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Turbiditylenchus corticeus n. gen., n. sp. (Rhabditida: Anguinidae) from the bark of Eucalyptus macrorhyncha from the Australian Capital Territory

In: Nematology
Authors:
Daniel C. Huston Australian National Insect Collection, National Research Collections Australia, CSIRO, PO Box 1700, Canberra, ACT 2601, Australia

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https://orcid.org/0000-0002-1015-4703
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Manda Khudhir Australian National Insect Collection, National Research Collections Australia, CSIRO, PO Box 1700, Canberra, ACT 2601, Australia

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https://orcid.org/0009-0004-8012-5643
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Mike Hodda Australian National Insect Collection, National Research Collections Australia, CSIRO, PO Box 1700, Canberra, ACT 2601, Australia

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Summary

A new genus and species of the Anguinidae, Turbiditylenchus corticeus n. gen., n. sp., was isolated from the bark of Eucalyptus macrorhyncha from southeastern Australia. Turbiditylenchus corticeus is readily differentiated from all recognised anguinid genera and is characterised primarily by a slender body, lateral field with six incisures, an anteriorly flattened lip region continuous with the body, delicate stylet 7.9-9.9 μm long, muscular median bulb containing a strongly refractive valve, post-vulval uterine sac 1.87-4.4 times vulval body diam., conical tail with pointed tip, and males with leptoderan bursa and spicules 20.5-25.8 μm in length. The phylogenetic relationships of the new species with other anguinid lineages were reconstructed using sequences of the small subunit ribosomal RNA (18S rRNA), the internal transcribed spacer region (ITS; comprising ITS1-5.8S-ITS2) and partial large subunit ribosomal RNA (28S rRNA D2-D3) genes based on Bayesian inference and maximum likelihood analyses. These analyses demonstrate the new species represents a lineage distinct from all other anguinids. Based on phylogenetic results we also transfer Ditylenchus parvicauda Gu, Ma, Castillo & Munawar, 2024 and Ditylenchus gracicauda Gu, Ma, Castillo & Munawar, 2024 to Ditylenchoides Subbotin & Ryss, 2024 as Ditylenchoides parvicauda n. comb. and Ditylenchoides gracicauda n. comb.

Prior to the recent revision of Subbotin & Ryss (2024), Ditylenchus Filipjev, 1936 was the largest genus in the family Anguinidae Nicoll, 1935 and comprised 71 species (Hashemi & Karegar, 2019; Aliverdi et al., 2021; Azimi & Abdolkhani, 2022; Hashemi et al., 2022; Hodda, 2022; Gu et al., 2023, 2024; Vyshali et al., 2023; Zeng et al., 2023). Using a combination of morphological and phylogenetic evidence, Subbotin & Ryss (2024) narrowed the concept of Ditylenchus to include only those plant-parasitic species that induce swelling and deformation of host tissues, ultimately retaining just 13 species in the genus. These authors also proposed two additional genera: Ditylenchoides Subbotin & Ryss, 2024, with Ditylenchoides destructor (Thorne, 1945) Subbotin & Ryss, 2024 as its type species and with 12 additional species transferred from Ditylenchus, and Paraditylenchis Subbotin & Ryss, 2024, with Paraditylenchus gallaeformans (Oliveira, Santin, Seni, Dietrich, Salazar, Subbotin, Mundo-Ocampo, Goldenberg & Barreto, 2013) Subbotin & Ryss, 2024 as its type and only species. Lastly, Subbotin & Ryss (2024) amended the diagnosis of Zeatylenchus Zhao, Davies, Alexander & Riley, 2013 and transferred Ditylenchus drepanocercus Goodey, 1953 to this genus as Zeatylenchus drepanocercus (Zhao, Davies, Alexander & Riley, 2013) Subbotin & Ryss, 2024.

The narrower definition of Ditylenchus, along with the transfer of many species to new and amended genera, represents a significant step towards a natural classification of the Anguinidae. Subbotin & Ryss (2024) stated that all species previously assigned to Ditylenchus but which did not align with their new concept of the genus (i.e., those species that did not induce swelling or deformation in a plant host) should be assigned to other genera. However, these authors did not formally assign all such species to new or known genera, leaving 45 species in an informal category of ‘Ditylenchus-like’ species. Phylogenetic placements of these ‘Ditylenchus-like’ species from Subbotin & Ryss (2024) suggest that many new genera may be required to accommodate these species.

