Three new species of Microlaimus (Nematoda: Microlaimidae) from the South Atlantic

Three new species of Microlaimus are described from the continental shelf of the Campos Basin, southwest Atlantic, Brazil. Microlaimus campiensis sp. n. differs from all other species in the presence of two anterior testes, slender spicules with enlarged proximal ends, 7–11 pre-cloacal papilliform supplements, and females with a pair of constriction structures, one on each branch of the ovary. Microlaimus alexandri sp. n. shows sexual dimorphism in the size of the amphidial fovea, which occupies 100% of the diameter of the corresponding area in the male; the buccal cavity provided with five teeth and a slightly cuticularized cuticular ring. Microlaimus vitorius sp. n. has four longitudinal-lateral rows of glands associated with small pores, one seta and three pores small pre-cloacal, and the gubernaculum has a triangular base. An amendment to the diagnosis of the genus is proposed, where the number of teeth was modified.

In a study of nematode biodiversity in sediments of the Campos Basin, South Atlantic, of which this contribution is a part, members of the genus Microlaimus was dominant on the continental shelf, and contributed significantly to community structuring at depths of 25-50 m on the inner shelf Fonsêca-Genevois et al., 2017).
De Coninck & Schuurmans Stekhoven (1933), Gerlach (1950), Chitwood (1951) and Wieser (1954) carried out the first studies on the genus and contributed lists of species and identification keys, as well as designating species that they considered dubious. Following the study by Wieser (1954), several investigators have provided revisions and

MATERIALS AND METHODS
The Campos Basin (23 30'S and 21 30'W) extends from the states of Rio de Janeiro to Espírito Santo, with the northern margin defined by the Espírito Santo Basin, and the southern margin defined by the Santos Basin. The basin covers an area of approximately 120,000 km 2 and reaches depths of 3,500 m. The region is influenced by the Brazil Current, which flows parallel to the coast and reaches depths of 200 m. The basin floor is covered with fine continental sediment and sand, composed mainly of foraminiferans (Soares-Gomes et al., 1999).
Sampling was conducted during the cruises carried out by the HABITATS project; May 2008 and July 2009. Sediment samples were collected with a Van Veen grab (dimensions 92 × 80 × 40 cm). The sediment samples were washed through sieves with openings of 500 and 45 mm, and the material retained on the smaller-mesh sieve was passed through the flotation technique with a solution of colloidal silica (Somerfield, Warwick & Moens, 2005).
Nematodes were gently picked out with a stainless-steel stylet, fixed in 4% formaldehyde, and transferred to glycerin (De Grisse, 1969). Drawings were made using an OLYMPUS CX 31 optical microscope fitted with a camera lucida. Photographs were taken with a Zeiss AxioCam ICc 5 digital camera. The software ZEN lite 2012 was used for image processing.
The diagnosis is an amendment to that of Decraemer & Smol (2006). The holotype and one paratype (female) of each species are deposited in the Nematoda Collection of Museum of Oceanography Prof. Petronio Alves Coelho (MOUFPE), Brazil. Other paratypes are deposited in the Meiofauna Laboratory, Zoology Department, Federal University of Pernambuco (NM LMZOO-UFPE). The nomenclature adopted for the body regions is presented in Table 1 and the measurements are expressed in micrometers.
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration Table 1 Abbreviations for body structures used in the tables, after Coomans (1979).

