Halanonchus scintillatulus sp. nov. from New Zealand and a review of the suborder Trefusiina (Nematoda: Enoplida)

We provide a review of the enoplid suborder Trefusiina Siddiqi, 1983, based on morphological considerations and analyses of new and published 18S rDNA sequences. We also describe Halanonchus scintillatulus Leduc sp. nov. from the Hauraki Gulf, northern New Zealand, as well as females of Trefusialaimus idrisi Leduc, 2013 from the continental slope of New Zealand. We show for the fi rst time that the structure of the female reproductive system of Trefusialaimus Riemann, 1974 consists of two opposed and outstretched ovaries, an unusual feature for the Enoplida. The Trefusiina did not form a monophyletic group in the 18S rDNA phylogeny due to the placement of Lauratonema Gerlach, 1953 and Trefusialaimus sequences well away from the main Trefusiina clade. However, due to generally weak Maximum Likelihood support values, we refrain from changing the classifi cation of these taxa until more comprehensive analyses can be conducted. Our phylogenetic analysis supports the inclusion of the Trischistomatidae Andrássy, 2007 within the Trefusiina, meaning that all of the enoplid suborders now include at least some terrestrial/freshwater representatives. The Trefusiina currently comprises fi ve families, 14 genera and 92 valid species.

The classifi cation of the Enoplida will likely keep evolving as more comprehensive molecular analyses are conducted. The SSU phylogenetic analyses of Bik et al. (2010), for example, suggest that most of the enoplid suborders listed in the widely used classifi cation of De Ley & Blaxter (2004) are not monophyletic. The only two exceptions are the closely-related Trefusiina and Tripyloidina; these two suborders, however, were found to form a larger monophyletic clade with the terrestrial/freshwater genera Trischistoma Cobb, 1913 andTripylina Brzeski, 1963, which were previously classifi ed within the order Triplonchida Cobb, 1920(family Tripylidae de Man, 1876Zullini 2006;Andrássy 2007). Several other SSU phylogenies and molecular studies have shown a close relationship between Trischistoma, Tripylina and the Enoplida, and the family Trefusiidae in particular (Holterman et al. 2006;Meldal et al. 2007;Zhao & Buckley 2009;van Megen et al. 2009;Zhao et al. 2012).
In order to take into account this new evidence regarding the placement of the genera Tripylina and Trischistoma, Zhao (2011) proposed an updated classifi cation where these two genera are placed within the family Trischistomatidae Andrássy, 2007 in the suborder Tripyloidina. This placement was considered conservative, as the Tripylidae had previously been classifi ed together with the Tripyloididae by Lorenzen (1981) and Siddiqi (1983), and the Trefusiina was exclusively marine. The classifi cation of the Tripylidae with the Enoplida by Lorenzen (1981) was partly based on his observation of metanemes in Tripylina glomerans Bastian, 1865;Zhao (2011) later also observed metanemes in four Trischistoma species he described from New Zealand. The presence of metanemes in Tripylina and Trischistoma appears to suggest closer affi nities with the Tripyloididae (Tripyloidina), which are characterised by the presence of metanemes, than the Trefusiidae (Trefusiina), which do not possess metanemes. The two genera also resemble Tripyloidina by the presence of teeth in the buccal cavity (absent in Trefusiina) and a monorchic male reproductive system (diorchic in most of Trefusiina). In addition, Tripylina is characterised by the outer labial and cephalic setae in a single circle, a feature of Tripyloidina, but which differs from the Trefusiina (mostly two separate circles). The arrangement of outer labial and cephalic setae in separate circles in Trischistoma, on the other hand, is the same as in most of the Trefusiina. Moreover, both Trischistoma and Tripylina have non-spiral (pocket-shaped) amphids and a monodelphic female reproductive system, which differ from the Tripyloidina (spiral amphids, didelphic female reproductive system) but are consistent with some of the Trefusiina. Therefore, while there are morphological (presence of metanemes) and molecular grounds (based on SSU phylogenies) to classify Trischistoma and Tripylina within the Enoplida, the morphological data is equivocal as to whether they should be placed with the Tripyloidina or Trefusiina. The molecular evidence, however, strongly suggests a closer relationship between Trischistoma and Tripylina and the Trefusiina than with the Tripyloidina (Van Megen et al. 2009;Bik et al. 2010).
The Trefusiina currently comprises four families: Simpliconematidae Blome & Schrage, 1985, Xennellidae De Coninck, 1965, Lauratonematidae Gerlach, 1953and Trefusiidae Gerlach, 1966(De Ley & Blaxter 2004. The Trefusiidae comprises two subfamilies, six genera and over 30 valid species (Bezerra et al. 2020). The Trefusiidae is common in shallow marine sediments worldwide and has also been found as deep as the abyssal plain (Miljutin et al. 2010). Changes to the classifi cation of the family were recently proposed by Shi & Xu (2017) based on analyses of 18S rDNA sequences, which led them to argue that the structure of the female reproductive system is a more meaningful taxonomic character for defi ning subfamilies than buccal morphology.
A reassessment of the Trefusiina is timely given the recent and rapid developments in molecular phylogenetics and considering that the latest comprehensive taxonomic treatment of the Trefusiina based on morphological characteristics was conducted decades ago (Lorenzen 1981(Lorenzen , 1994. Here, we provide a review of the Trefusiina based on an overview of each family, subfamily and genus of the suborder and review relationships based on morphological considerations and phylogenetic analysis of new and published 18S rDNA sequences of the Trefusiina and other Enoplida. We also describe Halanonchus scintillatulus Leduc sp. nov. (family Trefusiidae) from the Hauraki Gulf, northern New Zealand, as well as females of Trefusialaimus idrisi Leduc, 2013 from the continental slope off southeastern New Zealand.