Despite global richness, relatively few species of the Anguinidae are known from Australia. Of the economically important species, only Anguina agrostis (Steinbuch, 1799) Filipjev, 1936, Anguina tritici (Steinbuch, 1799) Chitwood, 1935, Ditylenchus dipsaci (Kühn, 1857) Filipjev, 1936 and Ditylenchoides myceliophagus (Goodey, 1958) Subbotin & Ryss, 2024 have been confirmed as occurring in Australia (Hodda & Nobbs, 2008). Just a single species of ‘Ditylenchus-like’ nematode has been described from Australia, Ditylenchus australiae Brzeski, 1984, which was isolated from heavy soils on the banks of the Darling River, New South Wales (Brzeski, 1983). As several widespread and economically damaging species, such as the rice stem nematode Ditylenchus angustus (Butler, 1913) Filipjev, 1936 and Ditylenchoides destructor (Thorne, 1945) Subbotin & Ryss, 2024, do not yet occur in Australia (Hodda & Nobbs, 2008), the capacity to identify these important species and differentiate them from native species is critical for the Australian biosecurity system. To this end, adequate characterisation of native species will reduce incidences of false-positive detection and will facilitate the use of new biosecurity surveillance methods such as environmental DNA (eDNA).

As part of a recent re-collection of Ptychaphelenchus eucalypticola Hodda, 2009 (see Huston et al., 2023), a species morphologically consistent with the previous concept of Ditylenchus was isolated from the outer bark of Eucalyptus macrorhyncha F.Muell. ex Benth (Myrtaceae) from the Australian Capital Territory. However, in the light of the recent revision of Ditylenchus by Subbotin & Ryss (2024), our morphological and molecular analyses indicate this nematode should be assigned to a new genus. The species is herein described as Turbiditylenchus corticeus n. gen., n. sp.

Materials and methods

Samples of fibrous bark were collected from a group of E. macrorhyncha growing on Mount Ainslie, ACT, Australia in 2022. Nematodes were extracted from bark samples using Whitehead-Hemming trays (Whitehead & Hemming, 1965). Live nematodes were collected from the bulk extract and studied on wet-mount slides using a Zeiss Axioscope light microscope; after study they were removed from the slides and placed directly into DNA extraction buffer. Additional nematodes from the bulk extract were killed using near-boiling water and preserved in 10% formalin.

Specimens used for further morphological observation were transferred from formalin to glycerol using the slow method (Hooper, 1986) and mounted in glycerol on wax-ring microscope slides. Photographs and measurements of specimens were taken using a Zeiss Axiocam 506 mono camera mounted on a Zeiss Axioscope light microscope and associated Zeiss Blue imaging software. Drawings were made using a drawing tube attached to the above microscope. Drawings were digitised and images were edited and annotated in Adobe Illustrator CS6. All measurements except morphometric indices are expressed in μm unless otherwise indicated.

Genomic DNA was extracted from specimens using a DNeasy Blood and Tissue kit (Qiagen®) following the manufacturer’s instructions. Three molecular markers were targeted: the small subunit ribosomal RNA (18S rRNA), the internal transcribed spacer region (ITS; comprising ITS1-5.8S-ITS2) and the large subunit ribosomal RNA (28S rRNA). The 18S region was amplified in two fragments, with the first fragment using the forward primer G18S4 (5′-GCT TGT CTC AAA GAT TAA GCC-3′) (Blaxter et al., 1998) and reverse R18Ty11 (5′-GGT CCA AGA ATT TCA CCT CTC-3′) (Chizhov et al., 2006) and the second fragment using the forward primer F18Ty12 (5′-CAG CCG CGG TAA TTC CAG C-3′) and reverse R18Ty12 (5′-CGG TGT GTA CAA AGG GCA GG-3′) (Chizhov et al., 2006). The ITS region was amplified using the forward primer TW81 (5′-GTT TCC GTA GGT GAA CCT GC-3′) and reverse AB28 (5′-ATA TGC TTA AGT TCA GCG GGT-3′) (Curran et al., 1994). The 28S region was amplified using the forward primer D2A (5′-ACA AGT ACC GTG AGG GAA AGT TG-3′) and reverse primer D2B (5′-TCG GAA GGA ACC AGC TAC TA-3′) (Nunn, 1992). PCR and clean-up followed Huston et al. (2023) and was the same for all three gene regions. PCR products were sent to the Biomolecular Resource Facility, Australian National University, Canberra, for Sanger sequencing and sequenced using the amplification primers. Resultant reads were assembled and edited using Geneious Prime® v2023.2.1 (Biomatters).

