New species of Thelonema , Metasphaerolaimus , and Monhystrella (Nematoda, Monhysterida) from Kermadec Trench, Southwest Paci ﬁ c

. Three new species of the order Monhysterida are described based on specimens obtained at depths of 8081 and 9177 m in the Kermadec Trench. Thelonema clarki sp. nov. is characterised by a large body size (3230–4461 μ m), short cylindrical buccal cavity, gubernaculum without apophyses, and long conico-cylindrical tail. This is the ﬁ rst record of the genus since its original description over two decades ago from the Peru Basin. Metasphaerolaimus constrictus sp. nov. is characterised by a relatively long body (1232–1623 μ m), slightly arcuate spicules without gubernaculum, and conico-cylindrical tail with inner cuticle conspicuously thickened immediately anterior to cylindrical portion. Monhystrella kermadecensis sp. nov. is characterised by a circle of papillose outer labial sensillae slightly anterior to the four short cephalic setae, gubernaculum with caudal apophyses, the presence of distinct cuticularised piece along anterior vaginal wall, and a relatively short conical (males) or conico-cylindrical tail (females) with conical, ventrally-curved spinneret. M. kermadecensis sp. nov. can be differentiated from all other species of the genus, and, indeed, the entire family, based on the variable position of the anterior gonad relative to the intestine. The new species is classi ﬁ ed within the Monhysteridae, and not the closely-related Xyalidae, based on the small body size, a smooth cuticle, and the presence of six outer labial papillae and only one testis. Further work is required to clarify the placement of M. kermadecensis sp. nov. relative to other monhysterid genera. A tabular key to all ten valid Metasphaerolaimus species is presented.


Introduction
The Order Monhysterida is well represented in hadal trench samples (> 6000 m depth), with the family Monhysteridae often dominant (Gambi et al. 2003;Vanhove et al. 2004).The families Sphaerolaimidae and Linhomoeidae are also common in hadal trench samples, but are typically much less abundant (e.g., Tietjen 1989).Whilst species of the families Sphaerolaimidae and Linhomoeidae are relatively large (> 1 mm in length) and easy to identify, species of the family Monhysteridae are typically small (< 0.8 mm in length) and often represented by many morphologically similar species in deep-sea samples (D.Leduc unpublished data).As a result, data on the distribution of Monhysteridae species in the deep sea are scarce despite their high abundance (Tietjen 1989;Miljutin et al. 2010).In addition, there has been some confusion related to the identifi cation of the small and morphologically similar genera Monhystrella Cobb, 1918and Thalassomonhystera Jacobs, 1987. Vanhove et al. (2004) and Gambi et al. (2003) recorded high densities of the genus Monhystera Bastian, 1865 in hadal trenches, despite a taxonomic revision by Jacobs (1987) almost two decades earlier resulting in all marine Monhystera species being transferred to either Thalassomonhystera or Monhystrella (Fonseca & Decraemer 2008).More work is clearly needed on the taxonomy of this family, as well as other Monhysterida, in order to identify patterns in species distribution within and among trenches.

Materials and methods
Kermadec Trench is situated in the Southwest Pacifi c Ocean and is formed by the subduction of the Pacifi c Plate under the Indo-Australian Plate.It extends from approximately 26° to 36º S near the northeastern tip of New Zealand's North Island.Samples were collected from the axis of Kermadec Trench at depths of 8081 and 9177 m during the Woods Hole Oceanographic Institute (WHOI) cruise TN309 (RV Thomas G. Thompson) in May 2014.The sediment cores were obtained using the submersible ROV Nereus (core internal diameter = 6.35 cm).Cores were sliced into 0-1, 1-2, 2-3, 3-4, 4-5 and 5-10 cm layers and fi xed in 10% buffered formalin.Samples were rinsed on a 20 μm sieve to retain nematodes.Nematodes were extracted from the remaining sediments by Ludox fl otation, stained with Rose Bengal, and transferred to pure glycerol (Somerfi eld & Warwick 1996).Species descriptions were made from glycerol mounts using differential interference contrast microscopy and drawings were made with the aid of a camera lucida.

