Description of Marylynnia puncticaudata n . sp . ( Nematoda , Cyatholaimidae ) from Bizerte Lagoon , Tunisia

Description of Marylynnia puncticaudata n. sp. (Nematoda, Cyatholaimidae) from Bizerte Lagoon, Tunisia.— A new free–living marine nematode species of Cyatholaimidae, Marylynnia puncticaudata n. sp. from Bizerte Lagoon (Tunisia) is morphologically described. Males are characterized by a slightly larger body than females, a cephalic ring followed by ten subcephalic setae, modified cuticular punctuation, caudal lateral differentiation of large dots, and strongly cuticularized gubernaculum with a unique shape and bidenticulated distal half. The cuticle ornamentation of females is similar to the males. However, their caudal lateral differentiation is composed of smaller and more spaced dots. An updated morphological key to species of Marylynnia is given.


Introduction
To date, 6,900 species of free-living marine nema� todes have been morphologically described (Apple� tans et al., 2012), most of which are from West Europe and North America.Very few have been described from the African continent (Semprucci & Balsamo, 2012).In the case of Tunisia, 249 species of free-living marine nematodes have been collected since 1977 from several lagoons, beaches and mudflats (Boufahja et al., 2014).Of these, 20 species are being consid� ered as new to science (Boufahja et al., 2014).So far, morphological descriptions have been published, by Aїssa & Vitiello (1977), for three validated species collected from the Northern Lake of Tunis (Tunisia): Chromadorina metulata, Metalinhomoeus numidicus and Synonchiella edax.However, 17 species have not yet been described (Boufahja et al., 2014).
Our specimens are recognized as Marylynnia, which belong to the second richest nematode family in Tunisia, the Cyatholaimidae (25 reported species) (Boufahja et al., 2014).The aim of the present paper is to provide new insights into the composition of nema� tode in the Tunisian area, to describe a new species of this genus, and to propose an updated identification key to the species of the genus.
The family Cyatholaimidae, included in the superfa� mily Chromadoroidea Filipjev 1917, was first described by Filipjev (1918) and comprises two subfamilies, 23 genera and 215 species (Hodda, 2011).The genus Marilynia was established by Hopper (1972) with type species Marilynia annae (synonym: Longicyatholaimus annae Wieser & Hopper, 1967), and thereafter modified to Marylynnia by Hopper (1977).According to Platt and Warwick (1988), Marylynnia species are characterized by cuticle with transverse rows of dots with a lateral differentiation of larger and more widely spaced dots.Two types of pores are present on the cuticle: simple rounded and longitudinally oval.The genus Marylynnia is closely related to Longicyatholaimus but differs by the presence of a larger buccal cavity, prominent dorsal tooth and paired ventrosublateral teeth, a less com� plex form of the gubernaculums, and lateral modified punctuations on the conoid portion of the tail.

Sediment collection and processing
Undisturbed sediment samples were taken on 1 VII 13 in the channel zone of Bizerte Lagoon (37° 15.906' N, 09° 52.052' E) (fig.1).At low tides, the sediment was removed following parallel tracks that were 5cm deep.The uppermost 2 cm of the intermediate zones were then collected using a large spatula (Boufahja & Sem� prucci, in press).Immediately, the sediment was fixed with neutralized 4% formalin for morphology details, and a few drops of Rose Bengal (0.2 g/l) were added to stain the specimens (Higgins & Thiel, 1988).
In the laboratory, all sediment samples were rinsed with a gentle jet of freshwater over a 1 mm and 40 µm sieves and nematode separation followed the clas� sic decantation-floatation method (Mahmoudi et al., 2007).Nematodes were taken from every sampled core under a 50x stereoscopic microscope (Model Wild Heerbrugg M5A) and fixed in neutralized 4% formalin.

Mounting and identification
Nematodes were transferred to a ‫1׃9‬ (V‫׃‬V) solution of 50% ethanol‫׃‬glycerol in block cavity to slowly evapo� ‫׃‬glycerol in block cavity to slowly evapo� glycerol in block cavity to slowly evapo� rate ethanol.They were then mounted in a drop of anhydrous glycerol on permanent slides (Seinhorst, 1959).
Type specimens were deposited in the collections of free-living marine nematodes at the Faculty of Sci� ences of Bizerte (collection code: FB-BL-NV1) (Bou� fahja et al., 2014).Drawings were done directly from the slide using (1) a Nikon DS-Fi2 camera coupled to a Nikon microscope (Image Software NIS Elements Analysis Version 4.0 Nikon 4.00.07 (build 787) 64 bit) and (2) a Olympus XC50 camera coupled to a Olympus BX53 microscope (Image Software CellSens Standard Version 1.6).All measurements (not ratios) are given in micrometers (µm) and all curved structures are measured along the arc (table 1).The abbreviations used in the text are as follows: a. Body length divided by maximum body diameter; b.Body length divided by pharyngeal length; c.Body length divided by tail length; c'.Tail length divided by anal body diameter; abd.Anal body diameter; cbd.Corresponding body diameter; hd.Head diameter at the level of the cephalic setae; L. Body length; M. Maximum body diameter; Spic.Spicule length along arc; V%.Position of vulva from anterior end expressed as a percentage of total body length; LMPs.Lateral modified ponctuations, that is, ring-shaped connections between the punctuations.