Sequences of the 18S, ITS and 28S gene regions for members of the Anguinidae Nicoll, 1935 and selected outgroup taxa (Allantonematidae Pereira, 1931, Howardula aoronymphium Welch, 1959, and Tylenchoidea Örley, 1880, Deladenus siricidicola Bedding, 1968) were obtained from GenBank. Alignments for the three gene regions were constructed separately using MUSCLE (Edgar, 2004) as implemented in MEGA 11 (Tamura et al., 2021). Best-fit nucleotide substitution models for phylogenetic analyses were evaluated for each marker in MEGA 11; the GTR + G model was selected for the 18S and ITS dataset and the GTR + G + I model was selected for the 28S dataset. Phylogenetic analyses were performed on XSEDE (Towns et al., 2014) accessed through the CIPRES portal (Miller et al., 2010). Majority-rule consensus trees were constructed using Bayesian inference (BI) and maximum likelihood (ML) analyses. Bayesian inference was performed using MrBayes v3.2.6 (Ronquist et al., 2012) with default priors and four chains sampled every 1000 of 1 × 107 generations; the first 2500 samples were discarded as burn-in. Maximum likelihood analyses were performed using RAxML (Stamatakis, 2014) with 1000 bootstrap pseudo-replicates. Combined BI/ML trees were edited and annotated in Adobe Illustrator CS6. We follow Subbotin & Ryss (2024) and use quotation marks to indicate ‘Ditylenchus-like’ species that are not presently considered to belong to Ditylenchus by those authors, but which have not yet been assigned to other genera.

Results

Turbiditylenchus n. gen.

Diagnosis

Anguinoidea. Body 738-1002 μm in length, slender, ventrally arcuate following heat fixation. Lip region continuous, low, cephalic framework weakly developed. Median bulb muscular with strongly refractive valve. Isthmus narrow. Basal pharyngeal bulb pyriform, slightly overlapping intestine. Excretory pore anterior to basal bulb. Lateral field with six incisures. Ovary straight without flexures, posterior with oocytes arranged in single row. Spermatheca elongate, 1.6-4.1 times length of ovary, sperm spheroid. Crustaformeria in form of quadricolumella, four rows of four cells each. Vulva a transverse slit, vagina perpendicular to slightly anteriorly directed relative to body. Post-vulval uterine sac 1.9-4.4 times longer than vulval body diam. Female tail conical, terminus pointed. Male bursa leptoderan, not reaching tail tip. No known plant-parasitic or insect associations; presumably fungal feeding. Known only from Australia.

Type species

Turbiditylenchus corticeus n. sp.

Etymology

The name is constructed by combining the Latin word ‘turbidus’, meaning ‘cloudy, muddy or opaque’ and the genus name to which the new species would otherwise been assigned had it not been recently revised, Ditylenchus. The name is chosen to maintain a connection with Ditylenchus while reflecting the uncertain phylogenetic relationships between the new species and other anguinid lineages. The ZooBank LSID code for this genus is: urn:lsid:zoobank.org:act:62B370F3-A4E4-4CF0-9F19-B4980BDA389E.

Relationships

Turbiditylenchus can be readily differentiated from Anguina Scopoli, 1777, Subanguina Paramonov, 1967, Nothoanguina Whitehead, 1959 and Paleoanguina Poinar, 2011 in lacking obese females and a plant-parasitic life cycle; from Synchnotylenchus Rühm, 1956, Neoditylenchus Meyl, 1961 and Neomisticius Siddiqi, 1986 in having leptoderan, rather than peloderan bursa in males and in lacking an insect association; from Cotylenchus Álvarez-Ortega & Subbotin, 2024, in having a smaller and less robust stylet (7.9-9.9 vs 12.0-13.5 μm), a basal bulb slightly overlapping the intestine vs one abutting, and in lacking a plant-parasitic life cycle; from Diptenchus Khan, Chawla & Seshadri, 1969, in having a post-vulval uterine sac (vs lacking one) and six, rather than five, incisures in the lateral field; from Ficotylus Davies, Ye, Giblin Davis & Thomas, 2009 in having six, rather than two or four, incisures in the lateral field, in having a muscular median bulb and refractive valve (vs lacking) and being free-living rather than having an intimate association with figs (Ficus spp.); from Halenchus Cobb, 1933 in having six, rather than four, incisures in the lateral field, in having a median bulb and valve (vs lacking), in having an excretory pore not strongly sclerotised (vs strongly sclerotised) and in being terrestrial and free-living rather than a parasite of marine algae; from Indoditylenchus Sinha, Choudhury & Baqri, 1985 in having six, rather than four, incisures in the lateral field and in having an elongate-conical, rather than filiform, tail; from Litylenchus Zhao, Davies, Alexander & Riley, 2011 in lacking a semi-obese female, having a low, continuous lip region (vs a distinctly offset head), a pharyngeal bulb slightly overlapping the intestine (vs abutting) and a free-living, rather than plant-parasitic, life cycle; from Orrina Brzeski, 1981, in having six, rather than four, incisures in the lateral field, in having a muscular median bulb (vs lacking), and in lacking a plant-parasitic life cycle; from Pseudhalenchus Tarjan, 1958 in having six, rather than four, incisures in the lateral field, a basal pharyngeal bulb slightly overlapping the intestine vs an elongate pharyngeal gland lobe significantly overlapping the intestine, and in having an elongate, rather than rounded, spermatheca; from Pterotylenchus Siddiqi & Lenne, 1984, in having six, rather than four, incisures in the lateral field, in lacking the large and prominent vulval flaps present in species of the former genus and in lacking a plant-parasitic life cycle; and from Safianema Siddiqi, 1980, in having a pyriform basal pharyngeal bulb only slightly overlapping the intestine, vs an elongate gland-lobe significantly overlapping the intestine, in having a pharyngeal gland nucleus anterior, rather than posterior, to the pharyngeal-intestinal junction, and an elongate-conical, rather than filiform, tail.