Sampling and morphological analyses
A multicorer was used to obtain samples from the Firth of Thames in the Hauraki Gulf, a large bay in the north of New Zealand's North Island, in December 2003. Samples were fi xed in 10% formalin and stained with Rose Bengal. Samples were subsequently rinsed on a 1 mm sieve to remove large particles and on a 45 μm mesh to retain nematodes. Halanonchus scincillatulus sp. nov. and other nematodes were extracted from the remaining sediments by Ludox fl otation and transferred to pure glycerol and mounted onto permanent slides (Somerfi eld & Warwick 1996).
A sediment sample was obtained in April 2007 using a multicorer on the eastern Chatham Rise off the east coast of New Zealand's South Island at a depth of 1029 m. The sample, which consisted of sandy silt sediment, was fi xed in 10% buffered formalin and stained with Rose Bengal. Samples were subsequently rinsed on a 45 μm mesh and extracted using the Ludox fl otation method. Trefusialaimus idrisi specimens were transferred to pure glycerol and mounted onto permanent slides (Somerfi eld & Warwick 1996).
A one litre sediment sample (0-10 cm sediment depth) was obtained by hand at low tide from the upper subtidal zone at Sesoko Beach, Okinawa, Japan on 13 December 2017. The sediment consisted of coarse carbonate sand. Nematodes were extracted by decantation on a 63 μm mesh immediately after sampling, and live nematodes were sorted under a dissecting microscope. One male Trefusialaimus specimen and one morphologically similar juvenile specimen were mounted in a drop of seawater on a temporary slide to confi rm their identity, and images of key morphological features were taken prior to molecular analyses (see below). Visual inspection of the male specimen at 400 × magnifi cation showed similarities with Trefusialaimus idrisi due to the presence of numerous round, golden inclusions along the body, the length and arrangement of cephalic sensilla and the structure of the spicular apparatus, but species identity could not be confi rmed without risking damaging or losing the specimen.
Descriptions were made from glycerol mounts using differential interference contrast microscopy and drawings were made with the aid of a camera lucida. The terminology used for describing the arrangement of morphological features such as setae follows Coomans (1979). All measurements are in μm, and all curved structures are measured along the arc. Type specimens are held in the National Institute of Water and Atmospheric Research (NIWA) Invertebrate Collection, Wellington, and the National Nematode Collection of New Zealand (NNCNZ), Landcare Research New Zealand Ltd, Auckland.
Abbreviations a = body length/maximum body diameter b = body length/pharynx length c = body length/tail length cʹ = tail length/anal or cloacal body diameter cbd = corresponding body diameter ceph. = cephalic cs = cephalic seta g = granule ils = inner labial sensilla L = total body length n = number of specimens ND = no data ols = outer labial sensilla V = vulva distance from anterior end of body %V = V/total body length × 100

DNA extraction, PCR and sequencing
Following observation and digital imaging under a compound microscope, one Trefusialaimus male specimen and one morphologically similar juvenile specimen were transferred to 50 μl of a guanidinium thiocyanate solution prepared following Sinniger et al. (2010). The DNA was further extracted as described in Sinniger et al. (2010), adjusting the volumes accordingly (i.e., using 50 μl of isopropanol for precipitation and eluting in 30 μl of ultrapure water). The rDNA small subunit (SSU) was amplifi ed using the primers from Holterman et al. (2006 for the fi rst SSU fragment and 1813F, 5'-CTGCGTGAGAGGTGAAAT-3' and 2646R, 5'-GCTACCTTGTTACGACTTTT-3' for the second fragment. The LSU fragment was amplifi ed using the primers D2A (5' ACAAGTACCGTG-AGGGAAAGT 3') and D3B (5' TGCGAAGGAACCAGCTACTA 3') (Nunn, 1992) with thermal cycles as described in Leduc & Zhao (2018). The PCR products were sequenced bi-directionally using the amplifi cation primers by Macrogen Japan (Kyoto, Japan). Sequences were assembled and edited in Geneious ver. 10.2.2 (Kearse et al. 2012).

Sequence alignment and phylogenetic inference
The ribosomal DNA SSU and D2-D3 of LSU sequences of Trefusialaimus sp. were deposited in GenBank under accession numbers MN689267, MN689268 and MN689269, MN689270, respectively. SSU phylogenetic analyses were conducted using sequences of representative genera of the Enoplida and rooted using Triplonchida sequences. The initial D2-D3 of LSU analyses confi rmed that the LSU rDNA gene is only informative at the species to family levels (De Ley et al. 2005) and could not determine the placement of Trefusialaimus. The D2-D3 of LSU sequences were therefore not used to determine phylogenetic relationships. The SSU of DNA sequences were aligned using the MUSCLE algorithm (Edgar 2004a(Edgar , 2004b with default parameters, and then the alignment was modifi ed by using Gblocks (Castresana 2000;Talavera & Castresana 2007) with relaxed gap setting (only positions where 50% or more of the sequences have a gap are treated as a gap position) to remove the sites of questionable alignment. After removing sites of questionable alignment, Gblocks gave a 1445 bp site alignment from the original SSU rDNA with 1638 bp alignment.
Phylogenies were built in Geneious ver. 10.2.6 (http://www.geneious.com, Kearse et al. 2012). MrModelTest ver. 2.3 (Nylander 2004) in conjunction with PAUP* ver. 4.0b10 (Swofford 2002 ) and jModelTest ver. 2.1.10 software (Darriba et al. 2012) were used to select the best model using the Akaike Information Criterion. The substitution model [GTR (general time-reversible) + I (proportion of invariable sites) + G (gamma distribution)] was selected by MrModelTest in conjunction with PAUP* as the best-fi t model, whereas the substitution model TVM + I + G was selected as the best model by jModelTest ver. 2.1.6. Because the model TVM + I + G cannot be implemented in Geneious ver. 10.2.6, Bayesian trees were constructed with MrBayes under the most similar best-fi t model [GTR + I + G] (Huelsenbeck & Ronquist 2001), which is not expected to have a signifi cant impact on tree topology. The trees were run with chain length of 1 100 000, and burn-in length of 100 000. The perimeter fi les from multiple runs were inspected for chain convergence in Tracer ver. 1.5 (Rambaut & Drummond 2007), and the trees were edited in FigTree ver. 1.4.2 (http://tree.bio.ed.ac.uk/software/fi gtree) and PowerPoint. These analyses were also conducted with PhyML ver. 3.0 using the default settings in Geneious ver. 10.2.6. The substitution model GTR, the NNI (default, fast) topology search and 1000 bootstrap replicates (Guindon et al. 2010) were selected for building the tree.

