Two new free-living nematode species ( Trefusiina : Trefusiidae ) from the Chatham Rise crest , Southwest Pacific Ocean

Two new species of the family Trefusiidae, viz., Trefusia piperata sp. nov. and Trefusialaimus idrisi sp. nov., are described from the crest of the Chatham Rise, Southwest Pacific Ocean (350 m water depth). The present study provides the first species records for this family in the region. Trefusia and Trefusialaimus comprise twenty and three valid species, respectively. A key to males of Trefusia is provided.


Introduction
Recent studies show high levels of nematode alpha (local) and beta (turnover) diversity on the continental slope of New Zealand (Leduc et al. 2012 a, b).The systematics of several free-living marine nematode taxa, however, remains unstudied in the region.One such taxon is the family Trefusiidae, for which no species records exist to date (Leduc & Gwyther 2008).Surveys of nematode communities on Chatham Rise and Challenger Plateau suggest the presence of at least twelve Trefusiidae morphospecies (Leduc et al. 2012a;D. Leduc unpublished data).The low density of most species in deep-sea habitats, however, often impedes the description of new species (e.g., Miljutin et al. 2010).Man, 1893 andTrefusialaimus Riemann, 1974 are from relatively coarse (sandy) sediments (e.g., Riemann 1974;Keppner 1992;Van Gaever et al. 2004;Riera et al. 2010).Trefusia has been recorded in a variety of deep-sea habitats such as manganese nodule deposits (Bussau 1993;Miljutin et al. 2010), canyons (Vitiello 1970), and mounds ( Van Gaever et al. 2004).Trefusialaimus is a rare genus and, until now, had only been recorded from the Arctic Ocean (Filipjev 1946) and Northeast Atlantic (Riemann 1974).Here, two new species, Trefusia piperata sp.nov.and Trefusialaimus idrisi sp.nov., are described from sandy sediments on the continental slope of New Zealand.

Methods
Sediment samples were obtained from chatham Rise, a submarine ridge that extends eastwards from the South Island of New Zealand, from depths of ca.250 to 3000 m.Samples were collected in February 2011 during National Institute of Water and Atmospheric Research (NIWA) cruise TAN1103, from a site near central Chatham Rise crest at a depth of 350 m (43.331° S, 178.288° E) (see Leduc & Pilditch 2013 for details of sampling site).
A total of 15 sediment samples was collected, using an Ocean Instruments MC-800A multicorer (MUC; core internal diameter = 9.5 cm).Samples were obtained from ten MUC deployments from the sampling site.Each sample consisted of one subcore of internal diameter 26 mm taken to a depth of 5 cm.Cores were split into 0-1, 1-3, and 3-5 cm sediment depth layers, fixed 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 sieve to retain nematodes.Nematodes were extracted from the remaining sediments by Ludox flotation and transferred to pure glycerol (Somerfield & Warwick 1996).Species descriptions were made from glycerol mounts using differential interference contrast microscopy (10-1000× magnification) and drawings were made with the aid of a camera lucida.Measurements (in µm) were made using image analysis software (cellSens Standard 1.6), and all curved structures are measured along the arc.Type specimens are held in the NIWA Invertebrate Collection (NIC), Wellington.

Diagnosis
Cuticle smooth or with faint striations.Metanemes absent.Amphid either spiral or non-spiral.Outer labial sensillae and cephalic setae usually in two separate circles (except in Trefusialaimus); jointed cephalic setae.Buccal cavity funnel-or barrel-shaped, teeth absent.Males usually with two testes (except Trefusialaimus) and females usually with two ovaries.

Diagnosis
Amphideal fovea cryptospiral, oval, or horse-shoe shaped.Funnel-shaped buccal cavity.Males with ventral (and sometimes subventral) longitudinal rows of cervical sensillae.Small papillose pre-cloacal supplements present, situated either in a single ventral row or two sub-ventral rows; post-cloacal papillae absent.Spicules usually with handle-shaped proximal ends.Females with two ovaries.Tail conical, conico-cylindrical, or filiform.

Remarks
The last treatment of the genus Trefusia was by Keppner (1992), who provided an updated key to the males of the 17 species of the genus based on Riemann (1966).T. varians Gerlach, 1955 was not included in the key because the original description was based on female specimens only.Since then, Trefusia monodelphis Bussau, 1990 was synonymised with Trichistoma gracile Andrássy, 1985by Andrássy (2007), and two new species were described by Bussau (1993).There are currently 20 valid Trefusia species (including T. piperata sp.nov.).