Turbiditylenchus is most similar to the recently proposed genera Ditylenchoides and Paraditylenchus (Subbotin & Ryss, 2024), the recently amended concepts of Ditylenchus and Zeatylenchus (Subbotin & Ryss, 2024) and the recently amended concept of Nothotylenchus Thorne, 1941 (Hashemi & Karegar, 2020). From Ditylenchoides, Turbiditylenchus differs in having a low, rather than high, lip region and in having a weakly, rather than moderately sclerotised cephalic framework. In addition, the basal bulb in Ditylenchoides is compact or irregular, rather than pyriform as in Turbiditylenchus, and the excretory pore in the former genus is located near the middle of the basal bulb, posterior to the isthmus, rather than anterior to the basal bulb as in the latter. Turbiditylenchus differs from Paraditylenchus in having a low, rather than high, lip region, in having a weakly, rather than moderately sclerotised cephalic framework, in having a lateral field with six, rather than four, incisures, in having a post-vulval uterine sac 1.87-4.4 time longer than the vulval body diam. (vs 1.4-1.8), and in having leptoderan, rather than peloderan, bursa in males; from Ditylenchus in having a low, rather than high, lip region and in having a weakly, rather than moderately sclerotised cephalic framework, and a post-vulval sac 1.87-4.4 times longer than the vulval body diam. (vs always more than three times the vulval body diam.). In addition, Ditylenchus is diagnosed as having four, rarely six, incisures in the lateral field, vs six in Turbiditylenchus. Lastly, Subbotin & Ryss (2024) amended the concept of Ditylenchus to include only species that induce tissue deformation in plants, thus excluding free-living fungal-feeding species as in Turbiditylenchus. From Zeatylenchus, Turbiditylenchus differs in having a lateral field with six, rather than three, incisures, a pyriform, rather than compact or irregular, basal pharyngeal bulb, a post-vulval uterine sac 1.87-4.4 times longer than the vulval body diam. (vs 1.0-1.5) and in lacking a plant-parasitic life cycle; and from Nothotylenchus in having a muscular median bulb and a strongly refractive median bulb valve; median bulbs and valves are lacking in species of Nothotylenchus (see Hashemi & Karegar, 2020).

Morphological data, coupled with a consensus of molecular phylogenetic information, indicate that Turbiditylenchus represents a novel lineage within the Anguinidae, although the relationship between this lineage and others is presently unclear.

Fig. 1.
Fig. 1.

Turbiditylenchus corticeus n. sp., illustrations. A: Adult male, entire body; B: Adult female, entire body; C: Male posterior end; D: Female posterior end; E: Male anterior region; F: Female anterior region; G: Female genital system. (Scale bars: A, B = 150 μm; C-F = 75 μm; G = 50 μm.)

Citation: Nematology 26, 10 (2024) ; 10.1163/15685411-bja10363

Fig. 2.
Fig. 2.

