Family Tylenchidae (Nematoda): an Overview and Perspectives

Organisms Diversity & Evolution (2019) 19:391–408 https://doi.org/10.1007/s13127-019-00404-4

ORIGINAL ARTICLE

Family Tylenchidae (Nematoda): an overview and perspectives

Xue Qing1 & Wim Bert1

Received: 7 February 2019 /Accepted: 11 May 2019 /Published online: 29 May 2019 # Gesellschaft für Biologische Systematik 2019

Abstract Nematodes in the Tylenchidae family are one of the most important soil-inhabiting species, yet little is known about this intriguing group. The present review examines newly collected samples of Tylenchidae from worldwide sources as well as slides from museum collections. Together with all available literature, detailed morphology among genera are summarized and compared, allowing us to explore the importance of each morphological character in a phylogenetic framework. An updated phylogeny inferred from concatenated 18S and 28S ribosomal RNA dataset is reconstructed; the results suggest that not all didelphic genera may be included in Tylenchidae. In fact, our analyses suggest Tylenchidae should be split into several families, although their phylogeny has not yet fully been resolved. Currently, the Tylenchidae family comprises 44 genera and 412 nominal species; however, diversity estimations for the group ranged from 2000 to 10,000 species, meaning that 75–95% of the species remains undiscovered. This is partially because the biased sampling in agro-ecosystems with most Tylenchidae may present in neglected habitats. Finally, we discussed current difficulties in morphology, taxonomy, and molecular phylogeny research of Tylenchidae and the need for multi-gene phylogeny or phylogenomic approaches to resolve the deep phylogeny in Tylenchidae.

Keywords Nematode . Phylogeny . Taxonomy . Tylenchoidea . Tylenchomorpha

Introduction their small body size and lack of clearly homologous characters prevent us from deriving a consistent systematic frame- Tylenchidae is one of the most important soil-inhabiting nem- work. As a result, the delimitation of taxa in this group re- atode families (Andrássy 1981), with species diversity often mains poorly documented and highly uncertain (Bongers and contributing up to about 30% of the nematode community in Bongers 1998; Ferris and Bongers 2006; Yeates 2003). any given soil sample (Ferris and Bongers 2006; Yeates and Molecular phylogeny inferred from ribosomal RNA Bird 1994). As early diverging Tylenchomorpha sensu De Ley (rRNA) genes suggests that Tylenchidae is polyphyletic & Blaxter, 2002 (i.e., tylenchids with supposedly ancestral (Bert et al. 2008; Holterman et al. 2006; Subbotin et al. characters), they do not comprise economically important 2006). Although several recent studies have improved our plant-parasites (Bert et al. 2008; Luc et al. 1987; Siddiqi understanding of this family (Atighi et al. 2013; Pereira et al. 2000). Knowledge of their feeding habits remains limited de- 2017; Qing and Bert 2017;Qingetal.2016, 2017; Yaghoubi spite their abundance and high diversity. Yet, this information et al. 2016), they have focused only on specific groups, and is vital for understanding trophic interactions and their role on information on this topic remains largely scattered and disor- soil foodwebs, which is key to assess soil health. Furthermore, ganized. In this study, we examine 43 additional species newly collected from worldwide sources, as well as species al- ready archived in museum slides and published studies. The Electronic supplementary material The online version of this article goals of the present study are (a) to give an overview of the (https://doi.org/10.1007/s13127-019-00404-4) contains supplementary material, which is available to authorized users. variation of morphological characters in Tylenchidae, (b) to incorporate all currently available sequences and reconstruct * Xue Qing an updated phylogeny of Tylenchidae, (c) to offer a summary [email protected] of Tylenchidae diversity and ecology, and (d) to outline the problems and obstacles faced in such research and therefore 1 Nematology Research Unit, Department of Biology, Ghent provide suggestions for future studies focused on this nema- University, Ledeganckstraat 35, B-9000 Ghent, Belgium tode group. 392 Qing X., Bert W.

Materials and methods empirical observations (Qiao et al. 2018;QingandBert2018; Qing et al. 2016, 2017, 2018). Sample collection and morphological analyses