Results
Class Chromadorea Inglis, 1983Order Monhysterida Filipjev, 1929Superfamily Siphonolaimoidea Filipjev, 1918Family Linhomoeidae Filipjev, 1922 Diagnosis (From Fonseca & Bezerra 2014) Cuticle often striated, sometimes smooth.Inner labial sensillae papillose or not observable.Amphideal fovea usually circular, rarely unispiral.Inner side of labial region formed by an annular, soft-skinned pad that narrows the buccal opening.Cardia long and conspicuous.Usually two outstretched and opposed gonads, rarely single gonad present.Anterior gonad to the left or right of intestine, posterior gonad to the opposite side.

See diagnosis of type and only genus Thelonema.
Thelonema Bussau, 1993 Diagnosis (modifi ed from Bussau 1993) Inner labial sensillae not observable with light microscopy.Six outer labial setae situated in separate circle from the four sensillae of the third circle; the latter consists of papillae located in depressions.Four sub-cephalic setae located immediately posterior to the cephalic papillae.Large circular amphideal fovea located far posteriorly, and with nerve process usually visible.Buccal cavity cylindrical or funnelshaped, cuticularised, without teeth.Pharynx with weak posterior bulb; cardia long and gradually widening posteriorly.Female with two outstretched and opposed ovaries and male with two outstretched and opposed testes.Tail conico-cylindrical.

Etymology
This species is named after Malcolm R. Clark, principal investigator of the HADES project (HADal Ecosystem Studies) who has made an outstanding contribution to the fi eld of deep-sea ecology and who kindly supported the involvement of the author on the Kermadec Trench voyage.

Male
Body cylindrical, tapering slightly towards both extremities.Cuticle striated from level of buccal cavity to near tail tip.Somatic setae absent except for a few 1 μm long setae in pharyngeal region.Lip region slightly concave; head region otherwise rounded.Internal labial sensillae not observed.Six outer labial setae, 5 μm long, situated in separate circle from the four sensillae of third circle; the latter consists of papillae located in depressions (Fig. 3B).Small, granular glands sometimes observed, apparently connected to base of outer labial setae (Fig. 1B); larger unicellular glands also sometimes observed and apparently connected to cephalic sensillae (Fig. 2C).Four sub-cephalic setae located immediately posterior to the cephalic papillae, 3-4 μm long, similar in length or slightly shorter than outer labial setae.Large circular amphideal fovea with cuticularised outline situated 1.7 cbd from anterior body extremity.Mouth opening narrow, surrounded by bulge of inner portion of lip region; buccal cavity cylindrical, 7 μm deep, 9 μm wide, with cuticularised walls, not surrounded by pharyngeal tissue.Pharynx narrow, muscular, with oval posterior bulb; pharyngeal glands and their orifi ces indistinct.Nerve ring slightly posterior to middle of pharynx length.Secretory-excretory system with two or three renette cells (third cell may sometimes be obscured) all situated just posterior to pharyngeal bulb; ampulla and pore between nerve ring and posterior pharyngeal bulb.Cardia conspicuous, 90 μm long, widening posteriorly, not surrounded by intestine walls.
Reproductive system diorchic with two opposed and outstretched testes, anterior testis to the left of intestine and posterior testis to the right of intestine.Mature sperm globular, nucleated, 4-5 × 6-7 μm.Spicules paired, 1.2 abd long, strongly arcuate, with well-developed capitulum and pointed distal end.Gubernaculum thin, pointed at both ends, without apophyses but with rounded glandular tissue extending dorsally.Rectal glands not observed; one ejaculatory gland situated between spicules.Pre-cloacal supplements absent.Tail long, conico-cylindrical, with rounded tip.Caudal glands not observed; short and sparse caudal setae present, no terminal setae.Intestine with numerous transparent crystalline structures, square to rhomboid-shaped, up to 14 × 14 μm (Fig. 3C-D).

Female
Similar to males but with lower values of a, b, and c, slightly smaller amphids (0.42-0.44 vs 0.65 cbd) and longer tail (9.0-10.4vs 6.4 abd).Buccal cavity 5 μm wide and 4-5 μm deep.Reproductive system didelphic; anterior branch outstretched, to the right of intestine, posteror branch poorly developed, to the left of intestine.Spermatheca present in anterior branch only, simple, not cuticularised.Vulva transverse, situated slightly posterior to mid-body; small vaginal glands present on either side of vagina; muscular pars proximalis vaginae.