Turbiditylenchus corticeus n. sp., light microphotographs. A-C: Adult male, anterior end; D-F: Adult female, anterior end; G-I: Adult male, posterior end; J, K: Adult female, posterior end; L-N: Adult female, genital system; O, P: Adult females, lateral lines. (Arrows in A, B, C, F indicate excretory pore; E: hemizonid; G: bursa; J: anus; L, N: vulva; M: post-vulval uterine sac; P: reflected light and shadow, which may lead to an erroneous count of eight, rather than six, lateral lines.) (Scale bars = 10 μm.)

Citation: Nematology 26, 10 (2024) ; 10.1163/15685411-bja10363

Table 1.
Table 1.

Measurements and morphometric indices of Turbiditylenchus corticeus n. gen., n. sp. from the Australian Capital Territory. Measurements are in μm and presented as mean ± standard deviation (range).

Citation: Nematology 26, 10 (2024) ; 10.1163/15685411-bja10363

Table 1.
Table 1.

(Continued.)

Citation: Nematology 26, 10 (2024) ; 10.1163/15685411-bja10363

Turbiditylenchus corticeus n. sp. (Figs 1, 2)

Measurements

See Table 1.

Description

Adult female

Body elongate, vermiform, ventrally arcuate following heat fixation. Cuticle distinctly annulated. Lateral field with six incisures, width 6-12 or 29-56% of maximum body diam. Lip region low, anteriorly flattened, lacking annulation, continuous with body contour. Cephalic framework weakly developed. Stylet delicate; knobs small, rounded. Dorsal pharyngeal gland orifice (DGO) just posterior to stylet. Pharyngeal procorpus cylindrical, relatively uniform as it passes posteriorly. Median bulb fusiform, muscular, containing distinct refractive valve. Isthmus narrow, cylindrical, encircled by nerve ring about mid-length. Basal pharyngeal bulb pyriform, posterior portion slightly overlapping intestine centrally. Rectum tubular, narrow; anal opening not protuberant. Hemizonid just posterior to nerve ring. Excretory pore just posterior to hemizonid, anterior to joining of isthmus with basal pharyngeal bulb. Reproductive system monodelphic-prodelphic. Ovary cylindrical, outstretched, extending anteriorly to about mid-body, oocytes arranged in single row. Oviduct tubular, narrow, winding posteriorly, joining elongated spermatheca containing spheroid sperm and enlarged maturing oocytes in anterior and posterior reaches, respectively. Crustaformeria distinct, quadricolumellate, posterior joining elongate, bulbous, anterior uterus. Vulva located in posterior quarter of body; lips slightly protruding. Vagina perpendicular to slightly anteriorly directed relative to body. Post-vulval uterine sac 1.9-4.4 times vulval body diam. in length, cylindrical to reniform. Tail elongate, conical, tail terminus pointed. Phasmids not observed.

Adult male

General morphology similar to that of female. Testis single, outstretched, reaching anteriorly to near mid-body, spermatocytes arranged in single row. Bursa leptoderan, extending from near head of spicules to about one anal body diam. from tail tip. Cloacal opening slightly protuberant. Spicules tylenchoid, ventrally arcuate, slightly cephalated anteriorly, transition from manubrium to calomus indistinct, calomus with subtle constriction, proximal part of lamina lacking distinct expansion or tumulus. Gubernaculum simple, crescentic.

Type habitat and locality

Fibrous outer bark of Eucalyptus macrorhyncha F.Muell. ex Benth, Mount Ainslie, ACT, Australia (35°16′S, 149°10′E; 720 m a.s.l.). The bark habitat along with no evidence of a close relationship with plant-parasitic anguinids in molecular analyses suggests this species feeds on fungal hyphae as is thought for most free-living anguinids (Sturhan & Brzeski, 1991; Siddiqi, 2000; Hashemi & Karegar, 2019).

Type material

The female holotype and 17 female and 11 male paratypes are deposited in the Nematology section of the Australian National Insect Collection, CSIRO, Canberra, ACT, Australia (slide nos 8918-8946). The ZooBank LSID code for this species is: urn:lsid:zoobank.org:act:870AC58F-827E-433A-9898-2AF3AA25F27A.

Etymology

The specific epithet ‘corticeus’ is derived from Latin, meaning ‘of bark’. The name is chosen to reflect the habitat where this species was found.

Diagnosis and relationships

Subbotin & Ryss (2024) amended the diagnosis of Ditylenchus and moved many species previously recognised in this genus into new or other genera, but not all. Because the new concept of Ditylenchus encompasses only those plant-parasitic species that induce tissue deformation (Subbotin & Ryss, 2024), some species previously assigned to Ditylenchus still require transfer to other genera, and thus need to be compared with Turbidtylenchus corticeus n. gen., n. sp.