The morphological analysis was based on newly generated Results and discussion data and a literature review. A total of 43 species were collected from 11 countries (Supplementary Table S1). Soil samples Brief review of historical and current classification were incubated for 48 h on plastic trays lined with paper towels, and nematodes were subsequently concentrated using The family Tylenchidae was originally proposed by Örley a sieve (25 μm opening). After removing water, nematodes (1880), and it contains tylenchids characterized by conoid- were fixed in formalin (4%, buffered to pH = 7), heated to 60– to filiform-shaped tails, non-overlapping pharynx, and short 80 °C, and consequently transferred to anhydrous glycerin for and delicate stylets. The list of genera for the family, according morphological analyses, following the protocol of Seinhorst to different authors, is summarized in Table 1. The main dis- (1962). Drawings from slides were prepared with a drawing pute regarding Tylenchidae classifications concerns the place- tube mounted on an Olympus BX51 DIC microscope ment of four didelphic genera (Antarctenchus, Atetylenchus, (Olympus Optical, Tokyo, Japan), equipped with a Nikon and Psilenchus) and the taxonomic ranking of subfamilies DS-FI2 camera (Nikon Corporation, Tokyo, Japan) for image Ecphyadophorinae and Tylodorinae sensu Geraert (2008). recording. The three didelphic genera are either considered within Morphological data from the literature were summarized Tylenchidae (Geraert 2008; Geraert and Raski 1987)oras on genus level, and relevant illustrations were edited using separated family Psilenchidae (subfamily Psilenchinae: Adobe Illustrator CS3 and Adobe Photoshop CS6 at the orig- Atetylenchus,andPsilenchus; subfamily Antarctenchine: inal scale. Antarctenchus) (Andrássy 2005; Siddiqi 2000). Ecphyadophorinae consists of nine genera (Chilenchus, Ecphyadophora, Ecphyadophoroidea, Epicharinema, Molecular phylogenetic analyses Lelenchus, Mitranema, Tenunemellus, Tremonema,and Ultratenella)(Geraert2008) or considered to be a separate The sequences used for phylogeny analyses are listed in family with eight genera (similar to that in the previous texts, Supplementary Table S2. Multiple sequence alignments were with the exception of Epicharinema)(Andrássy2005;Siddiqi built using the Q-INS-i algorithm as implemented in MAFFT 2000). Since no conclusive taxonomic assignment is possible v. 7.205 (Katoh and Standley 2013), and the different genes based on current available data, we use the most recent system (i.e., 28S and 18S rRNA) were concatenated using Geneious proposed by Geraert (2008) in this study. The pictorial glos- R6 (Biomatters; http://www.geneious.com). The best-fitting sary of terminology used in this study is given in substitution model GTR + I + G for both rRNAs was estimat- Supplementary Fig. S1. ed using AIC in jModelTest v. 2.1.2 (Darriba et al. 2012). Maximum likelihood (ML) analysis was performed with Morphology and their taxonomic significance 1000 bootstrap (BS) replicates under the GTRCAT model using RAxML 8.1.11 (Stamatakis et al. 2008). Bayesian in- Lip region ference (BI) was carried out with the GTR + I + G model using MrBayes 3.2.3 (Ronquist and Huelsenbeck 2003). Analyses Lip regions are usually round, but laterally elongated (dorso- were run for 5 × 106 generations, and Markov chains were ventrally flattened) in Malenchus, Lelenchus, Ecphyadophoroides, sampled every 100 generations. Burnin was arbitrarily chosen Epicharinema,andTenunemellus. Sensilla consist of six inner to represent 25% of the results. The ML and BI analyses were labial papillae + four outer labial papillae, but the former performed at the CIPRES Science Gateway (Miller et al. are not always visible. Amphidial apertures vary in their 2010). appearance, from pore to long slit. Amphidial foveas are considered taxonomically important at generic level (Qing Estimation of diversity and Bert 2017) and are mostly reduced, but they can be pouch-like in Malenchus, Lelenchus, Ecphyadophoroides, The unknown diversity of the family Tylenchidae was estimated and Tenunemellus. Currently, eight patterns are recog- based on the ratios-between-taxa method following Hawksworth nized, seven from Geraert and Raski (1987) and one (1991). The number of described Tylenchomorpha species was addedbyQingandBert(2018) (see the illustration of approximated based on Andrássy (1992) and Siddiqi (2000). The Fig.1inQingandBert(2018)). Siddiqi (2000)and ratio of Tylenchidae species to obligate plant-parasitic Andrássy (2005) rejected the lip pattern as being impor- Tylenchomorpha species number was estimated based on our tant, as it can vary intraspecifically. This view was also Family Tylenchidae (Nematoda): an overview and perspectives 393

Table 1 Comparison of the taxonomic content of Tylenchidae Authors Maggenti et al. (1987) Siddiqi (2000)Andrássy(2007) Geraert (2008) according to four widely used classifications Genera 33 genera 25 genera 29 genera 42 genera Subfamilies Atylenchinae, Boleodorinae, Boleodorinae, Atylenchinae, Boleodorinae, Duosulciinae, Duosulciinae, Boleodorinae, Tylenchinae, Tanzaniinae, Thadinae, Ecphyadorinae, Ecphyadorinae, Thadinae, Tylenchinae, Tylenchinae, Tylodorinae Tylenchinae Tylodorinae Tylodorinae

supported by a recent study showing that lip pattern VIII to Miculenchus (Figs. 1b, 2d); (c) coarsely annulated, with (superficially similar, although the internal anatomy re- longitudinal ridges: the cuticle surface outside the lateral fields mains unknown) is the result of convergence evolution shows minute squares or rectangles. The number of these (Qiao et al. 2018). Nevertheless, the main patterns longitudinal ridges is either fixed at genus level (e.g., (amphidial aperture shape and location, labial plate shape, Eutylenchus has 10, excluding ridges in lateral fields) or sensilla arrangement) are generally well-conserved and intragenerically variable and used as a species delimitation congruent with molecular-based phylogeny (Qing and character (e.g., Coslenchus has 10–34 and Neothada has 12– Bert 2018), thus being of taxonomic importance. 20, excluding ridges in lateral fields) (Figs. 1c, 2a); (d) smooth in light microscopy (LM) but faintly annulated in scanning Cuticle and lateral region electron microscopy (SEM). This has been used as a generic character for Polenchus (Figs. 1d, 2e); (e) pronounced annu- The cuticle in Tylenchidae generally has six patterns, which lation in the lip region only, annuli extending twice as far are as follows: (a) marked only with transverse annuli. The posteriorly in the lateral as in the dorsal and ventral zones, width and thickness of the annuli and the presence of grooves but not past the base of the stylet. The rest of the body is between two annuli vary among genera. This is the predomi- marked with longitudinal ridges and deep grooves in-between. nant pattern and present in most genera in Tylenchidae This pattern is known for Campbellenchus and Ridgellus (Figs. 1a, 2c); (b) deep, transverse zigzag striae. This is unique (Figs. 1e, 2b); (f) the surface of the cuticle has longitudinal

Fig. 1 The illustration of cuticle annulation patterns in Tylenchidae. a Cuticle only marked with transverse annuli: this is the most common pattern in Tylenchidae. b The zigzag transverse annuli: only found in Miculenchus. c Cuticle with longitudinal ridges or grooves that divide the surface into minute squares or rectangular blocks: this pattern is presented in Atylenchus, Coslenchus, Ecphyadophoroidea, Eutylenchus, Neothada, Pleurotylenchus,andTanzanius. d Cuticle appears smooth in LM, but with shallow annulation: this pattern is found in Allotylenchus, Polenchus,andLelenchus. e The distinct transverse annuli only in lip region, other part of body marked by longitudinal ridges: this pattern is only known for Campbellenchus. f Cuticle marked with transverse annuli but surface has shallow longitudinal striae: this is represented in some of Malenchus species, e.g., M. nanellus Siddiqi, 1979 394 Qing X., Bert W.

