 24. M. shaheenae. 25. M. solovjovae. 26. M. subtilis. 27. M. truncates. 28. M. undulates. 29. M. leiodermis. 30. M. parthenogeneticus. 31. M. williamsi.

Prophasmid

The phasmid usually occurs in the lateral region of the tail, but in Tylenchidae, it is situated postmedian, just outside the lateral fields and termed prophasmid (Siddiqi, 1978). In females, the position ranges from 2 to 8 annuli anterior to 4 to 5 posterior vulva. Andrássy (1981) considered the prophasmid position as taxonomically informative at species level, ignoring the considerable variation presented in the same paper. Similar variations are also observed in this present study (Fig. 12); the intraspecific variation is often as large as the interspecific variation. Even in the same specimen both prophasmids can differ in up to 5 annuli from one another. Hence, this character is not reliable to distinguish species, except for M. williamsi with an unusual but conserved prophasmid position (post-vulval vs anterior to vulva in other species, see Fig. 12).

Fig. 12.

Fig. 12

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The relative position of prophasmid in Malenchus. All prophasmids located dorsal side near lateral lines, bars here only shows range of phasmid locations measured by number of annulations anterior/posterior to vulva. A. M. acarayensis. B. M. andrassyi. C. M. bryanti. D. M. bryophilus. E. M. exiguus. F. M. sexlineatus. G. M. macrodorus. H. M. malawiensis. I. M. nanellus. J. M. nobilis. K. M. ovalis. L. M. pachycephalus (Andrassy’s population). M. M. pachycephalus C161. N. M. parthenogeneticus. O. M. parvus. P. M. pressulus. Q. M. solovjovae. R. M. williamsi. Based on data from Andrássy (1981), Geraert and Raski (1986), and Qing et al. (2016) and this study.

Reproductive system

Female reproductive system monodelphic, ovary outstretched with oocytes arranged in a single row. Uterine sac spacious with thickened wall, eggs only present exceptionally (nongravid) (Brzeski, 1988), postvulval uterine sac (PUS) about half of body width. Vagina has well-developed muscles, perpendicular to body or slightly anteriorly direct. Vulva sunken, cavity shape with epiptygmata and lateral flaps (=dikes in Andrássy [1981]).

On the basis of dissected gonoducts, the oviduct comprises two rows of three (M. pachycephalus, M. acarayensis, and M. ovalis) or four cells (Malenchus sp. C163), the spermatheca is offset, comprises 10 to 17 cells (Table 2) and is connected to the uterus by two cells (uterus except for M. ovalis), and the uterus cells are arranged in four regular rows (=quadricolumella) of five cells (Figs. 3F,13; Table 2). Our observations concur with other gonoduct studies of Tylenchidae (Bert et al., 2006); the oviduct and uterus rows have been considered as an evolutionary stable structure: two oviduct cell rows were considered as an apomorphy of the order Rhabditida and four rows in uterus were typical for Tylenchidae and Anguinidae (Geraert, 1983; Bert et al., 2006; Geraert, 2006; Bert et al., 2008). The cell number of the spermatheca is intraspecifically consistent in all examined specimens, supporting spermatheca number as a species-specific indicator (Bert et al., 2006, 2008). However, additional observations based on more species are necessary to validate this character for Malenchus species identification. According to in vivo observations, the spermatheca of Malenchus appear as rounded to elongated and offset or bilobed-offset. However, examination of the expelled M. ovalis gonoduct shows that the bilobed appearance is the result of the non-offset part of spermatheca being filled with sperm. This confirms the observations of Qing et al. (2016) who presumed that the observed bilobed structure is the effect of sperm cells in the proximal part of the uterus and further limits the use of this trait in species diagnosis (Geraert and Raski, 1986).

Table 2.

Detail counts of female gonad cellular architecture.a

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Fig. 13.

Fig. 13

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Line drawings of the cellular composition of oviduct, spermatheca and distal part of uterus of representative of the genus Malenchus. A. M. pachycephalus. B. M. acarayensis. C. Malenchus sp. C163. D. M. ovalis. Scale bar: A–C = 10 µm; D = 5 µm.