Hashemi & Karegar (2019) highlighted several key morphometrics and indices that were useful for differentiation of species of what now comprises Ditylenchus, Ditylenchoides, Paraditylenchus and many ‘Ditylenchus-like’ species, i.e., stylet length, V, V’, PUS/VBW, PUS/V-A%, shape of tail tip, c, c’ and length of spicules. Using these indices in the tables provided by Hashemi & Karegar (2019), Turbidtylenchus corticeus n. gen., n. sp. is readily distinguished from nearly all previously described ‘Ditylenchus-like’ species. Turbidtylenchus corticeus n. gen., n. sp. is most similar to ‘Ditylenchus’ azarbaijanensis Khakbaz, Gharibzadeh, Atighi & Pedram, 2021, but differs in having a greater ratio of body length to body diam. (a) on average (35.3-46.8 vs 19.5-35.5 and 33.4-50.9 vs 23.7-28.0 in females and males, respectively), a longer tail in the male (46.6-57.3 vs 60-64 μm), shorter spicules (20.5-25.8 vs 26.5-31 μm) and a shorter gubernaculum on average (7-8.8 vs 8-11.5 μm). Adult females of ‘D’. azarbaijanensis also have more posteriorly directed vaginas than in T. corticeus n. gen., n. sp., and rudimentary post-uterine sacs, rather than fully developed ones. In addition, there are biological and geographic differences: ‘D’. azarbaijanensis was collected from the rhizosphere of the sun spurge Euphorbia helioscopia from Iran (Khakbaz et al., 2021), vs T. corticeus n. gen., n. sp. from the bark of a Eucalyptus tree in the Australian Capital Territory, locations nearly 13 000 km apart.

Turbiditylenchus corticeus n. sp. can also be readily distinguished from the only other ‘Ditylenchus-like’ species described from Australia, ‘Ditylenchus’ australiae, via morphology and biological characteristics. From ‘D’. australiae, T. corticeus n. gen., n. sp. differs in adult females having a more posterior vulva (V 84-86% vs 80-82% and V’ 90-92 vs 88-90), adult females having a wider lateral field (29-56% of corresponding body diam. vs <25%), the basal pharyngeal bulb overlapping the intestine (vs not overlapping), the vagina being much longer (45-80% of corresponding body diam. vs 30-40%), the spicules being a different size and shape (length 21-26 vs 18-19 μm, tip pointed vs squarish and a ventral projection near the manubrium vs no projection), the gubernaculum being longer (7-9 vs 5-6 μm), and the bursa extending to near the tail tip (vs only 29% of the length). In addition, there are biological and geographic differences. Ditylenchus australiae was found in heavy soil rather than fibrous bark (Brzeski, 1983), in a semi-arid location nearly 700 km distant from the mesic Mt Ainslie, and at a much lower elevation (37 m a.s.l. vs 720 m a.s.l.).

Molecular characterisation and phylogenetic relationships

We generated two, six and four sequences of the 18S, ITS and 28S gene regions, for the newly isolated nematodes, respectively. Gene sequences lacked intraspecific variation; thus a single replicate of each was submitted to GenBank: PQ212511 (18S); PQ212512 (ITS); PQ212513 (28S). Analyses of sequences against the NCBI GenBank database via BLAST (Altschul et al., 1990) confirmed that the nematode belonged to the Anguinidae, with the 18S sequences having 97% similarity to a variety of anguinid taxa (e.g., Ditylenchus dipsaci MK292125), the ITS sequences having 85% similarity to Anguina paludicola Bertozzi & Davies, 2009 (AF396363) and 28S sequences having 86% similarity to Litylenchus crenatae Kanzaki, 2019 (MK292138).

Fig. 3.
Fig. 3.

Bayesian majority-rule consensus tree of the 18S dataset. Bayesian inference (BI) posterior probabilities (pp) for nodes represented by circles, maximum likelihood (ML) bootstrap support (bs) represented by squares. Support values less than 0.90 (pp) and 70 (bs) not shown. The scale-bar indicates the expected number of substitutions per site. GenBank accession number is presented beofre taxa name. New taxa and combinations are presented in boldface and highlighted; other taxa with placements of note are also highlighted.

Citation: Nematology 26, 10 (2024) ; 10.1163/15685411-bja10363

Fig. 4.
Fig. 4.