Fig. 2 Photomicrographs of cuticle and lateral patterns in the family hexalineatus (Geraert, 1962) Geraert & Goodey, 1964. g Filenchus Tylenchidae. a Coslenchus costatus. b Ridgellus elenae (Geraert & vulgaris. h Filenchus cylindricus (Thorne & Malek, 1968) Niblack & Raski, 1986) Siddiqi 2000. c Malenchus pachycephalus. d Miculenchus Bernard, 1985. i Malenchus acarayensis. j Malenchus pachycephalus salvus Andrássy, 1959. e Filenchus vulgaris. f Cephalenchus striae, although these are very faint (probably solely in the number of incisures in Tylenchidae is largely convergent: an epicuticle) and only visible in SEM. This is known in some equal number can be found in different genera, while it is of Malenchus species (Fig. 1f). common that one genus has a variable number of incisures The lateral fields, characterized by longitudinal incisures, (e.g., two or four incisures in Basiria, four or six incisures in are very heterogeneous in Tylenchidae but correspond re- Cephalenchus). markably well with clades in phylogenetic trees (Qing and Bert 2017;Qingetal.2017). Generally, there are five patterns, Stylet which are as follows: (a) four incisures, resulting from two elevated ridges separated by wide grooves (e.g., Fig. 3a, c, d) Stylets are generally thin and short, but they vary widely or three ridges separated by narrow grooves (e.g., Figs. 2g, h, (Figs. 4, 5, 6). The length ranges from 4 μm(Filenchus 3f, j, n), the latter being the most common pattern; (b) two spp.) to 120 μm(Tylodorus spp.). The conical part is usually incisures, resulting from one broad (e.g., Fig, 3m)ornarrow shorter than or equal to the shaft length, but it can be longer in (e.g., Fig. 3b, g, o) ridge; (c) lateral region undifferentiated Tylodorus. In most species, the cone is straight, connects to the (e.g., Fig. 3e, i) or invisible in LM, but with shallow incisures shaft with a comparable width, and tapers sharply anteriorly, in the lateral region visible under SEM (e.g., Fig. 3h); (d) one but it can also be cylindrical or a dorsally or ventrally bent offset ridge but several sub-ridges forming 14–22 incisures cone (e.g., Neopsilenchus spp., Fig. 4l). The knobs vary great- (two incisures are visible in LM): typical for most species in ly in absence/presence, size and shape: commonly round in Malenchus (Figs. 2i, j, 3l); (d) five ridges forming six shape and perpendicular to the shaft (e.g., Coslenchus, incisures: this pattern is present in most species of Aglenchus,andFilenchus spp.), directed backwards (e.g., Cephalenchus (Figs. 2f, 3k), some species in Boleodorus Malenchus,mostofTylenchus and Filenchus spp.) or anteri- and in Malenchus williamsi Geraert & Raski (1986). The orly directed (e.g., some of Aglenchus spp.); in some cases, Family Tylenchidae (Nematoda): an overview and perspectives 395

Fig. 3 Illustration of cross section in different Tylenchidae genera. a Aglenchus sp. b Atylenchus sp. c, d Coslenchus spp. e Pleurotylenchus sp. f Campbellenchus sp. g Tanzanius sp. h Lelenchus sp. i Ridgellus sp. j Basiria sp. k Cephalenchus sp. l Malenchus sp. m–o Filenchus spp. knobs are minute and flange-like (Basiria and Boleodorus); Basiria, Boleodorus,andCephalenchus, Coslenchus costatus the stylet can also be cylindroid and without knobs (de Man 1921) Siddiqi 1978). The spermatheca shows several (Psilenchus, Neopsilenchus, Chilenchus, Atetylenchus, and variations in cellular architecture within the Tylenchidae fam- Basiria gracilis (Thorne 1949) Siddiqi 1963); Cephalenchus ily but usually comprises 10–16 cells. Two large cells are and Tanzanius have a stylet with large, flatted/elongated, usually present connecting the spermatheca to the uterus. knobs; and, more rarely, Irantylenchus and Antarctenchus The uterus cells are usually arranged in four regular rows, each have amalgamated knobs. Although these differences are spe- comprising 4–9 cells (Bert et al. 2006). The posterior uterus cies-/genus-specific, their significance for species identifica- sac (PUS) is rudimentary, usually not greater than one vulval tion is limited due to low observational resolution in LM. body width, but absent in Aglenchus, Coslenchus, Nevertheless, with improvements in visualization technolo- Fraglenchus, Gracilancea,andcertainFilenchus species gies, variations in stylet morphology may become more im- (species belonging to the former Duosulcius and portant in the future. Zanenchus)—the absence of a PUS has been used as a generic character. Reproductive system Vulva in Tylenchidae are delimited by a gradual depression of the cuticle that forms a wide sink (e.g., Coslenchus, The ovary is outstretched with oocytes usually arranged in a Aglenchus, Malenchus, Fig. 7a–e), a sharp and narrow sink single row. In certain cases, the oocytes are arranged in two of one annulus (this being the most common type, e.g., rows, and this can be used as a species-specific character (e.g., Filenchus, Lelenchus, Basiria, Boleodorus, Fig. 7j), or an el- Boleodorus acurvus Jairajpuri 1982). The oviduct in evated cone (found only in Eutylenchus,Fig.7f). Epiptygmata Tylenchidae comprises two rows of three to seven cells. The are usually small, sometimes only visible in SEM (Aglenchus, oviducts of Tylenchus, Filenchus, Coslenchus,andAglenchus Coslenchus, Fraglenchus, Gracilancea,andMalenchus), ex- are composed of two rows of three or four cells. In Basiria, cept for Silenchus, whose epiptygma forms a distinct beak- Boleodorus, Neopsilenchus,andPsilenchus,five(occasional- like projection (Fig. 7g). The vulva is mostly open, but it can ly six) cells per row are present, with the most proximal ovi- also be covered by a longitudinal flap (Atylenchus)orbor- duct cells usually being slightly larger; Cephalenchus is char- dered by lateral flaps that are either wide (Aglenchus, acterized by a longer and slightly bent oviduct that comprises Coslenchus, Fraglenchus, Gracilancea, Eutylenchus,and two rows of five, six, or seven cells. The spermatheca is offset Cephalenchus)orsmall(Malenchus). The thickness of mus- (e.g., most species in Filenchus and Tylenchus, Boleodorus cles attached to the vaginal wall is an important generic de- thylactus Thorne 1941) or axial (e.g., most species in limitation character in some genera (Qing and Bert 2017). It is 396 Qing X., Bert W.