Molecular phylogenetic analyses
Two SSU (18S) sequences of 846 and 1587 bp and two D2-D3 of LSU (28S) sequences of 766 and 778 bp were generated from the juvenile and male Trefusialaimus specimens, respectively. The two SSU sequences were 100% identical over the 841 bp region of overlap. The two LSU sequences were Fig. 1. Bayesian tree of the Enoplida (and Triplonchida outgroup) inferred from SSU sequences, aligned using the MUSCLE alignment algorithm and with regions of questionable alignment removed using Gblocks, under the general time-reversible (GTR) + proportion of invariable sites (I) + gamma distribution (G) model. Trefusiina sequences are shown in underlined font, and new Trefusialaimus sequences are highlighted in grey. The fi ve enoplid clades described by Bik et al. (2010) are identifi ed on the right. Posterior probability (left) and bootstrap values (right) are given on corresponding clades. Dashes (-) indicate no support and aserisks (*) indicate <50% support. The scale stands for substitutions per site. largely identical, with the exception of two ambiguous base pairs (R vs A and Y vs T) and an additional guanine in a polyG stretch (5 Gs vs 4 Gs) in the juvenile sequence; however, these differences were not considered relevant as possibly resulting from PCR or sequencing artefacts. These results support the morphological observations of the male and juvenile specimens suggesting that they belong to the same species.
The SSU consensus tree recovered six main enoplid lineages, which largely correspond to the enoplid clades identifi ed by Bik et al. (2010), although with some discrepancies (Fig. 1). A major difference is that in our analysis, the Ironidae de Man 1876 (Ironus Bastian, 1865, Dolicholaimus de Man, 1888and Trissonchulus Cobb, 1920 and the Alaimina (Alaimus de Man, 1880 and Paramphidelus Andrássy, 1977) are not grouped together (clade 2 in Bik et al. 2010). However, the Maximum Likelihood support for the placement of the Ironidae and Alamina is weak or non-existent in both the present study and in Bik et al. (2010).
The Trefusiina did not form a monophyletic group in the SSU phylogeny due to the placement of Lauratonema Gerlach, 1953 and Trefusialaimus sequences well outside of enoplid clade 3 ( Fig. 1). Lauratonema sequences were grouped with Anoplostoma Bütschli, 1874 with weak to strong support (enoplid clade 5; 96% posterior probability and 50% bootstrap support), whereas Trefusialaimus sequences were grouped with sequences of the Suborder Campydorina (Rhabdolaimus de Man, 1880, Campydora Cobb, 1920 andSyringolaimus de Man, 1888) with no or weak support (enoplid clade 1; 71% posterior probability and 0% bootstrap support). Although this SSU phylogeny does not provide support for the inclusion of Lauratonema and Trefusialaimus within the Trefusiina, it does not provide conclusive evidence indicating which clade they should be assigned to because of generally weak Maximum Likelihood support values.

Phylum Nematoda Diesing, 1861
Class Enoplea Inglis, 1983 Subclass Enoplia Pearse, 1942Order Enoplida Filipjev, 1929Suborder Trefusiina Siddiqi, 1983 Diagnosis (modifi ed from Smol & Coomans 2006) Cuticle smooth or striated, except in Xennellidae De Coninck, 1965 where it is annulated. Metanemes absent, except in Trischistomatidae. Amphids usually non-spiral; spiral only in some Trefusiinae Gerlach, 1966 andHalanonchinae Wieser &Hopper, 1967. Outer labial and cephalic sensilla setiform and usually positioned in two well-separated circles (except in Trefusialaimus, Lauratonematidae and Tripylina). No cephalic capsule, except in Xennella which has a non-annulated cephalic capsule formed by thickening of body cuticle. Buccal cavity usually without teeth (except in Trischistomatidae and Lauratonematidae). Opening of pharyngeal glands unknown in most species, near buccal cavity in Trefusialaimus. Male reproductive systems usually with two testes (monorchic in Trefusialaimus and Trischistomatidae). Female reproductive system monorchic or diorchic; ovaries refl exed except in Cytolaimium exile Cobb, 1920 and Trefusialaimus idrisi where they are outstretched. Caudal glands (when present) lie completely within the tail or position unknown. Lorenzen (1981) erected the order Trefusiida, which was subsequently the lowered to the level of suborder within the Enoplida in the classifi cation of De Ley & Blaxter (2004) based on analyses of 18S rRNA sequences (Rusin et al. 2001) and following Siddiqi (1983). The Trefusiida was not considered monophyletic by Lorenzen (1981) because it is not characterized by any character which is apomorphic for that taxon. The order, as defi ned by Lorenzen (1981), originally comprised the following families: the Simpliconematidae, Xennellidae, Lauratonematidae, Trefusiidae and Onchulidae Andrássy, 1964. The Onchulidae, which is comprised of terrestrial and freshwater species but no marine species, has since been moved to the order Triplonchida Cobb, 1920 in the classifi cation of De Ley & Blaxter (2002;2004). The Onchulidae is characterised by spicules surrounded by a muscular pouch (or 'capsule'), a trait which differentiates the Triplonchida from the Enoplida (De Ley & Blaxter 2002).

Remarks
The family Simpliconematidae comprises a single genus with a single species described from a single male specimen. Simpliconema aenigmatoides Blome & Schrage, 1985 was placed within the Trefusiida by Lorenzen (1981Lorenzen ( , 1994 based on the presence of three lips, the pharyngeal glands seemingly opening in the frontal part of the pharynx, the absence of metanemes and having secretory-excretory gland located in the pharyngeal region. Lorenzen (1981Lorenzen ( , 1994, however, also noted similarities with the genus Linhystera Juario, 1974, family Xyalidae Chitwood, 1951, order Monhysterida Filipjev, 1929 in the arrangement of cephalic sensilla, amphid shape, location of secretory-excretory gland and single anterior testis to the left of the intestine. The family Xennellidae comprises two genera, Xennella Cobb, 1920 andPorocoma Cobb, 1920, comprising four and one species, respectively. This group is unusual in having a cephalic capsule (Xennella) and an annulated cuticle with longitudinal ridges (both genera) but is similar to most Trefusiina in having outer labial and cephalic setae in separate circles and pocket-shaped amphideal fovea, as well as lacking metanemes (Lorenzen 1981).
The family Lauratonematidae comprises three genera (Lauratonema, Lauratonemella Tchesunov, 1984 andLauratonemoides De Coninck, 1965, together comprising 13 species) which differ in the structure of the male copulatory apparatus and/or female reproductive system. The Lauratonematidae differ from most other Trefusiina taxa (except Tripylina and Trefusialaimus) in having the outer labial setae and cephalic setae in a single circle. The family is also characterized by unique features within the Enoplida, i.e., vulva either very close to anus or with female genital branch joining the cloaca, presence of only one posterior testis in some species, and ovary always to the left of the intestine and posterior testis always to the right of the intestine (Lorenzen 1981). The placement of this group in the SSU phylogenetic tree indicates no relationship with the Trefusiina; instead it appears to be closely related to Anoplostoma (see Fig. 1). However, due to the weak support for this placement, we leave the Lauratonematidae within the Trefusiina until more conclusive evidence for an alternative classifi cation is provided.
The family Trischistomatidae comprises two genera: Tripylina with 22 valid species and Trischistoma with 17 valid species. Phylogenies based on SSU sequences consistently show that Trischistoma and Tripylina form a monophyletic clade with the Trefusiidae, which, along with morphological similarities discussed below, strongly indicate that the Trischistomatidae should be included in the Trefusiina.