Bayesian majority-rule consensus tree of the 28S dataset. Bayesian inference (BI) posterior probabilities (pp) for nodes represented by circles, maximum likelihood (ML) bootstrap support (bs) represented by squares. Support values less than 0.90 (pp) and 70 (bs) not shown. The scale-bar indicates the expected number of substitutions per site. GenBank accession number is presented before taxa name. New taxa and combinations are presented in boldface and highlighted; other taxa with placements of note are also highlighted.

Citation: Nematology 26, 10 (2024) ; 10.1163/15685411-bja10363

Bayesian and ML analyses of the 18S dataset produced congruent topologies (Fig. 3). The Anguinidae resolved as two major clades, with Nothotylenchus acris (Thorne, 1941) Fortuner & Maggenti, 1987 and ‘Ditylenchusbrevicauda (Micoletzky, 1925) Filipjev, 1936 falling in outside basal positions. The first major clade was well supported and comprised ten species of Ditylenchoides, as well as Pseudhalenchus minutus Tarjan, 1958, and a sequence each of Nothotylenchus and Pterotylenchus. The second major clade formed was divided into one supported and one unsupported clade. The supported clade comprised ‘Ditylenchus’ adasi (Sykes, 1980) Fortuner & Maggenti, 1980 as sister to species of Ditylenchus, Litylenchus, Anguina and Subanguina. The unsupported clade formed a polytomy comprising several individual sequences, a supported clade comprising Paraditylenchus gallaeformans sister to species of Zeatylenchus, a supported clade comprising Cotylenchus cleo Álvarez-Ortega & Subbotin, 2024 sister to ‘Ditylenchus’ israelensis Gu, Ma, Castillo & Munawar, 2023 + Halenchus fucicola (de Man, 1892) Cobb, 1933 and a supported clade comprising a variety of ‘Ditylenchus-like’ species and insect and fig associated species. The placement of Ditylenchus angustus in this clade, a species that was retained in Ditylenchus by Subbotin & Ryss (2024), suggests this sequence may be erroneous. Although Turbiditylenchus coritceus n. gen., n. sp. was placed in the second major clade with strong support, the relationship between this species and others in the clade was unresolved.

Bayesian and ML analyses of the 28S dataset (Fig. 4) also produced congruent topologies. The clade comprising species of Ditylenchoides and the clade comprising species Ditylenchus, Litylenchus, Anguina and Subanguina were recovered with strong support as in analyses of the 18S dataset. The clade containing P. gallaeformans + species of Zeatylenchus was also recovered with high support. Cotylenchus cleo was again found sister to ‘Ditylenchus’ israelensis, but with the inclusion of ‘Ditylenchus’ valveus Thorne & Malek, 1968 and Nothotylenchus persicus Esmaeli, Heydari, Castillo & Palomares-Rius, 2016. The final supported clade comprised a variety of ‘Ditylenchus-like’ species, species of Nothotylenchus and insect- and fig-associated species. The placement of sequence MF996705 in this clade, putatively of Ditylenchoides myceliophagus, suggests that the species identification may be in error. Turbiditylenchus corticeus n. gen., n. sp., along with ‘Ditylenchusterricolus, was placed with no clear relationship to the other anguinid clades.

Fig. 5.
Fig. 5.

Bayesian majority-rule consensus tree of the ITS dataset. Bayesian inference (BI) posterior probabilities (pp) for nodes represented by circles, maximum likelihood (ML) bootstrap support (bs) represented by squares. Support values less than 0.90 (pp) and 70 (bs) not shown. The scale-bar indicates the expected number of substitutions per site. GenBank accession number is presented before taxa name. New taxa and combinations are presented in boldface and highlighted; other taxa with placements of note are also highlighted.

Citation: Nematology 26, 10 (2024) ; 10.1163/15685411-bja10363

Although BI and ML analyses of the ITS dataset (Fig. 5) produced largely congruent topologies, and the clade containing species of Ditylenchus, Anguina, Subanguina and Litylenchus was again supported as monophyletic, support for most remaining clades was generally poor with both Ditylenchoides and Nothotylenchus appearing polyphyletic. Turbiditylenchus corticeus n. gen. n. sp. + Nothotylenchus geraerti Kheiri, 1971 was supported as sister to the clade comprising species of Ditylenchus, Litylenchus, Anguina and Subanguina along with four other clades, but relationships between these groups were unclear.

Ditylenchus parvicauda Gu, Ma, Castillo & Munawar, 2024 resolved within Ditylenchoides in all present analyses and Ditylenchus gracicauda Gu, Ma, Castillo & Munawar, 2024 resolved within Ditylenchoides in the present 28S analyses (only 28S data are available for the latter species). These species names were published by Gu et al. (2024) only a few days before the revision of Subbotin & Ryss (2024) was published, and thus were not included in the work of the latter authors. Based on our molecular results, we transfer these two species to Ditylenchoides as Ditylenchoides parvicauda n. comb. and Ditylenchoies gracicauda n. comb.