The vulva is delimited by a depression of the cuticle, usually a gradual sinking that extends over two or three adjacent annuli, this in contrast to a sharp sink of one annulus in Filenchus. Lateral flaps (i.e. lateral dikes by Andrássy [1981] or vulval membranes by Carta et al. [2009]) are the cuticular outgrowths lateral and perpendicular to vulval slit. Two-annuli-long lateral flaps (Fig. 14B) is most common but they can be also indistinct (e.g., M. pachycephalus, M. solovjovae, and M. macrodorus) or extend to 7 to 8 annuli (M. williamsi) (Fig. 14C). Interestingly, lateral flaps usually reduced in species with wider annuli. Epiptygmata (Fig. 14A–C) are found in all studied specimens and are considered as cuticular protrusions of the vaginal wall (Siddiqi, 2000). Although indistinct in LM, they can be clearly distinguished in SEM (Fig. 7B). A vagina with swollen walls (Figs. 3I; 15D,E) is most promising character we recovered. Although it has been noticed by several authors (Siddiqi, 1979; Andrássy, 1981; Geraert, 2008), none of them used it as generic delimitation character. A swelling of the proximal or middle part of the vagina is presents in all examined Malenchus and we consider this character as an important generic character. In Aglenchus and Coslenchus, the vulva is also swollen but more in the distal part (Fig. 15) and this trait may have evolved independently.

Fig. 14.

Fig. 14

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Ventral view of typical vulval flap and epiptygmata in the genus Malenchus. A. Flap occupies one annulus without overlapping on vulva. B. Flap occupies about two annuli slightly overlapping vulva. C. Flap occupies more than four annuli covering half of vulva.

Fig. 15.

Fig. 15

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Vagina with different types of swollen walls. A–C. Vagina with swollen wall in distal part, present in Coslenchus and Aglenchus. D, E. Vagina with swollen wall in more proximal or middle part, present in Malenchus. F. Thin and straight wall, most common type in Tylenchidae.

The male is generally less frequent than the female. Testis single, spermatogonia normally arranged in one row, spermatids few, indistinct. Spermatozoa always round but size can differ among species, filling proximal part of vesicula seminalis. Cloacal opening bears prominent cone with protruding lips. Spicules are variable in size and shape and thus taxonomically important in some species (Nickle, 1970; Hechler, 1971; Geraert and De Grisse, 1982; Adams and Nguyen, 2002). Within Tylenchomorpha spicule is less informative in species diagnosis; however four characters are potentially useful on genus level: (i) curvature; (ii) the length/diameter ratio; (iii) the presence/absence of a velum; and (iv) the shape of spicule tip (Geraert and De Grisse, 1982; Geraert, 2006). The typical “tylenchid-like” shape of capitulum, shaft, and blade varies among the four examined species (Fig. 16). Remarkably, the spicule tip is twisted in M. pachycephalus and M. acarayensis, the edges curve in at level of blade but abruptly twist 180° and curve outward at the end of the blade, which appears as a C shape in the cross view of distal end (Figs. 3C,G; 16). Such a structure is unique to Tylenchidae. The gubernaculum is similar to other Tylenchomorpha (Clark et al., 1973; Wen and Chen, 1976; Wang and Chen, 1985), being centrally concave with ridge and two curved sides expanding laterally (Figs. 3D,H; 16E,F).

Fig. 16.

Fig. 16

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Spicules and gubernaculum in four Malenchus species. A, E, F. M. pachycephalus. B, C. M. acarayensis. D. Malenchus sp. C163. A, B, D. Lateral view of spicule. C. Lateral-ventral view of spicule. E, F. Distal end of spicule and gubernaculum. Scale bar = 5 µm.