Remarks
Simpliconema is characterized by cephalic and caudal regions similar to those of Marisalbinema Tchesunov, 1990 (family Xyalidae; Fig. 2), which was described after the treatment of the Trefusiida by Lorenzen (1981Lorenzen ( , 1994, while the long slender spicules are similar to those of Paramonohystera Steiner, 1916 (Xyalidae). More broadly, Simpliconema is characterized by features which resemble the Monhysterida more closely than the Trefusiina, including a circular amphideal fovea (within the Trefusiina, the amphideal fovea is circular only in some Trefusiidae genera, namely species of Cytolaimium, Trefusia, and Rhabdocoma), 6 + 10 arrangement of the anterior sensilla (usually 6 + 6 + 4 in the Trefusiina, except in the Lauratonematidae, Trefusialaimus and Tripylina), and the presence of  Tchesunov, 1990. Drawings modifi ed from Blome & Schrage (1985) and Tchesunov (1990). only one anterior testis (usually two testes in Trefusiina, except some Lauratonematidae, Trefusialaimus, and Trischistomatidae). Blome & Schrage (1985) also noted differences with the Trefusiidae, such as the structure of the sperm (drop-shaped vs elongated in the Trefusiidae) and differentiated vas deferens (vs undifferentiated in the Trefusiidae). It appears likely that the taxonomic placement of the Simpliconematidae will need to be updated. Morphological information on the structure of the female reproductive system should allow us to settle the placement of this genus.

Remarks
De Coninck (1965) erected the subfamily Xennellinae, which originally only contained the genus Xennella. He placed the subfamily within the family Dasynemellidae De Coninck, 1965, order Desmodorida De Coninck, 1965. The subfamily was later raised to family by Gerlach & Riemann (1973. The Xennellidae was placed within the Trefusiida by Lorenzen (1981) based on the pocket-shaped amphid and absence of metanemes. This classifi cation was followed by De Ley & Blaxter (2004).
Within the Enoplida, an annulated cuticle is found in the genus Cricohalalaimus Bussau, 1993(Oxystominidae, suborder Ironina Siddiqi, 1983, and the Lauratonematidae (suborder Trefusiina) are characterized by a "distinctly striated" cuticle. A cephalic capsule is present only within the suborder Enoplina, which is formed by the muscles of the anterior end of the pharynx attaching to the body cuticle. In Xennella, there is no evidence of any attachment between the pharynx and the body cuticle; instead, the cephalic capsule appears to be formed solely by the thickening of the cuticle. This would suggest that this genus may be better placed within one of the marine chromadorean orders, instead of within the Enoplida. It is possible that the placement of Xennella and Porocoma will need to be updated in the future as more morphological and molecular data become available.
Genus Xennella Cobb, 1920 Diagnosis Xennellidae. Cuticle annulated or smooth; longitudinal ridges on cuticle present or absent. Tapering cephalic capsule offset from rest of body by constriction, cuticular discontinuity and/or thickened cuticle. Female reproductive system with refl exed anterior ovary and rudiment of posterior genital branch. Male reproductive system monorchic (at least in X. suecica Allgén, 1935). Spicules short, arcuate; gubernaculum present or absent, precloacal supplements present or absent. Four species.

Remarks
In the classifi cation of Filipjev (1925;1934), Xennella was placed together with the genus Tycnodora Cobb, 1920, which has since been synonymized with Halalaimus de Man, 1888 by Lorenzen (1981), and Schistodera Cobb, 1920, which has since been synonymized with Oxystomina Filipjev, 1918(family Oxystominidae Chitwood, 1935 by Hope & Murphy (1972). This placement refl ected the apparently smooth cuticle of X. cephalata, although the cuticle of X. suecica and X. metallica is clearly annulated. De Coninck (1937) later provided a detailed description of the males of X. suecica, and indicated close similarities between Xennella and Dasynemoides Chitwood, 1936 based on the annulated cuticle with longitudinal ridges, structure of the cephalic capsule and arrangement of anterior sensilla.
The changing classifi cation of Xennella partly stems from uncertainty regarding the structure of the amphids. While Cobb (1920) shows a pocket-shaped amphideal fovea in his original description of X. cephalata (which indicates relationships with the Enoplida), De Coninck (1965) shows a rounded amphideal fovea in X. suecica (which, together with other features, could indicate relationships with either the Desmodoridae Filipjev, 1922, Ceramonematidae Cobb, 1933or Monoposthiidae Filipjev, 1934. Other authors only show an almond-shaped amphideal aperture without showing the structure of the amphideal fovea (Allgén 1935;Tchesunov 1988), which may have been obscured by the thick cuticle of the cephalic capsule (De Coninck 1937).
Genus Porocoma Cobb, 1920 Diagnosis (from Gerlach 1962 andCobb 1920) Xennellidae. Cuticle annulated with longitudinal ridges. Cephalic capsule absent. Secretory excretory pore lies on a setiform elevation. Amphideal fovea horseshoe-shaped. Female reproductive system with two posterior ovaries, one of which extends anterior to vulva and folds posteriorly. Number and structure of male genital branch(es) unknown. Spicules short, arcuate. Tail conicocylindrical. One species.