Fig. 4 Illustration of stylet in different Tylenchidae genera. a Filenchus n Tylenchus davainei Bastian, 1865. o Irantylenchus vicinus (Szczygiel, discrepans (Andrássy, 1954) Andrássy, 1972. b Ultratenella vitrea 1969) Brzeski & Sauer, 1983. p Coslenchus costatus. q Aglenchus Siddiqi, 1994. c Tenunemellus graminis (Husain & Khan, 1968) Siddiqi agricola. r Tylenchus maius Andrássy, 1979. s Basiria duplexa 1986. d Filenchus macramphis (Siddiqi & Lal, 1992) Brzeski, 1997. e (Hagemeyer & Allen, 1952) Geraert, 1968. t Basiria paragracilis Chilenchus elegans (Raski & Geraert, 1985) Siddiqi 2000. f Lelenchus Geraert & Raski, 1986. u Antarctenchus hooperi Spaull, 1972. v leptosome. g Atetylenchus abulbosus (Thorne, 1949) Khan, 1973. h Gracilancea graciloides (Micoletzky, 1925) Siddiqi, 1976. w Tanzanius coffeae Siddiqi, 1991. i Malenchus exiguus (Massey, 1969) Epicharinema keralense Raski, Maggenti, Koshy & Sosamma, 1980. x Andrássy, 1980. j Malenchus andrassyi Merny, 1970. k Filenchus Tylodorus acuminatus Meagher, 1964. Scale bar: A–W=10 μm, X = thornei (Andrássy, 1954) Andrássy, 1963. l Neopsilenchus magnidens 20 μm (Thorne, 1949) Thorne & Malek, 1968. m Cephalenchus hexalineatus. thin in most species, but swollen either in the more distal part Tail (Aglenchus and Coslenchus,Fig.7a–c) or in the proximal to middle part of the vagina (Malenchus,Fig.7d, e). The filiform tail is quite heterogeneous (Fig. 9). Being filiform In male specimens, the most remarkable character is the is most likely a result of convergence evolution and hence less variation of male bursa (Fig. 8), ranging from long informative. (Silenchus, considered as a generic character, Fig. 8b)toshort Tail morphology displays the greatest variation in (Fig. 8a), absent (Miculenchus, Atylenchus, and Tanzanius, Filenchus: tail length ranges from around 30 to 300 μmand Fig. 8c) to lobed (e.g., Epicharinema, Tenunemellus, the general shape varies from attenuated (c = 8.2–8.9, c′ = Tremonema,Fig.8e, f, g). The variation in spicules has not 5.3–7) to extremely filiformed (c = 2.4–2.6, c′ =32–37). In been used to characterize Tylenchidae species as, except for the majority of Boleodorinae, the tail is shorter, mostly rang- Ecphyadophorinae with relatively straight and long spicules, ing from 50 to 80 μmwithac value ranging from 5 to 13. no useful morphological differences can be observed. Similarly for Malenchus and Tylenchus, a tail ranging between However, a recent study shows that the dissection of the spic- 12 and 60 μminlength,withc values between 4 and 7 are ule can allow the visualization of informative structures (e.g., common. Conversely, species belonging to spicule tips) that are invisible in vivo (Qing and Bert 2017). Ecphyadophorinae are extremely slender, their tails thin and This suggests that morphological characters may be long, reaching up to 350 μm(e.g.,Chilenchus, Epicharinema) overlooked in regular LM observations. in length, with c values that can be less than 2 (some Family Tylenchidae (Nematoda): an overview and perspectives 397

Fig. 5 LM pictures from different genera in family Tylenchidae. a Coslenchus alacinatus Siddiqi, 1981. q Coslenchus turkeyensis Siddiqi, Cephalenchus hexalineatus. b Cephalenchus leptus Siddiqi, 1963. c 1981. r Coslenchus costatus. s Coslenchus maritus Andrássy, 1991. t Malenchus acarayensis. d Malenchus pachycephalus. e Miculenchus Ridgllus elemae (Geraert & Raski, 1986) Siddiqi 2000. u Tenunemellus salvus. f Irantylenchu vicinus. g Tylenchus davainei. h Basiria aberrans sheri (Raski, Koshy & Sosamma, 1982) Siddiqi 1986. v Aglenchus (Thorne, 1949) Siddiqi, 1963. i Basiria graminophila Siddiqi, 1959. j ainakamurae Mizukubo, 1989. w Aglenchus agricola. x Labrys Boleodorus thylactus. k Neopsilenchus magnidens. l Psilenchus chinensis. The sources of species were listed in Electronic aestuarius Andrássy, 1962. m Lelenchus leptosome. n Lelenchus supplementary material. Scale bar = 10 μm schmitti Bernard, 2005. o Coslenchus lateralis Andrássy, 1982. p 398 Qing X., Bert W. Family Tylenchidae (Nematoda): an overview and perspectives 399

Fig. 6 LM pictures from different Filenchus species showing the Phylogeny and evolution of Tylenchidae variation of anterior body. a Filenchus terrestris Raski & Geraert, 1986. b Filenchus andrassyi. c Filenchus annulatus (Siddiqui & Khan, 1983) Siddiqi 1986. d Filenchus sp. 1. e Filenchus sp. 2. f Filenchus sp. 3. g Our concatenated phylogeny (Fig. 10) based on 28S and 18S Filenchus sp. 4. h Filenchus filipjevi Andrássy, 1988. i Filenchus sp. 5. j rRNA genes concurs with previous studies of Tylenchidae Filenchus balcarceanus Torres & Geraert, 1996. k Filenchus quartus (Pereira et al. 2017;Qingetal.2017). The results highlight (Szczygiel, 1969) Lownsbery & Lownsbery, 1985. l Filenchus misellus. the difficulties associated with this taxonomically notorious m Filenchus hamatus (Thorne & Malek, 1968) Raski & Geraert, 1986. n Filenchus sandneri (Wasilewska, 1965) Raski & Geraert, 1986; o group: most genera are not monophyletic (except for Filenchus butteus (Thorne & Malek, 1968) Raski & Geraert, 1986. p Coslenchus, Aglenchus, Boleodorus, and Miculenchus) and Filenchus angustatus Brzeski, 1997. q Filenchus sindhicus Shahina & clades are weakly supported. The former is likely caused by Maqbool, 1994. r Filenchus vulgaris. s Filenchus discrepans. t Filenchus inappropriate generic definition characters owing to a lack of sp. 6. u Filenchus sp. 7. v Filenchus cylindricus. w Filenchus sp. 8. x Filenchus orbus Andrássy, 1954. The sources of the pictured species are detailed morphological analysis (Qing and Bert 2017). The listed in Electronic supplementary material. Scale bar = 10 μm latter may be a result of the coverage limitation (scarcity of homologous sites in an alignment of current available sequences) (Qing et al. 2017) or a complex evolutionary history Lelenchus leptosoma (de Man 1880) Andrássy, 1954 popula- in Tylenchidae (e.g., reticulate evolution, recombination). tions), with only a few shorter exceptions (e.g., the tail in Tylenchidae shows many supposedly ancestral morpholog- Ecphyadophora caelata Raski & Geraert 1985 is around ical characters (e.g., weak stylets and median bulbs, basal 50 μm long). Tylodorinae also have long tails; in bulbs with a full complement of non-glandular cells, long Campbellenchus, the tail can reach up to 450 μm, which is tails, uterus cells that are arranged in 4 rows) (Baldwin et al. the longest tail in Tylenchidae. 2001;Bertetal.2008; Siddiqi 2000). They display apparently