Juvenile
Similar to females, but with shorter body and lower values of a and b.

Remarks
Thelonema clarki sp.nov.can be differentiated from the only other species of the genus, T. majum, by the larger body size (3230-4461 vs 1000-1460 μm), short cylindrical buccal cavity (vs long and funnelshaped in T. majum), sub-cephalic setae of similar length or shorter than outer labial setae (sub-cephalic setae longer than outer labial setae in T. majum), secretory-excretory system with two or three renette cells (secretory-excretory system not observed in T. majum), absence of gubernacular apophyses (vs dorso-caudal apophyses present in T. majum), and longer tail (6.4-10.4vs 2.7 abd).This is the fi rst time that Thelonema is recorded outside the type locality in the Peru Basin (~ 4150 m depth) since the original description of the genus by Bussau (1993) The presence of crystalline structures in the intestine is intriguing, as many appear too large to have been ingested (the structures are up to 14 μm wide, and the width of the cuticularised buccal cavity is 4-9 μm).It seems unlikely that the buccal cavity can stretch to accommodate such large particles given the presence of cuticularised walls.The crystalline structures may have grown through accretion whilst in the intestine, but this process would presumably require a relatively long period of time.gubernaculum, tail conico-cylindrical with inner cuticle conspicuously thickened immediately anterior to cylindrical portion and with three short terminal setae.

Etymology
The species name is derived from the latin constrictus (= drawn together or contracted), and refers to the distinctive shape of the tail with thickening of the inner cuticle.

Description Male
Body cylindrical, tapering slightly towards both extremities.Cuticle faintly striated along entire body.Eight rows of somatic setae, relatively long (4-7 μm) and numerous in pharyngeal region, short and sparse elsewhere.Head rounded, with well-developed lip region.Inner labial sensillae not observed; six outer labial setae, 1-2 μm long, and four cephalic setae, 2-3 μm long, in one circle.Eight groups of three to four sub-cephalic setae, 3-8 μm long.Large circular amphideal fovea with strongly cuticularised outline, 1.4 cbd from anterior body extremity.Buccal cavity large, 24 μm deep and 13 μm wide; six H-shaped mandibles hooked anteriorly and with wide base articulating onto cuticularised rim posteriorly.Posterior portion of buccal cavity surrounded by pharyngeal tissue.Pharynx muscular, cylindrical, widening very slightly towards posterior extremity, with strongly cuticularised lumen.Cardia extend into intestine lumen.Nerve ring situated at middle of pharynx length.Secretory-excretory system not observed.
Reproductive system diorchic with two outstretched testes; anterior testis to the left and posterior testis to the right of intestine.Mature sperm cells nucleated, spherical to globular, 9 × 9-13 μm.Spicules 1.3 abd long, slightly arcuate, with swollen proximal ends and pointed distal ends.Gubernaculum and pre-cloacal supplements absent.Tail conico-cylindrical with conspicuous thickening of inner cuticle (maximum thickness ~7 μm) immediately anterior to cylindrical portion (Fig. 5D); cylindrical portion shorter than conical portion.A few short, sparse caudal setae present sub-ventrally and sub-dorsally; three short terminal setae, 2 μm long.Caudal glands not observed.

Female
Similar to male, but with longer body and substantially smaller amphids (0.11-0.14 vs 0.48 cbd) situated 1.0-1.4cbd from anterior body extremity.Reproductive system monodelphic with outstretched anterior branch to the left or right of intestine.Spermatheca present, simple and not cuticularised.Vulva transverse, situated at almost two thirds of body length from anterior.Vaginal glands not observed.Three caudal glands observed in one specimen.Cuticle immediately anterior to cylindrical portion of tail up to 9 μm thick.

Remarks
Metasphaerolaimus constrictus sp.nov.can be differentiated from all other species of the genus by the distinctive tail shape with conspicuous thickening of cuticle (up to 7-9 μm thick) immediately anterior to the cylindrical portion.M. constrictus sp.nov. is most similar to M. gerlachi Jensen, 1992, M. hadalis (Freudenhammer, 1975), and M. inglisi Gourbault & Boucher, 1981.The new species can be differentiated from M. gerlachi based on larger body size (1232-1623 vs 893-971 μm), larger amphids in males (0.48 vs 0.30-0.40cbd), and presence of short terminal setae (vs long terminal setae in M. gerlachi); from M. hadalis by higher values of a (30-38 vs 26-27), larger amphids in males (0.48 vs 0.35 cbd) and shorter tail (2.9-3.1 vs 3.4-3.8abd); from M. inglisi by the absence of setae immediately anterior to amphids in females (vs three long setae in M. inglisi), and presence of terminal setae (absent in M. inglisi).