Systematics
Class Chromadorea Inglis, 1983Subclass Chromadoria Pearse, 1942Order Microlaimida Leduc, Verdon & Zhao, 2018Superfamily Microlaimoidea Micoletzky, 1922Family Microlaimidae Micoletzky, 1922Genus Microlaimus De Man, 1880(Syn Microlaimoides Hoeppli, 1926Paracothonolaimus Schulz, 1932) Diagnosis. (emended from Decraemer & Smol, 2006) Cuticle transversely striated, punctuations or longitudinal bars may be present. Lateral differentiation in the form of lateral alae occurs in M. falciferus Leduc & Wharton, 2008. Cephalic region often set off. Epidermal glands associated or not with pores or setae, small somatic setae occur in some species. Anterior sensilla arranged according to pattern 6 + 6 + 4: six inner labial setae, usually papilliform; six external labial setae, papilliform or setiform; and four cephalic setae. Cephalic setae longer than external labial setae, except for M. discolensis Bussau, 1993, in which setae are equal in length. Amphidial fovea cryptocircular or unispiral (= cryptospiral), usually located near cephalic setae. Sexual dimorphism in amphidial fovea size present or absent, when present it is larger in male. Buccal cavity small to medium-sized, with three small or well-developed teeth, especially dorsal tooth, except for M. alexandri sp. n., where five teeth are observed (one larger dorsal tooth located in anterior portion of buccal cavity, another dorsal tooth situated in median region; one of ventrosublateral teeth located at same level as previous dorsal tooth, and others located at basis of oral cavity), making it necessary to amend the diagnosis of the genus. Transverse cuticularized band or ring may be present in buccal cavity. Most species with two testes extending in opposite directions; some with two anterior testes, others with only one testis, positioned anteriorly or posteriorly. Pre-cloacal supplements absent or present (papilliform, tubular, or small pores). Spicules usually arcuate (1-2x cloacal diameter). Gubernaculum without dorso-caudal apophysis. Female didelphic-amphidelphic, with outstretched ovaries. Tail conical, predominantly.  Table 2). Body 813 µm long. Maximum body diameter corresponding to 1.8× cephalic diameter. Cuticle striated posteriorly to cephalic setae insertion. Anterior sensilla arrangement consisting of six inner labial papillae, six external labial papillae and four cephalic setae, in different cycles. Cephalic setae corresponding to 46% of cephalic diameter or 7.5× length of external labial setae. Amphidial fovea cryptocircular, located immediately posterior to buccal cavity; occupying 28.5% of corresponding body diameter. Buccal cavity cuticularized, cheilostoma with twelve longitudinal folds. Three cuticularized teeth, one large dorsal tooth and two smaller ventrosublateral, teeth. Pharynx dilated around buccal cavity and terminal oval bulb occupying 78% of corresponding body diameter. Nerve ring located at level of secretory-excretory pore, corresponding to 60% of pharynx length from anterior end. Ventral gland located immediately posterior to pharynx. Reproductive system with two anteriorly directed testes of approximately equal size. Spicules slender and enlarged proximally, about 1.3× cloacal diameter. Gubernaculum slender, about 2.2× spicule length. Seven pre-cloacal papilliform supplements, associated glands were observed only in papillae close to the cloaca. Tail conical, about 3× cloacal diameter. Three caudal glands.
In paratype males, the external labial papillae and nerve ring were difficult to observe. The buccal cavity, cuticularization of teeth, spicules shape and pre-cloacal supplements were similar among paratype males.
Full   these structures were observed to cause ovary constriction, which may be related to egg expulsion.
Diagnosis. Anterior sensilla arrangement consisting of six inner labial papillae, six external labial papillae and four cephalic setae (6+6+4). Cephalic setae corresponding to 26-60% of cephalic diameter. Amphidial fovea, located immediately posterior to buccal cavity, accounting for 26-37% of the corresponding body diameter. Buccal cavity with three teeth, one large dorsal and two smaller ventrosublateral. Males are characterized by two testes anteriorly positioned, slender spicules with cephalized proximal region (1.1-1.4× cloacal diameter) and 7-11 papilliform pre-cloacal supplements, of which some appear to be connected to the glands. Females with a pair of constriction structures, one on each branch of the ovary.
Microlaimus campiensis sp. n. differs from all the above-mentioned species in having two testes anteriorly positioned, one to the right of the intestine; it was difficult to observe the position of the other testis relative to the intestine. Two anteriorly positioned testes is a feature shared only with M. cyatholaimoides De Man, 1922,  In females of M. campiensis sp. n., each ovary has a constriction structure that constricts the egg, suggesting a strategy for facilitating the expulsion of eggs. These constriction structure were not observed in females of other Microlaimus species.
Microlaimus alexandri sp. n.  Table 4). Body 1,146 µm long. Maximum body diameter corresponding to 2.7× cephalic diameter. Cephalic set off. Cuticle thin, transversely striated from cephalic setae insertion. Cuticle striations more visible in anterior portion of body. Anterior sensilla arrangement consisting of six inner labial papillae, six external labial papillae and four cephalic setae, in different cycles. Cephalic setae corresponding to 57% of cephalic diameter. Amphidial fovea unispiral, located posterior to buccal cavity, and occupying 100% of corresponding body diameter. Buccal cavity cuticularized, with folds in its first portion. Five teeth, two dorsal and three ventrosublateral: larger dorsal tooth located in anterior portion of buccal cavity, another dorsal tooth situated in median region; one of ventrosublateral teeth located at same level as previous dorsal tooth, and others located at basis of oral cavity. Buccal cavity with slightly cuticularized cuticular ring, positioned at the level of larger dorsal teeth base. Pharynx with terminal oval bulb. Nerve ring located at 67% of pharynx length. Secretory-excretory pore not observed. Male reproductive system consisting of two    3.8× anal body diameter. Caudal glands present. Reproductive system didelphicamphidelphic, with outstretched ovaries located to right of intestine. Anterior and posterior genital branches measuring 170 and 177 mm, respectively. Vulva located at 52% of body length.