Fig. 7 Vulval regions in different genera of Tylenchidae. Illustration distinct beak-like projection, vagina with swollen muscle in proximal or partly modified from Qing and Bert (2017). a–c Vulva with wide flap, middle part, e.g., Silenchus. h Vulva covered by a longitudinal flap, e.g., vagina with swollen muscle in distal part, e.g., Aglenchus and Atylenchus. i Vulva with wide flap, vagina not or slightly swollen, e.g., Coslenchus. d, e Vulva with small or without flap, vagina with swollen Cephalenchus. j Vulva without flap, thin and straight wall without muscle in more proximal or middle part, e.g., Malenchus. f Vulva swollen muscle attached. This is the most common type in Tylenchidae, elevated, with flap, e.g., Eutylenchus. g Epiptygmata large, forming a e.g., Filenchus and Tylenchus 400 Qing X., Bert W.

Fig. 8 Male tail region showing the variation of bursa in different genera Atylenchus and Tanzanius. d, e Bursa flaps rectangular, lobed, of Tylenchidae. a The short adanal bursa, present in most genera of projecting outward and backward, e.g., Tenunemellus. f Bursa narrow, Tylenchidae. b Large and long bursa, reaching almost to midway on lobed with narrow tip, projecting outward and backward, e.g., tail, e.g., Silenchus. c Male without bursa, e.g., Miculenchus, Tremonema. g Bursa large, elongate-oval, flap-like, e.g., Epicharinema primitive feeding habits (algal and moss feeding) (Siddiqi are generally low (Bert et al. 2008; Holterman et al. 2006; 1986;Siddiqi2000), and the embryology of Tylenchidae is Qing et al. 2017; van Megen et al. 2009). Our concatenated similar to that of the bacteria feeder Cephalobidae (Dolinski phylogeny (Fig. 10) with limited sampled taxa suggests et al. 2001). Consequently, tylenchid nematodes have been Anguinidae and Sphaerularioidea to be monophyletic, albeit divided into early diverging tylenchids (i.e., tylenchids with with moderate support, but their relationships with supposedly ancestral characters)—group including Tylenchidae remain unresolved. Furthermore, these new anal- Tylenchidae, Anguinidae, and Sphaerularioidea sensu De yses concur with several studies on the divergent placements Ley and Blaxter (2002) and more derived group (tylenchids of Psilenchus, Atetylenchus, Cephalenchus, Eutylenchus, with supposedly derived characters, e.g., strong and well- Malenchus, Lelenchus, Miculenchus, Labrys, Ecphyadophora, developed stylet, which allows them to be obligated plant- and Tenunemellus (Pereira et al. 2017;Qiaoetal.2018;Qing parasites, i.e., Meloidogynidae, Pratylenchidae, Heteroderidae, and Bert 2018;Qingetal.2017, 2018). Consequently, there is Hoplolaimidae) that includes the remaining tylenchid taxa (e.g., no phylogenetic support for the placement of these genera in Siddiqi 2000). With limited taxa sampling, current molecular Tylenchidae. phylogenies inferred from 18S rRNA gene have divergent conclusions: either congruent with classical views (Bert et al. 2008) Genera placements: an update based or with Tylenchidae as sister to Criconematoidea within other on concatenated analyses of 18S and 28S rRNAs derived nematodes (Holterman et al. 2006;vanMegenetal. 2009). Our phylogeny (Fig. 10) suggests that the early diverg- Tylenchinae is the most heterogeneous subfamily in ing tylenchids are well-separated from tylenchids with more Tylenchidae, since several genera are polyphyletic (Fig. 10). derived traits. However, the support values for the backbone Filenchus is clearly a polyphyletic genus, as shown by sub- in all the aforementioned studies are low, and deeper nodes of stantial molecular evidence (Atighi et al. 2013; Bert et al. early diverging Tylenchomorpha remain unresolved. 2008; Holterman et al. 2006; Pereira et al. 2017;Qingetal. The relationship between Tylenchidae, Anguinidae, and 2017). This is not surprising, as Filenchus is characterized by Sphaerularioidea is subject to discussions. Morphology- the absence of characters rather than based on the presence of based phylogenetic concepts suggest Tylenchidae to be either apomorphies. Current molecular phylogeny suggests that all more closely related to Anguinidae and not to the four-incisured Filenchus form a well-supported clade Sphaerularioidea (Andrássy 2005; Brzeski 1998; Maggenti (Qing et al. 2017) and in addition, four-incisured et al. 1987; Siddiqi 2000), sister to both Sphaerularioidea Filenchus + Tylenchus + Irantylenchus forms another well- and Anguinidae (Ryss 1993; Siddiqi 1986) or in a broader supported clade (Fig. 10). Since the type species (Filenchus concept of the Tylenchidae that includes Anguinidae and at vulgaris (Brzeski 1963) Lownsbery & Lownsbery 1985) is least part of the Sphaerularioidea (Raski and Maggenti 1983). nested within this clade, in each possible review, this clade Phylogenies based on 18S and 28S rRNA genes reject mono- should retain the genus name. The two-incisured Filenchus phyly for these three scenarios. Additionally, branch support species (Ottolenchus) have more complex relationships as values to underpinning the relationships between these groups they form several clades and potentially need to be splitted Family Tylenchidae (Nematoda): an overview and perspectives 401