Superfamily Monhysteroidea Filipjev, 1929
Family Monhysteridae de Man, 1876 Diagnosis (modifi ed from Fonseca & Decraemer 2008) Small, slender nematodes usually less than 2.5 mm long.Cuticle smooth or striated.Anterior sensillae in two circles: anterior circle with six inner labial sensillae (usually papilliform) and posterior circle with six outer labial sensillae and four cephalic sensillae (both usually setiform).Amphideal fovea circular or cryptocircular.Buccal cavity small to medium-sized, bipartite or single, with or without denticles.Pharynx with or without posterior bulb.Secretory-excretory system present or absent.Female reproductive system monodelphic with outstretched gonad to the right of intestine; male monorchic with anterior testis to the right of intestine (except Monhystrella kermadecensis sp.nov.where position of anterior gonad relative to intestine is variable).Spicules usually simple and arcuate.Tail conical to conico-cylindrical; terminal setae absent.
European Journal of Taxonomy 158: 1-19 (2015) usually 1.5-2.0 or more head diameters from anterior end.Posterior part of pharynx enlarged forming a single or double bulb without valves.Secretory-excretory system usually absent.Female reproductive system short to medium sized; vulva near mid-body.Spicules mostly arcuate and short (< 2 abd).Tail usually with ventrally curved conical anterior portion and dorsally curved cylindrical fi liform posterior portion.Spinneret a long and slender cone or cylinder.

Remarks
The taxonomy of all marine Monhystrella species was revised by Fonseca & Decraemer (2008), who provided a detailed diagnosis of the genus and a key to all thirteen valid marine species; no new marine species have been described since.

Etymology
This species is named after the type locality.

Description Male
Body cylindrical, tapering slightly towards both extremities.Cuticle smooth.Anterior end rounded or slightly truncated.Six minute inner labial papillae, diffi cult to observe; circle of six small outer labial papillae slightly anterior to circle of four short cephalic setae, 0.23 cbd long.Amphideal fovea mediumsized, circular, situated 1.9 cbd from anterior end, outline not cuticularised.Two short setae situated in sublateral row posterior to amphideal fovea.Buccal cavity small, funnel-shaped, surrounded by pharyngeal tissue.Pharynx gradually widening posteriorly, apparently forming a weak posterior bulb in some cases.Nerve ring situated slightly posterior to middle of pharynx.Secretory-excretory system not observed.Cardia small, partly surrounded by intestinal tissue.Progaster not observed.Distinct layer of glycocalyx present in intestinal lumen.Reproductive system monorchic with anterior outstretched testis situated to left or right of intestine.Mature sperm nucleated, globular, 1.3-2.0× 1.5-2.4μm.Spicules paired, arcuate, without capitulum, widest at proximal end and with pointed distal tip.Gubernaculum with caudal apophyses and wide lateral crurae.Pre-cloacal supplements or setae not observed.Tail conical, with two pairs of sub-ventral setae and one pair of sub-dorsal setae.Well-developed, ventrally curved, conical spinneret; caudal glands not observed.

Female
Similar to males but body widest immediately anterior to vulva, then markedly smaller immediately posterior to vulva; body width decreases gradually towards both extremities.Amphideal fovea slightly smaller than in males (0.33-0.43 vs 0.43-0.50cbd), only one seta present posterior to amphideal fovea; tail longer than in males (7.8-11.0vs 6.2-6.7 abd), conico-cylindrical, without setae.Reproductive system monodelphic with outstretched anterior branch to the left (two specimens) or right of intestine (three specimens).Mature egg dimensions up to 12 × 39 μm.Vulva located at almost two thirds of body length from anterior.Vagina transverse or oblique, with distinct cuticularised piece situated along anterior wall (Fig. 6D).No vaginal glands visible.Post-vulvar sac not observed.