Diagnosis. Microlaimus alexandri sp. n. is characterized by the size of the amphidial fovea, occupying 100% of the corresponding area in males and 32% in females; the amphidial fovea is unispiral, in male, and cryptocircular, in female, and located more posteriorly from the anterior end, 2.1-2.4× cephalic diameter. The cephalic setae corresponding to 50-57% of the cephalic diameter. The buccal cavity is provided with five teeth, of which the anterior dorsal tooth is the most prominent, larger and heavily cuticularized than the other teeth; the second dorsal tooth, inserted in the median region of the buccal cavity, is very reduced in size. Of the ventrosublateral, teeth, one is situated at the level of the dorsal anterior tooth and two at the base of the oral cavity. The cuticular ring is slightly cuticularized and situated at the level of larger dorsal teeth base.
Differential diagnosis. (Table 5) The main diagnostic characteristic of Microlaimus alexandri sp. n. is the buccal cavity, which has five teeth, being a unique characteristic.   Etymology. The specific epithet vitorius is given in honor of the late Professor Verônica Gomes da Fonsêca-Genevois. Veronica is the latinized form of the name Berenice, which in Macedonian means bearer of victory (Greek: phere-nikē).
Description. Holotype male (Figs. 7 and 9; Table 6). Body 1696 µm long. Maximum body diameter 59 µm, corresponding to 3.7× cephalic diameter at level of cephalic setae. Cuticle striated from insertion of cephalic setae. Four lateral rows of hypodermal glands associated with small pores that extend longitudinally from pharynx to beginning of tail. Anterior sensilla arrangement in general pattern of genus, in three distinct cycles; inner labial and external labial papilliform, cephalic setae measuring 52% of corresponding body diameter. Amphidial fovea cryptocircular, located in posterior portion of buccal cavity, occupying 52% of corresponding body diameter. Buccal cavity cuticularized, with folds in its first portion; provided with three cuticularized teeth, one dorsal and two ventrosublateral, about same size. Pharynx involving buccal cavity and terminating in oval bulb. Nerve ring not observed. Secretory-excretory pore, distance from anterior end equivalent to 41.5% of pharynx length. Ventral gland not observed. Male reproductive system with two testes extending in opposite directions, larger anterior testis to right of intestine and smaller posterior testis to left of intestine. Sperm fusiform (16-33 mm long and 6-8 mm wide). Spicules arched, 1× cloacal body diameter. Gubernaculum lamellar,  with triangular base. One pre-cloacal seta and three small pre-cloacal pores. Tail conical (134 µm) with three glands.
Paratypes females (Figs. 8 and 9; Table 6). Female similar to male. Body 1,590-1,745 µm long and maximum diameter 70-86 mm. Four lateral longitudinal rows of hypodermal glands associated with small pores, in same arrangement as in male. External labial papillae   difficult to view. Cephalic setae corresponding to 50-57% of cephalic diameter. Amphidial fovea cryptocircular, smaller than in male, occupying 32-44% of corresponding body diameter, located in similar position to male. Buccal cavity and teeth also similar to those of male. Pharynx similar to that of male, with terminal bulb occupying 63-68% of corresponding diameter. Nerve ring not observed. The secretory-excretory pore in similar position to male, 7-83 mm from anterior end and equivalent to 40% of pharynx length. Ventral gland located immediately posterior to pharynx. Tail with same shape and measurements as in male. Three caudal glands. Reproductive system didelphicamphidelphic, outstretched ovaries located to right of intestine. Anterior and posterior genital branches measuring 280-320 and 265-324 mm, respectively. Sperm present in uterus. Vulva located 840-920 µm from anterior end, corresponding to 53% of body length.