Fig. 9 The tail in different genera of Tylenchidae. a, b Filenchus flagellicaudatus Bernard, 2005. c Tenunemellus sheri. d, e Epicharinema keralense. f, g Malenchus exiguus. h, i Boleodorus clavicaudatus Thorne, 1941. j Psilenchus hilarus Siddiqi, 1963. k, l Basiria tumida (Colbran, 1960) Geraert, 1968. m, n Filenchus misellus. o, p Lelenchus elegans Raski & Geraert, 1985. q, r Ecphyadophoroides annulatus Corbett, 1964. s, t Filenchus terrestris Raski & Geraert, 1986. u, v Aglenchus Agricola. w, x Tylenchus davainei. Scale bar = 50 μm

into several genera. Future studies should be well-advised to Filenchus clade, indicating that nematode size may be more pay further attention to detailed morphology; for example, the important than previously suggested. Malenchus represents a newly described two-incisured genus Labrys is clearly mor- divergent linage from other Tylenchinae genera (Fig. 10). This phologically different from Filenchus, but based on superfi- genus appears to be related to some of the Ecphyadophorinae, cial observations this genus could be misidentified as supported by a shared pouch-like amphidial fovea. However, Filenchus. Tylenchus is also a polyphyletic genus, since tail molecular tree topologies (Qing et al. 2017) provide different shape and stylet cone/shaft proportions do not define a natural or even contradictory conclusions and their actual placement clade. Large-sized (above 700 μm) Tylenchus are related to remains unresolved. Recent molecular phylogenies suggested certain Filenchus (Filenchus aquilonius (Wu 1969) Miculenchus either as sister of Malenchus, Lelenchus, and Lownsbery & Lownsbery 1985 and Filenchus andrassyi Tenunemellus or Labrys fujianensis (Qiao et al. 2018) depend- (Szczygiel 1969) Andrássy 1979) of similarly large size (such ing on the gene and the alignment methods used (Qing et al. asizeisrareinFilenchus). Similarly, small-sized Tylenchus 2018). Although long-branch attraction is unlikely to explain spp. (Tylenchus arcuatus Siddiqi 1963) are placed in a the divergent placement of Miculenchus (Qiao et al. 2018), 402 Qing X., Bert W. Family Tylenchidae (Nematoda): an overview and perspectives 403

Fig. 10 Phylogenetic analysis of the family Tylenchidae inferred from the phylogenetic importance of the number of genital concatenated sequences of the D2–D3 domains of the 28S and 18S rRNA branches (Bert et al. 2008). Moreover, the vulval flap and gene. For separated 28S and 18S phylogenies, see Qing et al. (2017). Both Bayesian inference (BI) and maximum likelihood (ML) methods epiptygmata do not display the same pattern observed in were used for tree reconstruction, but only ML topology is shown. Branch Coslenchus and Aglenchus. support values are providing as BI/ML. The sequences used for this The Boleodorinae is relatively well-defined by the pres- phylogeny are listed in Electronic supplementary material.Asterisk(*) ence of a distinctive slit-like amphidial aperture. denotes significance level of the support values: *, 50–80; **, 80–100 for ML bootstrap; *, 50–90, **, 90–100 for BI posterior probability; −,not Furthermore, each of the genera has an obvious genus- supported. The most important morphological supports for each clade are specific trait. All monodelphic genera (represented by illustrated on right side of the tree. 1 Labial plate deeply and broadly Boleodorus, Basiria, Neopsilenchus, Neothada) form a well- indented dorsally and ventrally. 2 Labial region offset from body by supported monophyletic clade (e.g., Yaghoubi et al. 2015). distinct constriction, four lobed, and each lobe with a seta. 3, didelphic reproductive system. 4 Four incisures in lateral region. 5 Stylet with However, Psilenchus and Atetylenchus have a didelphic repro- pronounced knob. 6 Amphidial apertures bound on peri-oral disc, cuticle ductive system and molecular phylogeny separate these gen- coarsely annulated with longitudinal ridges. 7 Amphidial apertures bound era from other Tylenchidae (Bert et al. 2008; Holterman et al. on peri-oral disc, cuticle without longitudinal ridges. 8 Vagina with swol- 2006). The genus Psilenchus is in fact the subject of len wall in distal part. 9 Amphidial apertures are broad oblique slits, starting posterior to the level of four cephalic papillae and continuing to longstanding discussions as to whether it is an early diverging sides of head. 10 Tail short and conical. 11 Stylet with flange-like knobs. genus of all Tylenchids, (Luc et al. 1987)orinsteadagenus 12 Lateral field with offset ridge, comprising small sub-ridges. 13 Vagina with a derived position in Tylenchids, only with ancestor char- with swollen wall in proximal or middle part. 14 Head can be dorso- acters of Hoplolaimids (Siddiqi 2000). Psilenchus has been ventrally compressed or more rounded, with pouch-like amphidial fovea. 15 Bursa lobed, rectangular. 16 Cephalic region strongly flattened dorso- removed from Tylenchidae in several studies (Andrássy ventrally, lacking labial disc, long amphidial aperture, large pouch-like 2005; Siddiqi 2000), in agreement with the importance of amphidial fovea. 17 Lateral region with one offset ridge forming two the presence of a didelphic vs a monodelphic reproductive incisures. 18 Cephalic region continuous, very less framework, stylet system in tylenchid phylogeny (Bert et al. 2008). knob minute and separate. 19 Long slit-like amphidial aperture, large pouch-like amphidial fovea. 20 Labial plate that has four narrow lobes Species belonging to Ecphyadophorinae are among the with tips detached from the adjacent cuticle. 21 Amphidial aperture elon- most remarkable of all Tylenchidae. So far, little is known gated, slender, dorso-ventrally oriented entirely on labial plate, cuticle about this subfamily, but several morphological traits (variable with zigzag pattern. 22 Male without bursa. head, amphidial aperture, vulva, and bursa shape) and molecular data (Lelenchus and Ecphyadophora are separated in 18S tree reconstruction and alignment errors are possible because rRNA phylogeny) suggest that Ecphyadophorinae is a hetero- of the extremely divergent sequences. Tanzanius is unique in geneous group. Currently, no specific trait has been found for Tylenchomorpha in the shape and structure of the stylet and this subfamily except the extremely slender body (Geraert pharynx. The pharynx and the short tail length of Tanzanius 2008;Siddiqi2000). Ecphyadophora is probably related to are divergent from other Tylenchinae or Tylenchidae and quite Tremonema and Mitranema based on its similar pore-like probably related to Paratylenchidae as suggested by Andrássy amphidial aperture and lobed bursa. It is differentiated from (2005). However, molecular data for this genus are currently other Ecphyadophorinae by a pore-like amphidial aperture unavailable, and, therefore, a taxonomic assignment is not yet and the absence of a pouch-like amphidial aperture. possible. Molecularly, Ecphyadophora is grouped with Filenchus Within the Atylenchinae, only Aglenchus and Coslenchus misellus (Andrássy 1958) Raski & Geraert 1986, Filenchus are monophyletic (Fig. 10). The monotypic genus Atylenchus chilensis Raski & Geraert 1986, and Labrys chinensis Qing is rendered unusual by the cephalic setae, the male being ab- and Bert 2018.However,Ecphyadophora is the type genus for sent of bursa and the vulva covered by long flap. The relative- the subfamily, and, thus, the validation of Ecphyadophorinae ly long stylet (16–19 μm) and slit-like amphidial aperture should be reconsidered; Ecphyadophoroides, Lelenchus,and entirely located in the labial plate suggest that this genus Tenunemellus may be closely related, based on a similar long may be more closely related to Cephalenchus and slit-like amphidial aperture and distinct amphidial fovea, a view Eutylenchus.WhilePleurotylenchus is also characterized by that is further supported by molecular phylogeny (Qing et al. being coarsely annulated and having prominent longitudinal 2018). Epicharinema has an unknown position, displaying a ridges, similar longitudinal ridges have originated several dorso-ventrally fattened cephalic region (probably because of times independently in Tylenchidae (e.g., Coslenchus, pouch-like amphidial aperture) and long slit-like amphidial ap- Neothada, Eutylenchus, Ridgellus). Furthermore, the vaginal erture, which resemble those of Ecphyadophoroides, wall is not swollen, thus disputing its close relation to Lelenchus,andTenunemellus. However, the pronounced medi- Coslenchus. Since the nature of the lip region is unknown an bulb with a valve in the pharynx and well-developed stylet and molecular data are missing, the position of this genus suggests that it may represent a different lineage. The genus remains uncertain. The didelphic Antarctenchus is more likely Ultratenella was assigned to Ecphyadophorinae, only by virtue to be related to other didelphic general in Tylenchidae, due to of its exceedingly thin body. However, it is probably related to 404 Qing X., Bert W.