Remarks
This genus was considered closely related to Oxystomina by Wieser (1953), presumably due to the shape and arrangement of the cephalic sensilla, the minute buccal cavity and body shape. Porocoma was included in the Oxystominidae in the classifi cation of Hope & Murphy (1972) but was later moved to the family Xennellidae by Lorenzen (1981) based on similarities with Xennella in the shape and arrangement of cephalic sensilla and presence of longitudinal cuticular ridges.
Family Lauratonematidae Gerlach, 1953 Diagnosis (emended from Lorenzen 1981) Cuticle distinctly striated. Metanemes absent. Outer labial setae and cephalic setae arranged in one circle. Amphideal fovea non-spiral, pocket-or club-shaped. Buccal cavity usually cuticularized; funnel-shaped pharyngostoma, sometimes with small teeth, cheilostoma cylindrical, shallow or deep. Secretory-excretory system either restricted to pharyngeal region or extends further posteriorly. Female reproductive system monodelphic with anterior refl exed ovary to the left of the intestine; vulva located very close to anus (Lauratonemoides) or ending in the cloaca (Lauratonema and Lauratonemella). Male reproductive system monorchic or diorchic, posterior testis always to the right of the intestine; precloacal supplements absent. Spicules short, straight or only slightly bent; gubernaculum present or absent. Caudal glands lie completely within the tail. Tail conical or conicocylindrical.

Type genus
Lauratonema Gerlach, 1953. Lorenzen (1981) states that this family is characterised by a monorchic male reproductive system; however, more recent species descriptions (Tchesunov 1984;Fadeeva 1989;Chen & Guo 2015) show the presence of two opposed testes in some Lauratonema, Lauratonemoides, and Lauratonemella species. The family was revised by Tchesunov (1984), who provided a key to species of the family.

Remarks
A recent key to valid species of the genus was provided by Chen & Guo (2015).
Genus Lauratonemella Tchesunov, 1984 Diagnosis Lauratonematidae. Female gonad and intestine ending in the cloaca. Male reproductive system with two opposed testes. Asymmetric male copulatory apparatus with left spicule larger than right spicule and left gurbenacular apophysis larger than right apophysis. One species.
Family Trischistomatidae Andrássy, 2007 Diagnosis (from Zhao 2011) Cuticle smooth, thin, not annulated. Metanemes present. Labial region divided into three lips. Outer labial setae and cephalic setae either in one circle or two separate circles. Amphideal fovea pocket-shaped with slit-like amphideal aperture. Buccal cavity narrow, surrounded by pharyngeal musculature, with three teeth (often only one is visible) in one or two stomatal chambers. Pharynx muscular, cylindrical; cardia present or absent. Female monodelphic with anterior refl exed ovary, with or without post-vulval uterine sac; vulva located at > 59 % of body length from anterior extremity. Male reproductive system monorchic. Spicules narrow, may or may not be enclosed within a muscular pouch; when present, muscle pouch almost completely surrounds spicules. Papillose precloacal supplements present or absent. Spermatozoa elongated or globular, usually with visible nucleus. Tail with three glands and terminal spinneret.

Remarks
The classifi cation of the family was last revised by Zhao (2011). Phylogenies based on SSU sequences consistently show that while Trischistoma and Tripylina form a monophyletic clade with the Trefusiidae, the two genera do not form a monophyletic clade, and Trischistoma is more closely related to the Trefusiidae than Tripylina (Holterman et al. 2006;Meldal et al. 2007;Zhao & Buckley 2009;van Megen et al. 2009;Bik et al. 2010present study). Morphologically, Trischistoma may be considered more similar to Trefusiina than Tripylina due to its having the same arrangement of the anterior sensilla (outer labial and cephalic setae in separate circles), as well as having the buccal cavity with minute denticles only (no teeth in Trefusiina); Tripylina differs more strongly in the arrangement of the anterior sensilla (outer labial and cephalic setae in one circle) and buccal cavity with larger teeth.
Trischistoma also shares an unusual spermatozoa morphology (relatively large and/or elongated, elliptical or fusiform, with central rod and/or nucleus near one extremity) with several Trefusiidae species. In Trischistoma, spermatozoa have been described for T. equatoriale Andrassy, 2006 and T. tenuissimum Andrassy, 2011, and they both exhibit this unusual morphology. In the Trefusiidae, similar spermatozoa have been observed in species of Trefusialaimus (Riemann 1974;Leduc 2013), Rhabdocoma (Ott 1977;Vincx & Vanreusel 1989), and Trefusia (Bussau 1993). This kind of spermatozoa morphology has not been observed in Tripylina.
The Trischistomatidae is characterised by having a buccal cavity with teeth, a feature not found in any other Trefusiina family except some Lauratonema. We therefore propose to retain this family for the time being, despite the morphological differences between the two genera, and SSU phylogenies indicating that Trischistoma and Tripylina do not form a monophyletic group.
Other valid species
Family Trefusiidae Gerlach, 1966 Diagnosis (emended from Lorenzen 1981) Cuticle smooth or faintly striated. Labial region generally divided into three lips. Inner labial sensilla usually papillose, rarely setose, either in separate circle or very close to outer labial sensilla. Outer labial sensilla and cephalic setae usually in separate circles (except Trefusialaimus); outer labial setae usually jointed, cephalic setae often located far posteriorly, sometimes posterior to amphids. Amphids either spiral or non-spiral (round or pocket-shaped). Buccal cavity without teeth, either minute to medium size, funnel-shaped and not cuticularised, or large, barrel-shaped and cuticularised. Secretory-excretory system restricted to pharyngeal region (often not observed). Spicules short, curved or straight, with or without capitulum; gubernaculum present or absent, without apophyses (except in Africanema). Female reproductive system monodelphic or didelphic; ovaries refl exed (except in Cytolaimium exile and Trefusialaimus idrisi). Male reproductive system usually diorchic (monorchic in Trefusialaimus). Papilliform, setiform or discoid precloacal and pharyngeal supplements may be present. Caudal glands restricted to tail region.