Diagnosis. Microlaimus vitorius sp. n. has four lateral longitudinal rows of hypodermal glands that open through small pores and extend from the pharynx to the beginning of the tail in both sexes. Cephalic setae comprise 50-72% of corresponding body diameter. Amphidial fovea in posterior portion of buccal cavity, accounting for 37.5-52% of corresponding body diameter in male and 32-44% in female. Buccal with three cuticularized teeth, one dorsal and two ventrosublateral, about same size. Male with one pre-cloacal seta and three small pre-cloacal pores. Gubernaculum lamellar, with triangular base.
Differential diagnosis (Table 7). Males of Microlaimus vitorius sp. n. and M. acinaces Warwick & Platt, 1973 are similar in the large, heavily cuticularized buccal cavity and well-developed dorsal tooth, and the presence of a pre-cloacal seta and small pre-cloacal supplements. Microlaimus vitorius sp. n. differs from M. acinaces in cephalic sensilla arrangement (external labial circle is papilliform in the new species vs setiform in M. acinaces), longer cephalic setae (79% vs 50% of the corresponding cephalic diameter in M. acinaces) and the presence of four lateral longitudinal rows of glands associated with small pores in M. vitorius sp. n. Additionally, M. acinaces has four longitudinal rows of somatic setae, two ventrosublateral, rows of pre-cloacal ducts associated with subcuticular glands, and the tail with ventrosublateral, rows of setae, characteristics not observed in M. vitorius sp. n. Microlaimus vitorius sp. n. and M. cyatholaimoides are similar in the pore-like pre-cloacal supplements and the four longitudinal rows of lateral glands. However, in this latter species, the glands are associated with setae. These species also differ in the amphidial fovea, which is located more anteriorly in the new species (amph. pos/hd = 0.4-0.7 in M. vitorius sp. n. vs 1.5-1.7 in M. cyatholaimoides) and the length of the cephalic setae (79% vs 40% of the cephalic diameter in M. monstrosus).
Another important difference between the two species is the presence of testes positioned in opposite directions in Microlaimus vitorius sp. n. vs two anterior testes in M. cyatholaimoides. Hopper & Meyers (1967) and Muthumbi & Vincx (1999) considered the rows of hypodermal glands along the body as a species-level diagnostic character. Microlaimus vitorius sp. n. shares this character with M. cyatholaimoides and three other species: M. discolensis Bussau, 1993, M. porus Bussau, 1993and M. parviporosus Miljutin & Miljutina, 2009. However, M. vitorius sp. n. differs from these last three species in the longer body

DISCUSSION
The shape of the setae (papilliform or setiform) of the second and third circles of the cephalic arrangement, the relationship between the length of the cephalic setae and the cephalic diameter, the diameter of the amphidial fovea in the corresponding region of the body (%) and its position in relation to the anterior extremity of the body provided important taxonomic information that was used to distinguish Microlaimus species. The percentage occupied by the amphidial fovea was previously indicated as an interspecific morphological variation for Microlaimus by Armenteros, Vincx & Decraemer (2010). Furthermore, Armenteros et al. (2009) argued that morphometry could be used in studies of phylogenetic relationships. These authors also suggested the possibility of evaluating the relationship between morphological plasticity and ecological success in free-living marine nematodes, based on a model proposed by Hollander (2008).
Interspecific differences in the species of the genus Microlaimus were observed for the number of testes, their position in the body (anterior or posterior) and their shape (outstretched or reflexed). Most species have two testes extending in opposite directions, and of the same or different sizes (the posterior testis may be shorter). Two anterior testes may be present, as in M. cyatholaimoides De Man, 1922, as noted by Pastor de Ward (1989), and in M. campiensis sp. n. and M. alexandri sp. n. Only six species have one testis, which is anterior in M. martinezi (Miljutin & Miljutina, 2009), M. nanus Blome, 1982, M. nympha (Bussau, 1993, M. texianus Chitwood, 1951 andM. westindicus (Kovalyev &Miljutina, 2009), and posterior and reflexed in M. capillaris Gerlach, 1957, as noted by Jensen (1989) for this last species. No information on these structures was provided in the species descriptions for approximately 30 species of Microlaimus. Tchesunov (2014) stated that this is common in the literature on Microlaimidae. However, despite this lack of information, we included the interspecific variability of this structure in the diagnosis of the genus.
Finally, it should be noted that the species M. alexandri sp. n., M. campiensis sp. n., and M. vitorius sp. n. were recorded only on the continental shelf (depth 25-50 m) of the Campos Basin, during a taxonomic study that also sampled on the continental slope (up to 3,000 m) of the basin Fonsêca-Genevois et al., 2017). This observation may indicate a possible bathymetric restriction of these species.