Ecphyadophora based on its similar vulval region and working on this group, two new genera have been reported amphidial aperture (described not observed for Ultratenella (Qing and Bert 2018; Yaghoubi et al. 2016) in the last two but probably pore-like as such apertures are hardly visible under years alone. It is very likely that only a fraction of the species LM). of Tylenchidae is known: (a) most have been described from Limited information is available for the Tylodorinae. Our the rhizosphere of economically important crops, while natu- phylogeny concurs with a previous study (Pereira et al. 2017) ral ecosystems (e.g., forest, meadow, and swamp) harbour a that Cephalenchus and Eutylenchus are related, but in a diver- significantly higher diversity of Tylenchidae compared to gent phylogenetic position with respect to other Tylenchidae. agro-ecosystems; (b) nearly all Tylenchidae species are de- The genus Campbellenchus is probably also related to scribed from soil habitats, while both morphological and Cephalenchus and Eutylenchus because of the similar stylet metabarcoding studies (Porazinska et al. 2010; Qing et al. and amphids and forms a unique clade related to 2015) have found a high diversity of Tylenchidae in leaf litter Criconematoidea and some Tylenchidae. However, additional and/or forest canopy (for example, 80% of the species found molecular data are needed to confirm this view. Tylodorus and in temperate forests reside in the soil, compared with only Arboritynchus have extremely long stylets and a different 20% of the species found in the tropics); (c) metabarcoding pharynx (procorpus bulbous vs elongated in most studies suggest that tropic nematode diversity is significantly Tylenchidae), and these two genera may in fact be more close- higher compared to much more frequently sampled temperate ly related to Criconematoidea. environments (Porazinska et al. 2010, 2012); and (d) the family Tylenchidae comprises cryptic species (e.g., Malenchus Feeding habits pachycephalus, Malenchus acarayensis Andrássy 1968), and, therefore, some of the nominal species can actually be Allocation of the feeding habits in Tylenchidae is one of the species complexes. most important discussion points among nematologists Several approaches have been developed to estimate spe- (Bongers and Bongers 1998). They are usually treated as root cies diversity, such as those based on body size frequency hair feeders (Bongers and Bongers 1998) or algal, moss, and distributions (May 1988), host-specificity and spatial ratios fungal feeders (Okada et al. 2002;Siddiqi2000). Although (Erwin 1982), time-species accumulation curves (Bebber they may also be parasites of lower and higher plants et al. 2007), patterns of higher taxonomic classification (Andrássy 2005; Siddiqi 1986; Siddiqi 2000), they do not (Bartels et al. 2016;Moraetal.2011), and those based on cause economic losses to crops. The available studies show metabarcoding (Ni et al. 2013). However, these methods ei- contrasting information about their feeding behaviors: ther require massive data collection and subsequent analyses Malenchus bryophilus (Steiner 1914) Andrássy 1981, or are based on assumptions that do not necessarily hold true Aglenchus agricola (de Man 1884) Andrássy 1954, for Tylenchidae. Although a definitive estimation is not pos- Coslenchus costatus, Cephalenchus emarginatus (Cobb sible based on the data obtained in this study, we have 1893) Geraert 1968, and Tylodorus fisheri Reay 1991 have endeavoured to provide a rough estimation of Tylenchidae been described as feeding on roots of higher plants species number based on the ratios-between-taxa method fol- (Andrássy 1976;Gowen1971; Hooper 1974;Kheraand lowing Hawksworth (1991). Based on the samples examined Zuckerman 1963; Sutherland 1967; Wood 1973); during this and previous studies (Qiao et al. 2018;Qingand Ottolenchus cabi Siddiqi & Hawksworth, 1982 is associated Bert 2018;Qingetal.2016, 2017, 2018) (from tropical re- with a lichen (Siddiqi and Hawksworth 1982); Malenchus gions, temperate Europe, and temperate and subtropical Asia), pachycephalus Andrássy, 1981 probably feed on moss (Qing we observe that in a given sample, Tylenchidae species are 1– and Bert 2017); and Filenchus spp. can be cultured on several 5 times more diverse compared to obligate plant-parasitic species of fungi (Okada et al. 2005). Based on this evidence, Tylenchomorpha (PPT). Given that the infraorder feeding behavior in Tylenchidae, although largely completely Tylenchomorpha contains 2240 (Andrássy 1992)or2828spe- unknown, is clearly genus- or even species-specific. cies (Siddiqi 2000) while ca. 400 and 300 species are from the family Tylenchidae and the superfamily Sphaerularioidea (in- Diversity: estimating a species number sect parasitic and/or mycophagous nematodes), respectively, for Tylenchidae the PPT comprise 1500–2100 species. Furthermore, in the context of economic importance for crops and higher plants, Currently, the family Tylenchidae comprises a total of 44 gen- PPT are relatively well-studied. Assuming that PPT species era and 412 nominal species. The most cosmopolitan genus is are relatively well-described and given that, based on our ob- Filenchus, having been reported from all continents except servations, Tylenchidae are up to five times more diverse com- Antarctica, while 17 genera are monotypic to very limited pared to PPT, around 2000–10,000 Tylenchidae species can locations or endemic. The actual diversity of this group is far be estimated, with 75–95% remaining undescribed. However, from being settled. Despite very few taxonomists worldwide this estimation remains very conservative, given the fact that Family Tylenchidae (Nematoda): an overview and perspectives 405