Type genus
Trefusia de Man, 1893. Gerlach (1966) erected the subfamily Trefusiinae, which he placed within the Oxystominidae and which comprised the genera Trefusia, Rhabdocoma, Cytolaimium and Halanonchus. While there had been general agreement by various authors about the placement of Trefusia with the Oxystominidae (Filipjev 1934;De Coninck & Schuurmans Stekhoven 1933;Chitwood & Chitwood 1937), the placement of Cytolaimium, Rhabdocoma and Halanonchus was more controversial and relationships had been proposed with the families Monhysteridae de Man, 1876, Linhomoeidae Filipjev, 1922and Tripyloididae Filipjev, 1928(Filipjev 1934Chitwood 1936Chitwood , 1951Wieser 1956;de Coninck 1965;Riemann 1966). Riemann (1966) noted that although Rhabdocoma and Trefusia share many similarities, they differ in the structure of the female reproductive system (monodelphic in Rhabdocoma and didelphic in Trefusia); however, he followed the classifi cation of Gerlach (1966). Wieser & Hopper (1967) subsequently moved Rhabdocoma, Cytolaimium and Halanonchus into the freshly erected subfamily Halanonchinae, which they placed within the family Tripyloididae. They justifi ed this change based on the large buccal cavity (in Halanonchus), the spiral amphids (in Cytolaimium and Rhabdocoma) and the presence of jointed setae (all three genera). They also argued that the presence of deeply incised lips, a trait also found in some Tripyloididae, indicates relationships with Tripyloididae. This argument appears to be mostly based on their observations of deeply incised lips in Halanonchus macrurus Cobb, 1920; however, we argue that they have misinterpreted the presence of a cuticular discontinuity in the buccal cavity as deeply incised lips (see below). It is not clear why Trefusia was not also moved to the Halanonchinae as it is very similar to Rhabdocoma and Cytolaimium except for features of the reproductive system. Gerlach & Riemann (1973/74) modifi ed the classifi cation of Wieser & Hopper (1967) by bringing the Halanonchinae together with the Trefusiinae and raising the latter to family status. They also moved Cytolaimium and Rhabdocoma back to the Trefusiinae, leaving Halanonchus as the sole genus within the Halanonchinae. No reason was given for this change, but it seems likely that the subfamilies were re-organised to refl ect differences in the buccal cavity (i.e., small and not cuticularized in Trefusiinae vs large and cuticularized in Halanonchinae). Trefusialaimus, a genus with a minute buccal cavity, was subsequently described by Riemann (1974) and placed within the Trefusiinae. Vincx & Furstenberg (1988) later described Africanema, a genus with a large cylindrical buccal cavity, which they placed within the Halanonchinae. Shi & Xu (2017) recently proposed moving Rhabdocoma to the Halanonchinae based on the presence of only one ovary in both Rhabdocoma and Halanonchus, and based on the result of phylogenetic analyses of 18S rDNA sequences. They argue that the structure of the female reproductive system is a more taxonomically informative trait for determining relationships among higher taxa than the buccal cavity.

Remarks
Cytolaimium is similar to Trefusia but can be distinguished from the latter by the presence of pairs of discoid supplements in both pre-and post-cloacal regions. Gerlach (1962) synonymized C. obtusicaudatum Chitwood, 1936 with C. exile. This was not accepted by Ott (1977) who considered the two species to be morphologically distinct. Gerlach & Riemann (1973/74) moved Trefusia conica Gerlach, 1957 to Cytolaimium; however, this species lacks the discoid supplements and is therefore considered to belong to Trefusia (Ott, 1977). Ott (1977) reviewed the genus and provided a key to species. The latter author also erected C. gerlachi to accommodate the specimens described by Gerlach (1962), which he deemed morphologically distinct from C. exile. Ott (1977) moved Rhabdocoma articulata to Cytolaimium, but because this species is known from a juvenile only, we consider it species inquirenda.

Diagnosis (modifi ed from Leduc 2013)
Trefusiinae. Cuticle smooth or striated. Six jointed outer labial setae situated in separate circle from the cephalic setae; the latter situated either slightly anterior to, at same level as, or posterior to amphids. Amphideal fovea circular, oval, unispiral, spiral, elongated or pocket-shaped. Buccal cavity small or minute, funnel-shaped, not cuticularized. Males with mid-ventral row of pharyngeal supplements that may be papilliform, setiform, or complex; sometimes two additional subventral rows are also present. Precloacal supplements usually present, papilliform or setiform. Arcuate or almost straight spicules, with or without capitulum; gubernaculum present or absent. Male reproductive system diorchic. Female reproductive system with two opposed and refl exed ovaries. Tail conico-cylindrical or fi liform. Eighteen species.

Remarks
Genus Trefusialaimus Riemann, 1974 Diagnosis (modifi ed from Riemann 1974) Trefusiinae. Cuticle smooth. Sub-cephalic and somatic setae absent. Four jointed cephalic setae and six jointed outer labial setae in one circle; amphid pocket-shaped. Buccal cavity minute, funnel-shaped, not cuticularized. Male with one anterior outstretched testis (monorchic) and peri-cloacal papillae. Elongated sperm cells with central rod and light-refractive nucleus at one extremity. Female reproductive system (known only for T. idrisi) with two opposed and outstretched ovaries. Tail conico-cylindrical or fi liform. Three species.

Remarks
Trefusialaimus and Tripylina are the only genera of the suborder Trefusiina characterized by having the outer labial sensilla and cephalic setae in a single circle. This character agrees with the diagnosis of the Tripyloidina; however, Trefusialaimus differs from the latter in the absence of metanemes (vs metanemes sometimes present in Tripyloidina), and in having pocket-shaped amphids (vs spiral amphids in Tripyloidina) and a toothless buccal cavity (vs teeth common in Tripyloidina). The current placement of Trefusialaimus within the Trefusiinae, which follows Riemann (1974) and Lorenzen (1981Lorenzen ( , 1994, is only tentative and may need to be revised as suggested by SSU phylogenetic analyses (present study). Leduc, 2013

Description
Female Body cylindrical, slender, tapering slightly towards anterior extremity, with slight golden colouration due to the presence of numerous round, ca 1 μm diameter, golden inclusions. Cuticle smooth. Cephalic region rounded, slightly set off from body due to thickened cuticle and constriction immediately posterior to cephalic setae. Three lips, each bearing two small, conical inner labial papillae. Six outer labial setae and four cephalic setae in one circle, all with single joint; cephalic setae slightly longer than outer labial setae (6-9 vs 7-10 μm). Sub-cephalic and somatic setae absent. Amphid pocket-shaped with transverse aperture, ca 6-9 μm wide by 2 μm high. Buccal cavity funnel-shaped, without teeth. Pharynx cylindrical, slightly wider posteriorly, completely surrounding buccal cavity. Nerve ring situated at 49-66% of pharynx length. Secretory-excretory system not observed. Cardia small, surrounded by intestine. Numerous sperm cells are present throughout the pseudocoelom between pharynx and anus, as well as in the uterus. Reproductive system with two opposed and outstretched ovaries, both to the right or left of intestine. Vagina at about two thirds of body length from anterior. Tail long, ca 8-10% of total body length, narrow, gradually tapering, without setae; spinneret not observed.