Sampling date, type locality and habitat
The specimens were collected on 1 VII 13 in a po� orly sorted sediment with a mixture of coarse sand and mud from the channel zone of Bizerte Lagoon (37° 5.906' N, 09° 52.052' E) (fig.1).

Mediterranean Sea
Tunisia 1000 km

Sampling site
Bizerte Lagoon 2 km N N Etymology This species is named after the characteristic larger dots in the lateral differentiation on the lower 75% conical region of the tail which is visible even at 10x magnification, especially for males.

Measurements
See table 1.

Description
Males.Organism relatively large.Body cylindrical, nar� rowing towards the cervical region (75-85 µm from the anterior end) where a front portion slightly set off by constriction is noted (30-40%).Buccal cavity cylindrical (width 14-18 µm, height 15-20 µm) with prominent large dorsal tooth and two ventrosublateral teeth.Thick cylindrical pharynx, slightly wider at base.No distinct cardia.Nerve ring at 40-45% of pharyngeal length from anterior end.Lower half of the head region character� ized by a cephalic ring (figs.2A, 4B) with 10-12 µm wide and 6-9 µm height.The arrangement of sensorial organs is: six inner labial sensilla 1-2 µm, followed by six outer labial sensilla 7-8 µm and four shorter cephalic sensilla 3-5 µm; the latter two arranged in a circle 5-6 µm from the anterior end.Ten subcephalic setae 16-18 µm (six submedian and four lateral) in a circle at 12-16 µm from the anterior end.Amphids, 12-15 µm far from the anterior end, multispiral, with ~4.5 turns in ventral direction, and 7-13 µm wide (~40% of the cbd) by 7-8 µm long (fig.2).Immediately below the cephalic ring there begin four rows of cuticular hypodermal pores and a lateral differentiation with an average width of 27 µm (~50% cbd).These rows divide the entire body into lateral fields and extend from behind the amphideal fovea to the end of the conical portion of tail.The lateral differentiation of 45-50 µm width is only present along 70-85 µm (i.e. until the middle of the distance from the anterior end of the nerve ring).It consists of larger dots which change to a honeycomb-like structure when the fine focus is used.Honeycomb-like structures are made with separated double-crosses; this variable lateral differentiation seems to be the result of the presence of thickened crossings.The cervical lateral differentiation is replaced with fine punctuation (and double-crosses) following by horizontal and parallel lines (0.8-1 µm annules) right through the middle region of the body.Approximately in the middle of the body, the punctuation becomes different and horizontal rows of fine dots border irregular fields of smaller dots.This type of punctuation overlaps from time to time with the lateral differentiation made with bigger dots.The fat dots (and double-crosses) characteristic of the cervical region reappear just up to the cloaca and become very clear in the lower 75% portion of the conical part of the tail.LMPs are not observed.
Reproductive system diorchics.Spicules 0.8-0.95abd along arc, ventrally curved, with two unequal cephalate proximal tips.Gubernaculum larger than spicule, strongly cuticularized and highly complex.It consists of two blades proximally and distally separated but ventrally juxtaposed at their median parts.The proximal parts curved and expanded at base by conical thickenings.Bidenticulated distally: one large and lateral (11-15 μm) and the other one curved inward (6-8 μm).Absent precloacal supple� μm).Absent precloacal supple� m).Absent precloacal supple� ments.Cylindrical portion of tail generally folded over the conical portion.In the caudal position, setae located immediately posterior to cloacal aperture and three caudal glands.Spinneret present.Three small setae (2.5-3.5 µm) alternately present on cylindrical part of tail and separated by ~15 µm.One slightly  Females.In most respects, the cuticle ornamentation is similar to the males.Lesser body maximum width with longer body and tail.Lower anal width, making the tail shape filiform.Caudal lateral differentiation with smaller and more spaced dots.No setae on the conical portion of the tail.Reproductive system didelphic, ovaries reflexed; anterior ovary situated subventrally to the right of the intestine, posterior ovary subventrally to the left of the intestine.Vagina well sclerotized.

Fig. 1 .
Fig. 1.Study area and location of sampling site.

Fig. 2 .
Fig. 2. Marylynnia puncticaudata n. sp.: A. Male head showing the lateral differentiation in the cervical region; B-C.Variable cuticle punctuation in pharyngeal region; D. Modified punctuation and lateral differentiation in the middle of the body; E. Male tail and lateral differentiation; F. Female tail and lateral differentiation.

Fig. 6 .
Fig. 6.Male genital armatures of the 21 reported Marylynnia species in comparison with those of Marylynnia puncticaudata n. sp.For the known species, the drawings have been modeled on those provided by the original descriptions.

Table 2 .
Comparative table of biometric and morphological data for holotype (or syntype) males of known species and Marylynnia puncticaudata n. sp.: L. Total body length; a, b, c.De Man's ratios; Sp.Spicule; abd.Anal body diameter; Gub.Gubernaculum; CHP.Row number of hypodermal pores; AT.Amphideal turns; CLD.Caudal lateral differentiation; PS.Number of precloacal supplements; CS.Number of cephalic setae; ?Not specified by authors.(For abbreviations of species see figure 6.)