Remarks
Monhystrella kermadecensis sp.nov.differs from most other Monhystrella species (except M. marina Timm, 1964) by the relatively short tail lacking a cylindrical fi liform posterior portion.This trait may suggest affi nities with Thalassomonhystera Jacobs, 1987, but species of this genus always possess a well-developed secretory-excretory system and lack a posterior pharyngeal bulb (Fonseca & Decraemer 2008).M. kermadecensis sp.nov. is also unusual in the position of the vulva at almost two thirds of body length from the anterior extremity instead of near mid-body, a trait which suggests affi nities with Halomonhystera Andrássy, 2006.The vulva in M. kermadecensis sp.nov.is, however, situated more anteriorly than in Halomonhystera (62-64 vs 76-92% of body length from anterior extremity); the new species also differs from Halomonhystera in the absence of secretory-excretory system and shape of the buccal cavity (Fonseca & Decraemer 2008).Elucidating the taxonomic relationships between M. kermadecensis sp.nov.and other Monhysteridae will require detailed molecular analyses which are not possible at present based on available specimens.
The holophyly of the Monhysteridae was established based on the anterior gonad always positioned to the right of the intestine (Lorenzen 1981).Monhystrella kermadecensis sp.nov.can be differentiated from all other species of the genus, and indeed the entire family, based on the variable position of the anterior gonad relative to the intestine (to the right or left of intestine in both sexes).The holophyly of the closely related family Xyalidae was established based on the anterior gonad always positioned to the left of the intestine and the posterior gonad (when present) always to the right of it (Lorenzen 1981).The new species therefore shows affi nities with both the Mohysteridae and Xyalidae; within the Xyalidae it resembles the genus Theristus Bastian, 1965 most due to the conical shape of the tail and lack of terminal setae.The following traits, however, suggest closer affi nities with the Monhysteridae: small body size (< 500 μm in length), smooth cuticle (always striated in Xyalidae; Lorenzen 1981), presence of only four setae in second circle (always ten setae in Theristus), and presence of only one testis (two testes often present in Xyalidae).

Discussion
Monhystrella kermadecensis sp.nov.was among the most common species at the 8081 m site in Kermadec Trench and represented 8% of total nematode abundance (D.Leduc unpublished data).This species, however, was not present at the 9177 m site.Thelonema clarki sp.nov.and Metasphaerolaimus constrictus sp.nov.were relatively common at the 9177 m site, representing 3 and 5%, respectively, of LEDUC D., Kermadec Trench Monhysterida total nematode abundance.These two species were also present at the 8081 m site but in lower numbers (≤ 1% of total nematode abundance).The present study increases the number of nematode species known from hadal trench environments from fi fteen to eighteen.Many more species are yet to be discovered and described, and future sampling in trench environments should ensure that preservation and fi xation methods allow for both morphological and molecular analyses to be conducted (Yoder et al. 2006).
All measurements are in μm, and all curved structures are measured along the arc.Type specimens are held in the NIWA Invertebrate Collection, Wellington, New Zealand.Abbreviations in the text are as follows: a = body length / maximum body diameter abd = anal body diameter b = body length / oesophagus length c = body length / tail length cbd = corresponding body diameter %V = vulva distance from anterior end of body × 100 / total body length European Journal of Taxonomy 158: 1-19 (2015)
Cuticle striated or annulated.Six outer labial setae and four cephalic setae in one circle, with cephalic setae longer than outer labial setae.Eight groups of sub-cephalic setae present (four groups in Megalamphis).Buccal cavity wide, barrel-shaped (except for SubsphaerolaimusLorenzen, 1978, which is conical), with longitudinal ribs, surrounded only at the base by pharyngeal tissue.Pharyngeal lumen with thick cuticle.Renette cell usually present, opening behind nerve ring.Females with one outstretched anterior ovary at left or right of intestine.Males usually with two testes; anterior one either to the left or right of intestine, posterior one at opposite side.Six inner labial papillae; six outer labial setae at same level as four longer cephalic setae; eight groups of sub-cephalic setae.Amphideal fovea circular, situated posteriorly to buccal cavity.Buccal cavity strongly cuticularised with six H-or X-shaped mandibles, hooked anteriorly and articulating on cuticularised rim posteriorly.Pharynx cylindrical with strongly cuticularised lumen.