Remarks
The female specimens described here agree well with the male specimen described from the central Chatham Rise (350 m depth) in the arrangement of anterior sensilla, size and position of the amphids, presence of numerous golden inclusions and tail shape. The female specimens, however, were characterized by longer bodies (5004-5947 vs 4539 μm) and shorter tails (cʹ = 18-21 vs 38). This is the fi rst time that female Trefusialaimus specimens are described. The structure of the female reproductive system in this species, which consists of two opposed and outstretched ovaries, is unusual for the Enoplida, although it has been observed in Cytolaimium exile (Trefusiidae), and Mediolaimus Tahseen, Sultana, Khan & Hussain, 2012and Rogerus Hoeppli & Chu, 1934(Enoplida, family Rhabdolaimidae Chitwood, 1951. It is unclear how sperm had entered the pseudocoelom of the female specimens we observed; however, the same observation was made previously for a juvenile of the same species (Leduc 2013).

Remarks
This subfamily is not monophyletic according to the SSU consensus tree (see Fig. 1). However, it is retained at least for now because Halanonchinae is unique within the Trefusiina in having a female reproductive system with a single posterior ovary.

Species inquirendae
R. articulata (Gerlach, 1955). R. brevicauda Schuurmans Stekhoven, 1950. R. cylindricauda Schuurmans Stekhoven, 1950. R. macrura Cobb, 1920. Ott (1977) reviewed the genus and provided a key to species. He considered R. macrura to be insuffi ciently described and therefore species inquirenda. Ott (1977) also considered R. cylindricauda and R. brevicauda to have been wrongly assigned to Rhabdocoma and instead likely to belong to the Siphonolaimidae. Rhabdocoma articulata was transferred to Cytolaimium by Ott (1977), but the species is doubtful since it is based on a juvenile description only. Rhabdocoma riemanni Jayarsee & Warwick, 1977 was described subsequently to the review by Ott (1977) but it was later synonymized with R. americana by Vincx & Vanreusel (1989).

Diagnosis (modifi ed from Pavlyuk 1984)
Halanonchinae. Cuticle smooth. Brown or golden granules often present along lateral, ventral and dorsal chords. Inner and outer labial sensilla either very close to each other or in separate circles. Inner labial sensilla papillose or setose; outer labial setae sometimes jointed; cephalic setae situated in separate circle further posteriorly but anterior to amphids. Buccal cavity large, barrel-shaped; both gymnostoma and stegostoma with cuticularized walls, often with curved cuticular discontinuity between gymnostoma amd stegostoma, which has been interpreted as "oval structures" supporting the buccal cavity. Amphideal fovea pocket-shaped, at level of buccal cavity or posterior to buccal cavity; oval or rounded amphideal aperture. Mid-ventral row of papillose pharyngeal supplements (complex supplements in H. bullatus Gerlach, 1964) usually present in males. Male copulatory apparatus consists of short, arcuate or straight spicules, and small gurbenaculum without apophyses; precloacal supplements present. Tail long and fi liform. Seven species.

Remarks
Latilaimus was synonymized with Halanonchus by Gerlach (1964). Pavlyuk (1984) considered H. renatus invalid due to the incomplete description, which did not include male specimens. We consider H. macramphidus and H. zosterae to be species inquirendae for the same reason. No females have yet been described for H. longicaudatus or H. papilatus.
Although the presence of pharyngeal supplements is given as a genus character by Pavlyuk (1984), this feature is absent in H. longicaudatus. Furthermore, only one inconspicuous pharyngeal supplement is present in H. cornutus and pharyngeal supplements were not observed in all male H. scintillatulus sp. nov. specimens. We also note that, within the genus, inner labial sensilla can be either papillose or setose, and can be situated either very close to the outer labial sensilla or in a separate circle. Wieser & Hopper (1967) listed deeply incised lips as a diagnostic feature of the Halanonchinae. The latter authors included Cytolaimium, Rhabdocoma and Halanonchus in the subfamily, which they classifi ed with the Tripyloididae. This family includes the genus Bathylaimus Cobb, 1894, which is characterized by deeply incised lips. This feature was later included in the diagnosis of Halanonchus provided by Pavlyuk (1984). We did not observe this feature in the Halanonchus specimens from the Hauraki Gulf, however, and did not see it clearly described in any description of Halanonchus species. We postulate that the presence of deeply incised lip was a misinterpretation of buccal cavity structures. We also believe that the description of oval structures supporting the buccal cavity resulted from a similar misinterpretation, as explained below.
Oval structures supporting the buccal cavity were fi rst illustrated in the description of H. macrurus by Cobb (1920). Unfortunately, the nature of these structures was not described or interpreted by the latter author. In his description of H. macramphidus Chitwood, 1936, Chitwood (1936 described a buccal cavity similar to that described by Cobb (1920), but without any oval structure. Chitwood's illustration shows instead a fi ne line demarcating the gymnostoma and stegostoma, which is consistent with a cuticular discontinuity in the buccal cavity wall. Gerlach (1964) included oval structures in his illustration of the buccal cavity of H. bullatus Gerlach, 1964, the posterior edge of which coincides with a cuticular discontinuity between the gymnostoma and stegostoma as seen in cross-section. He did not describe or interpret, however, the nature of these oval structures beyond illustrating them. Oval structures were fi rst explicitly discussed by Wieser & Hopper (1967) in their decription of H. macrurus. They stated that: "Each lip seems to be supported by a large oval structure which apparently was mistaken for the amphids by Allgén (1933) in his description of Latilaimus zosterae" (Wieser & Hopper 1967: 249). Their illustration shows a single oval structure, with a posterior margin coinciding with the limit between gymnostoma and stegostoma. Their illustration also shows two thin longitudinal lines spanning the length of the gymnostoma and stegostoma, which touch one side of the oval structure. These lines may have been interpreted by these authors as indicating the presence of deeply incised lips in this species. Our own observations, as well as the descriptions by Cobb (1920), Chitwood (1936), andGroza-Rojancovski (1972) in particular, indicate to us that the gymnostoma and stegostoma in Halanonchus are delimited by a curved cuticular discontinuity. In the case of H. scintillatulus sp. nov., the two buccal cavity compartments are also delimited by the more thickly cuticularized anterior edge of the stegostoma, which projects slightly into the buccal cavity (Fig. 5). In addition, our own observations, as well as the illustrations of Wieser & Hopper (1967) and Pavlyuk (1984), indicate that the ventrosublateral and dorsal sectors of the gymnostoma and stegostoma are delimited by longitudinal cuticular discontinuities. We hypothesise that it is the presence of these cuticular discontinuities which led previous authors to describe the presence of three oval structures supporting the buccal cavity in Halanonchus species, as well as the presence of deeply incised li ps.