Diagnosis and relationships
Trefusia piperata sp.nov. is characterised by six double-jointed outer labial setae with conspicuous clusters of dark granules at their base, four cephalic setae at level of amphid, one seta posterior to each amphid, and long filiform tail.Male is characterised by six cervical papillae, eight papillose pre-cloacal supplements, slightly bent spicules, and gubernaculum with funnel-shaped distal portion and pointed projections.
Trefusia piperata sp.nov.most closely resembles T. helgolandica Riemann, 1966, described from subtidal sediments in the German Bight.The two species are similar in the shape and position of the outer labial setae and cephalic setae, as well as in the structure of the spicules and gubernaculum.The new species, however, differs from T. helgolandica in head diameter (14-15 vs. 22-25), maximum body width (21-22 vs. ≥ 33), spicule length (24 vs. 38), number of setae posterior to the amphids (one vs. two or three), the number and shape of cervical papillae (six papillae with wide base vs. thirteen papillae without wide base), and number of pre-cloacal supplements (eight vs. fourteen).Trefusia piperata sp.nov.also differs from T. helgolandica in the presence of conspicuous clusters of dark granules at the base of the outer labial setae.

Etymology
The species name is derived from the latin word piperatus (= peppered), and refers to the conspicuous clusters of dark granules at the base of the cephalic setae.
Reproductive system diorchic with outstretched testes.Position of testes relative to intestine difficult to ascertain.Sperm cells drop-shaped with rod-shaped nucleus; vas deferens ca.445 µm long.Paired, equal spicules, slightly bent near distal one third, without obvious central cuticularised projection (i.e., lamella or median rib).Gubernaculum with narrow proximal region and funnel-shaped distal region with large anterior pointed projection and smaller posterior pointed projection (Figs 1D, 2B).Eight small papillose pre-cloacal supplements situated ventrally, 6-15 µm apart except for anterior-most supplement which is situated 40 µm from next supplement.Tail very long, ca.half of total body length, filiform, without setae, tightly coiled.

Female
Similar to male, but with slightly shorter cephalic setae and slightly smaller amphid (Fig. 1A).Female reproductive system didelphic, amphidelphic with reflexed ovaries.Position of ovaries relative to the intestine difficult to ascertain.Vulva situated at one third of body length (Fig. 1C).

Discussion
Trefusia piperata sp.nov.was rare at the study site, with only four specimens (the two type specimens and two juveniles in poor condition, each from a different subcore) recorded out of the 4412 individuals that were identified by Leduc & Pilditch (2013).Two individuals were found in the 1-3 cm sediment depth layers, and two were found in the 3-5 cm sediment depth layer (D.Leduc, unpublished data).

Diagnosis
Sub-cephalic and somatic setae absent.Ten cephalic setae in one circle; amphid pocket-shaped.Male with one testis and peri-cloacal papillae.elongated sperm cells with central rod and light-refractive nucleus at one extremity.Tail conico-cylindrical or filiform.

Etymology
The species is named after Idris Matai Kljucanin Brun, the author's godson.

Discussion
The presence of sperm cells in the pseudocoelom of the juvenile Trefusialaimus idrisi sp.nov.specimen is unusual.Some nematode species, such as Oncholaimus oxyuris, can transfer sperm through traumatic insemination (Coomans et al. 1988), a process whereby the male injects sperm directly into the body of a female (or potentially even a male or juvenile) by piercing the cuticle with the spicules.The presence of sperm cells in the juvenile specimen could be explained if a similar process occured in T. idrisi sp.nov.
The existence of such a reproductive strategy, however, is highly conjectural because no Trefusialaimus females have ever been observed and (to my knowledge) traumatic insemination has not been described in the suborder Trefusiina.
Trefusialaimus idrisi sp.nov.was rare at the study site, with only four specimens (the two type specimens and two juveniles in poor condition, each from a different subcore) recorded out of the 4412 individuals that were identified by Leduc & Pilditch (2013).All individuals were found in the surface (0-1 cm) sediment layer (D.Leduc, unpublished data).A single juvenile specimen (out of 4550 specimens identified from 30 locations on the New Zealand continental margin) was recorded from a site on the northern flank of Chatham Rise at a depth of 1000 m (178.500°E, 44.333° S; silt/clay content 95%) (Leduc et al. 2012a;D. Leduc unpublished data).
specimens %V = vulva distance from anterior end of body × 100/total body length