Revised generic definitions

Genus Malenchus Andrássy, 1968

Syn. Neomalenchus Siddiqi, 1979

Mukazia Siddiqi, 1986

Paramalenchus Sumenkova, 1988

Body straight or ventrally arcuate, dorso-ventrally flatted in cross view. Cuticle thick, most species have prominent folded annuli, occasionally with faint annuli, 0.76 to 2.38 µm, conspicuous even under low magnification. Head can be dorso-ventrally compressed or more rounded, with pouch-like amphideal fovea. Amphideal aperture usually S shaped, but can also straight. Basal plate of cephalic framework is not flat (appears as M shaped). Stylet weakly sclerotized, cone part of stylet always heavier sclerotized but distinctly shorter (1/3–1/2 vs shaft) and thinner than shaft. Basal knobs flattened, directed backwards, forming a triangle-like base in stylet. Lateral field with offset ridge, comprising 6-22 small sub-ridges, starting from stylet to level of median bulb and ending at middle of tail. Pharynx slender, median bulb from very weak to moderately developed, valvular apparatus present. Basal bulb short, pyriform. Female reproductive system monodelphic, prodelphic, straight, PUS about half of body width. Prophasmid dorso-lateral, usually anterior but rarely posterior to vulva. Vulva sunken, usually in a definite vulval cavity. Lateral flaps often present in species with narrow annuli (less than 1.8 µm), but reduced or absent in species with wider annuli (more than 1.8 µm). Epiptygmata present but may obscure in LM. Vagina with swollen wall in proximal or middle part. Body behind vulva markedly tapering so that width at anus is about half of that at vulva in most species, but can also be elongated behind vulva. Tail similar in both sexes. Male less frequent than females. Cloacal lips protruding. Bursa adanal, short, heavily curved. Spicule ventrally curved, tip is twisted. Gubernaculum small.

Comments on amended generic definitions

Based on characters recovered in the present study as well as available molecular evidence (Qing et al., 2016, 2017), we propose an amended definition of the genus Malenchus emphasizing on amphideal fovea, lateral region and vaginal structure. The most important traits of Malenchus, in comparison with earlier definitions, are presented in Table 3.

Table 3.

Comparison of generic definitions of Malenchus.a

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Ottolenchus are intimately related to Malenchus and Filenchus clades group 2 by sharing two incisures. Indeed, such a similarity has been noticed and repeatedly discussed (Siddiqi, 1979; Brzeski and Sauer, 1982; Raski and Geraert, 1986b; Brzeski, 1998; Siddiqi, 2000; Geraert, 2008). The two prevailing opinions are either Ottolenchus as a valid genus distinguished from Filenchus spp. by two incisures and ventral curved amphideal aperture (Siddiqi, 2000) or a synonym of Filenchus due to the high variability of lateral incisures (some species show faint interrupted inner lines in SEM) and an amphideal aperture similar with other known Tylenchus spp. and Filenchus spp. (Raski and Geraert, 1987; Andrássy 2007; Geraert, 2008). Molecular analysis indicates the two-incisures Filenchus (Fig. 6A) is separated from four-incisures Filenchus (Figs. 6B,17) and suggests the lateral region is an important character to define taxa (Qing et al., 2017). In such a scenario, we consider Ottolenchus as a valid genus and revised definitions are listed in Table 4. Given that SEM and other informative character are largely unknown in Filenchus or Ottolenchus, any action allocating species to one of the genera is difficult. Here we forward three taxonomic proposals for current Filenchus/Ottolenchus species: (i) species that fit definitions listed in Table 3 should move to Malenchus, (ii) species with two clear incisures, no pouch-like amphideal fovea, and nonswollen vaginas should move to Ottolenchus (further splits into more genera are still possible, as several molecular lineage present, but so far without morphological support), (iii) type species of Filenchus (F. vulgaris) bear four incisures, thus all four-incisures species should stay in Filenchus. Probably some Tylenchus species also need to be included in the latter group.

Fig. 17.

Fig. 17

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Phylogenetic position of the genus Malenchus in Tylenchomorpha. A. Inferred from 28S rDNA. The genus Malenchus is separated in two separated clades without morphological support. Filenchus clade1 has four lateral incisures. Filenchus clade 2 has one ridge forming two incisures. B. Inferred from 18S rDNA, genus Malenchus is monophyletic, Filenchus has three clade/clade groups. Trees modified from Qing et al. (2017). Malenchus clades indicate in blue, Filenchus in red.

Table 4.

Comparison of generic definitions of Ottolenchus.a

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Duosulcius and Zanenchus also have two incisures. The examined paratype D. acutus Siddiqi, 1979 (slide No. UGMD 100227) morphologically fits our proposed Malenchus definition, except for an unknown lateral region (no SEM available) and the absence of a PUS. The paratype of Z. nemorosus (slide No. UGMD100645) was also examined, but no nematode found in the slide. Since no further data were available, the validations for these two genera remain unknown.