Differential diagnosis
Halanonchus scintillatulus Leduc sp. nov. is the smallest species of the genus, with a body length less than 2000 μm (

Etymology
The species name is derived from the latin term 'scintillula', diminutive of 'scintillo' (= sparkle, glitter), and refers to the numerous small, light refractive granules present along the body of most specimens of this species.

Discussion
In the present study, we provided the fi rst overview of the Trefusiina since the work of Lorenzen (1981), which was later updated (Lorenzen 1994). The suborder now comprises fi ve families, 14 genera and 92 valid species, most of which belong to either the Trischistomatidae (39 species) or the Trefusiidae (34 species). We provide the fi rst record of the genus Halanonchus from the New Zealand region, describe the female reproductive system of Trefusialaimus for the fi rst time and provide the fi rst molecular sequences for this genus. Our SSU phylogenetic analyses confi rm that the Trischistomatidae are closely related to the Trefusiidae and provide strong support for including this family within the Trefusiina. Our results, however, puts into question the placement of Trefusialaimus and Lauratonema, which appear to have affi nities with taxa outside of the Trefusiina.
Trefusialaimus is characterized by some unusual morphological features within the Trefusiina, namely outer labial and cephalic setae in a single circle and female reproductive system with two outstretched ovaries. The former feature is also shared by the Lauratonematidae and Tripylina, but the latter feature is rare both within the Trefusiina (currently only known for Cytolaimium exile) and the Enoplida, where it is found in some Rhabdolaimidae, suborder Campydorina (Tahseen et al. 2012;Holovachov 2019). The SSU consensus tree indicates a possible relationship between Trefusialaimus and the Campydorina, although support for this placement was weak. The two taxa exhibit some similarities, including nonspiral amphids, monorchic male reproductive system, and outstretched ovaries (in some Campydorina taxa), but also show some dissimilarities in the structure of the cephalic sensilla (setose in Trefusialaimus vs usually papilliform in Campydorina), buccal cavity (without teeth in Trefusialaimus vs with teeth in Campydorina) and pharynx (no posterior bulb in Trefusialaimus vs bulb present in Campydorina). Trefusialaimus is also characterized by a somewhat unusual shape and structure of the sperm, which is elongated with a central rod and a cone-shaped nucleus at or near one end (Riemann 1974;this study). A similar sperm morphology has been observed in some species of Syringolaimus, order Campydorina, such as S. loofi Gourbault &Vincx, 1985 andS. renaudae Gourbault &Vincx, 1985, which also bears a fl agella at the nucleated end of the sperm. We have observed numerous sperms in the pseudocoelom of both Trefusialaimus idrisi juveniles (Leduc 2013) and females (present study). While it is unclear how sperm reached the pseudocoelom in these non-males, their presence outside the male and female genital tracts indicate that they are highly mobile. Further work is required to understand our observations of sperm in the pseudocoelom of juveniles and females of T. idrisi, as well as the potential taxonomic signifi cance of this unusual sperm morphology.
Our consensus SSU tree suggests that Lauratonema is closely related to Anoplostoma, family Anoplostomatidae Gerlach & Riemann, 1974, suborder Enoplina, with weak Maximum Likelihood support but strong posterior probability support. The Lauratonematidae and Anoplostoma share some morphological similarities, namely outer labial and cephalic setae in a single circle and pocketshaped amphids with small opening. In addition, the buccal cavity of some Lauratonema species (i.e., L. macrostoma, L. reniamphidum and L. mentulatum) is spacious and heavily cuticularized as in Anoplostoma. The Lauratonematidae, however, are characterized by the absence of metanemes (present in Anoplostoma), and a different structure of the female reproductive system (monodelphic with vulva close to anus or forming a cloaca in Lauratonematidae vs didelphic in Anoplostoma) and of the male reproductive system (monorchic or diorchic with outstretched testes in Lauratonematidae vs diorchic with refl exed posterior testis in Anoplostoma). Nevertheless, our fi ndings indicate that the placement of the Lauratonematidae may need to be changed in the future as more comprehensive molecular analyses are conducted.
Our SSU phylogenetic analysis supports the inclusion of the Trischistomatidae within the Trefusiina, as well as a close relationship between the Trefusiina and Tripyloidina, as indicated in previous SSU phylogenies (Holterman et al. 2006;Meldal et al. 2007;Zhao & Buckley 2009;van Megen et al. 2009;Zhao et al. 2012). Because metanemes are present in most enoplids, and indeed both the Tripyloidina and Trischistomatidae, which occupy a basal position within enoplid clade 3, it appears likely that metanemes were lost in the ancestor of the Trefusiidae. Metanemes may in fact have been lost repeatedly if Lauratonema and Trefusialaimus, which both lack metanemes, are confi rmed as belonging outside of the Trefusiina in future phylogenetic studies. Similarly, teeth are present in both the Tripyloidina and Trischistomatidae (but not the Trefusiidae), suggesting that they were lost in the ancestor of Trefusiidae. The inclusion of Trischistomatidae within the Trefusiina means that the suborder now includes terrestrial/ freshwater taxa in addition to marine taxa, and that all of the enoplid suborders now include at least some terrestrial/freshwater representatives. This lack of separation between marine and terrestrial taxa refl ects the multiple transitions that have occurred in both directions across the phylum between terrestrial and marine habitats (Holterman et al. 2019).