Discussion

We do not currently recognise any additional species in Turbiditylenchus n. gen. Although the morphological concept of this genus may encompass several ‘Ditylenchus-like’ species, we prefer not to create an additional ‘dumping-ground’ genus as was the case for Ditylenchus prior to the revision of Subbotin & Ryss (2024). Although this may lead to many additional genera being proposed to accommodate previously described and yet-to-be discovered ‘Ditylenchus-like’ species, this represents progress towards a natural classification of the lineage (Subbotin & Ryss, 2024). We recommend species only be assigned to genera in this group where there is molecular data to support their inclusion.

The only strongly supported relationship found between the new species and another in the present molecular phylogenetic analyses was a sister relationship between T. corticeus and Nothotylenchus geraerti in analyses of the ITS dataset. Although N. geraerti was not described or figured in the paper where the sequences were first published (Hashemi et al., 2022), we have little doubt that the specimens from which these sequences were derived are correctly identified as they come from the country of type-description (Kheiri, 1970), and the sequence authors have collected and reported on this species, and other species of Nothotylenchus and Ditylenchus sensu lato, on multiple previous occasions (Hashemi et al., 2018, 2022; Hashemi & Karegar, 2019, 2020). The only difference between the previous concept of Ditylenchus and Nothotylenchus is that species of the latter genus have non-muscular and valveless median bulbs vs muscular and valved median bulbs in the former. Notably, Nothotylenchus was polyphyletic in all present phylogenetic analyses. We again do not think this relates to reliability of identification, most sequences for the genus coming from integrative taxonomic publications (e.g., Esmaeli et al., 2016; Jalalinasab et al., 2018; Munawar et al., 2022). This suggests that that the absence of a muscular median bulb and a valve may not accurately reflect shared evolutionary origin among those species currently assigned to Nothotylenchus. Several other anguinid genera lack median bulbs and/or valves (i.e., Ficotylus, Halenchus, Orrina), so it is possible that the free-living species of Nothotylenchus represent multiple evolutionary lineages exhibiting morphological convergence due to derived loss of the median bulb and valve. Thus, resolving the polyphyly of Nothotylenchus is likely to represent a significant challenge.

As Nothotylenchus appears polyphyletic, we think it best not to place our new species into Nothotylenchus, nor transfer N. geraerti into Turbiditylenchus. Doing either would create a broad genus concept that could include species with and without median bulbs and valves. This could lead to a new ‘catch-all’ genus nearly immediately after efforts to create more narrow definitions of natural anguinid lineages (Subbotin & Ryss, 2024).

Multiple independent instances of the loss of the median bulb and valve may explain the seemingly erroneous position of several species of Nothotylenchus in previous and present phylogenetic analyses (e.g., Nothotylenchus andrassyi Jalalinasab, Hosseini, and & Heydari 2018 appears in the Ditylenchoides clade in the present 28S analyses). Alternatively, the gene sequences we have utilised for our phylogenetic analyses may not always have the power to distinguish between anguinid genera (e.g., Aliverdi et al., 2021; Hashemi et al., 2022; Zeng et al., 2023). There is some discordance between phylogenies derived from different gene sequences. The monophyly of Ditylenchoides was well supported in our 18S and 28S analyses, yet in the ITS analyses this genus was polyphyletic with generally poor support. ‘Ditylenchus’ dactylonae Zeng, Dant & Roberts, 2023 was placed in the Ditylenchoides clade in the tree derived from analyses of the ITS dataset, yet it was not found in this clade in either the 18S or 28S analyses and does not fit the morphological and ecological concept of Ditylenchoides (Subbotin & Ryss, 2024). Similarly, Ditylenchoides persicus (Esmaeili, Heydari, Castillo & Palomares-Rius, 2017) Subbotin & Ryss, 2024 is placed properly in the 18S and 28S analyses, while it is placed sister to Nothotylenchus medians Thorne & Malek, 1968 in the central polyphyletic clade in the ITS tree. Considering that the 18S and 28S data are generally congruent, this suggests that ITS data are unlikely to be robust for differentiating genera in this lineage. Sequencing different molecular markers such as mitochondrial genes may pave the way to taxonomic resolution for the Anguinidae.

*

Corresponding author, e-mail: daniel.huston@csiro.au

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