Phylogenetic implications

The main limitation of the current molecular phylogenies is the discordance of 18S rDNA and 28S rDNA genes. Specifically, Malenchus appears as a monophyletic genus according to 18S rDNA but splits into two well-supported clades in 28S rDNA (Qing et al., 2017). Although 18S rDNA is expected to be more informative and reliable since for 28S rDNA substations are saturated, the low coverage of the 18S alignment limits its conclusive power (Qing et al., 2017). Furthermore, Lelenchus is closely related to Malenchus and Filenchus clades group 2 according to 28S rDNA phylogenies but sister to Filenchus clades group 1 in the 18S rDNA phylogeny (Yaghoubi et al., 2015; Qing et al., 2016, 2017). However, the latter position is not fully supported (PP = 99, BS = 62) and also possibly influenced by long-branch attraction (Qing et al., 2017). Here we assume Lelenchus together with Ecphyadophoroides and Tenunemellus are sister groups to Malenchus (Fig. 18), based on a combination of morphology and molecular evidence: (i) Lelenchus and Tenunemellus are closely related to Malenchus based on 28S rDNA (Qing et al., 2017, unpubl. data); (ii) both share prominent pouch-like amphideal fovea; (iii) both present a similar S-shaped amphideal aperture and labial sensilla arrangement (see SEM from Raski and Geraert [1985], Brzeski and Sauer [1982]). Interestingly, Ecphyadophora tenuissima de Man, 1921 is, albeit with low support, closely related to Malenchus (Holterman et al., 2008; van Megen et al., 2009), and although this genus has a pore-like amphideal aperture, its inner fovea is very likely to be pronounced (see drawings from Tarjan [1957], Raski et al. [1982]), thus further emphasising the importance of amphideal fovea.

Fig. 18.

Fig. 18

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Cladogram based on informative morphology trait and molecular phylogeny of Qing et al. (2017). The tree shows possible phylogeny relationship of genus Malenchus and other related genera based on morphology characters. Ottolenchus is treated as valid genus (vs synonym of Filenchus [Raski and Geraert, 1986b; Brzeski, 1998; Geraert, 2008]) as two and four incisures Filenchus nested in divergent lineages (see Table 5 and Fig. 17). Character states are arranged as A/B. Character 1. Filiform tail. 2. Monodelphic female. 3. Conspicuous pouch shape amphideal fovea/indistinct amphideal fovea. 4. Vagina wall thin/vagina wall well developed. 5. Bursa rectangular/bursa simple with convex margins. 6. Lateral region one ridge forming two incisures/lateral region with four incisures. 7. Cuticle coarsely annulated/cuticle relatively smooth. 8. Cuticle with longitudinal lines/cuticle without longitudinal lines. 9. Heavily sclerotized stylet with cone half of total length/weakly sclerotized stylet with cone less than half. 10. Large, round amphideal aperture confined to labial plate/slit-like aperture extending 3–4 annuli beyond labial plate.

In conclusion, the phylogeny of Malenchus cannot be resolved based on current approaches. Given that LM-derived characters are relatively less informative for Malenchus and related taxa, any further taxonomic action devoid of SEM and informative molecular data should not be made. Hence, other available or new technologies are needed to extract more informative genes and/or morphological data.

Observations on ecology

The species in genus Malenchus generally appear in an undisturbed environment, preferably forest soil, often associated with moss or litter or aquatic sediments (all known species have at least once reported from these habitats). Occasionally, Malenchus is also found in agricultural fields (few populations from M. acarayensis, M. andrassyi, M. labiatus, M. laccocephalus, and M. ovalis, for details see Table 5). Allocation of the feeding behavior in Tylenchidae is a recurrent discussion point among nematologists (Bongers and Bongers, 1998). Normally, Malenchus species are considered as epidermal and root hair feeders (Bongers and Bongers, 1998) or algal, lichen and moss feeders and parasites of lower and higher plants (Siddiqi, 1986, 2000; Andrássy, 2007). The feeding studies in Tylenchidae (Okada et al., 2002; Okada and Kadota, 2003; Okada et al., 2005) suggested a fungal-feeding habit for three Filenchus species. Our feeding test on four different fungal species and one moss species failed to culture either M. pachycephalus or M. acarayensis. However, we observed numerous brown to green granules consistently presented in the anterior intestine of two analyzed Malenchus species, but not for other fungal feeding nematodes from the same sample (Fig. 3E). Interestingly, such pigments resemble to moss and/or soil algae and this is consistent with the most reported habitats of Malenchus, indicating that moss and/or algae are likely to be a natural food resource. However, the direct feeding on moss or algae was not observed, thus further study is necessary to understand the exact feeding behavior of Malenchus as well as other Tylenchidae.

Table 5.

Recovered habitats of different species in genus Malenchus

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