PPT number is still increasing, more species are expected to in GenBank reference sequences result in a scarcity of homol- be discovered and taking into account the presence of cryptic ogous sites for accurate alignment (due to coverage limita- species in PPT (Palomares-Rius et al. 2014). tions). Moreover, 18S rRNA has an inadequate amount of The diversity of Tylenchidae is best known in European informative sites to resolve early diverging Tylenchomorpha arable land, while the diversity in natural ecosystems in trop- (e.g., Tylenchidae); even based on full-length sequences, the ical and subtropical areas remains largely unknown. We ex- resolution among early-diverging groups is low (Bert et al. pect, although this is no more than an educated guess based on 2008; Holterman et al. 2006; van Megen et al. 2009), and our wide-ranging experience, that 70% of the undiscovered these resolution problems are not likely to be resolved by species will originate from Bneglected^ regions, 20% from adding more taxa (Qing et al. 2017). Furthermore, discor- well-studied regions, leaving 10% belonging to cryptic dances have been found between 18S and 28S rRNAs (e.g., species. monophyly/polyphyly of Malenchus and placement of With the growing application of metagenomics and inte- Lelenchus), and considerable variations of support values grative taxonomical approaches, we predict that putative new (PP/BS), even for the same gene, have also been observed in species will be discovered at an increasing rate. However, this study. owing to the lack of specialists/taxonomists and the minor Polymorphism is an additional obstacle to reconstruct the economic impact of Tylenchidae, most of these species will Tylenchidae phylogeny. rRNA polymorphism within a spe- surely have to wait to be formally described. It is therefore cies is expected to be very low or absent due to concerted likely that not more than 2–3speciescanbeexpectedtobe evolution (Dover 1982). For nematodes, polymorphisms have describedeachyear,andrealisticallythemajorityof been found in Halicephalobus (Yoshiga et al. 2014)and Tylenchidae species will remain completely undocumented Rotylenchulus (Nyaku et al. 2013;vanDenBergetal.2016) for years to come. and two genera of Tylenchidae: Cephalenchus (Pereira and Baldwin 2016)andMalenchus (Qing et al. unpublished re- Problems and perspective sults). However, these elevated intragenomic variations may exist in more taxa of Tylenchidae and their impact on phylog- DNA extraction and PCR eny remains to be evaluated. Gene selection is also of great importance for future phy- The majority of Tylenchidae species are not cultivable under logenetic studies. 18S and 28S rRNAs do not provide ade- laboratory conditions, and soil samples often contain different quate phylogenetic signals but are certainly still valuable, as similar species of the same genus, meaning that accurate sam- they represent the majority of the Tylenchidae reference se- ple extraction in combination with a successful PCR can be quences in GenBank. Multi-gene-based phylogeny ap- challenging. In some cases, a single soil sample contains proaches have been used for many other taxa, showing many somewhat more than five very similar species from same ge- advantages over other techniques, and several candidate genes nus (e.g., Filenchus), a fact that makes species identification are potentially invaluable: e.g., Hsp90, EF1a alpha, ATPase, difficult. A proper identification and photo/video vouchering ATPsyn, MAT, IF, CAT, Tropo, ALD, GAPDH, PFK, Mio, is of great importance prior to DNA extraction. Moreover, the and H3 (Anderson et al. 2004; Kim and Lee 2008;Papsetal. small quantities of DNA templates obtained from single spec- 2009; Shultz and Regier 2000; Yurchenko et al. 2006). imens allow for only very limited PCR attempts and thus the Evidently, whole-genome (including mitogenome) phylogeny risk of running without templates is relatively high if the bind- has a great capacity to resolve long-standing phylogenetic ing of the universal primer fails. problems (Park et al. 2011;Jarvisetal.2014), and with the ever-increasing development of next generation sequencing Gene selection techniques, this method will be both more affordable and practicable in the near future. Both 28S and 18S rRNAs have some limitations in the analysis of Tylenchidae phylogeny. The 28S rRNA has a high substitution rate that can cause long-branch attraction and ob- Taxa density scure the phylogenetic relationships among sequences. Despite this, the 28S rRNA gene has been widely used in The sampled taxa used for phylogenetic studies are limited: 22 Tylenchomorpha phylogeny (Subbotin et al. 2005, 2006, out of 44 genera do not have any sequence representative. 2007, 2008, 2011), and three of the last five studies on Furthermore, genera with associated sequences are either rep- Tylenchidae have been solely based on 28S rRNA resented only by a single sequence or by few very short frag- (Panahandeh et al. 2015a, b;Qingetal.2016; Yaghoubi ments (less than 800 and 500 bp for 18S and 28S rRNA, et al. 2015). On the other hand, 18S rRNA data have an ap- respectively). The lack of taxa has subsequently hampered propriate substitution rate, but considerable length variations our understanding of Tylenchidae phylogeny, especially for 406 Qing X., Bert W. those divergent genera with many unique characters (e.g., in Bert, W., Leliaert, F., Vierstraete, A. R., Vanfleteren, J. 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