Description of Spinocephalus tessellatus n. gen., n. sp. (Rhabditida, Cephalobidae) from Iran, a nematode with a new morphological pattern at lip region

Abstract A new genus and new species of the family Cephalobidae, subfamily Cephalobinae, named Spinocephalus tessellatus n. gen., n. sp. is described from Iran. Body 0.55–0.67 mm long, cuticle with tessellations, lateral field with two tessellated longitudinal wings, lip region with six triangular lips, primary axils deep and V-shaped with two conoid-elongate guard processes that originate from each lip, secondary axils deep and U-shaped with one thorn-like process (labial probolae?) in lateral view with a small rounded protuberance fused to the oral plate having triradiate symmetry more developed at the acute margin toward each primary axil, oral opening hexagonal, amphids large and clearly rounded to slightly oval, stoma cephaloboid with cheilostom with minute and rounded rhabdia, pharynx cephaloboid with corpus subcylindrical and isthmus very long being 1.4–1.7 times corpus length, nerve ring surrounds the isthmus, excretory pore at the level of the isthmus. Female monodelphic-prodelphic, spermatheca as long as the body diam., post-vulval uterine sac 0.8–1.0 times body diameter, tail conoid with small rounded terminus. Male monorchic, spicules 24–26 µm long, gubernaculum 11–14 µm long, tail conical and ventrally curved with small rounded terminus. Morphological, including SEM observations, and molecular (based on 18S and 28S rDNA) analyses revealed its relationship with the genera Acromoldavicus and Nothacrobeles.

In the present study, a new genus and species are described from Iran that has morphological and molecular features that are in between the families Cephalobidae and Elaphonematidae. Additionally, a related species, Acromoldavicus skrjabini (Nesterov, 1970) Nesterov and Lisetskaya, 1965 from Spain (Elaphonematidae, Kirjanoviinae), is described from SEM studies to compare the morphology of the two species.

Light microscopy (LM)
Photomicrographs were taken with a Nikon Eclipse 80i (Nikon, Tokyo, Japan) microscope with a differential interference contrast (DIC) optics mounted with a Nikon Digital Sight DS-U1 camera and processed with Adobe ® Photoshop ® CS. Demanian indices (de Man, 1881) and other ratios were calculated. The terminology used to describe the morphology of stoma and spicules-gubernaculum follows De Ley et al. (1995) and Abolafia and Peña-Santiago (2017), respectively.

Scanning electron microscopy (SEM)
Specimens preserved in glycerine were selected and prepared for observation with a SEM according to Abolafia (2015). They were cleaned in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany).

DNA extraction, PCR, and sequencing
Nematode DNA was extracted from single individuals, previously fixed in 70% ethanol, using a modified DNA extraction and PCR assays described by Castillo et al. (2003) somewhat modified (Archidona-Yuste et al., 2016). The specimens were cut in small pieces using the acute tip of a sterilized dental anesthesia needle on a clean slide with 18 ml of TE buffer (10 mM Tris-Cl + 0.5 mM EDTA; pH 9.0), transferred to a microtube and adding 2 μ l proteinase K (700 μ g/ml −1 ) (Roche, Basel, Switzerland), and stored to -80°C within 15 min (for several days) until processing. Finally, the microtubes were incubated at 65°C (12 hr), then at 95°C (15 min) and the solution were use as DNA template. For DNA amplification, 3 μ l of the extracted DNA was transferred to a microtube containing: 0.6 μ l of each primer (10 mM), 3 μ l Master Mix Taq DNA Polymerase (5x Hot FirePol Blend Master Mix) and ddH2O to a final volume of 20 μ l. The primers used for amplification of the region of 18S rRNA gene were the forward primer SSU F_04 (5′-GCTTGTCTCCAAAGATTAAGCC-3′) and the reverse primer SSU R_26 (5′-CATTCTTGGCAAATGC TTTCG-3′) (Blaxter et al., 1998). The primers used for amplification of the D2-D3 region of 28S rRNA gene were the D2A (5′-ACAAGTACCGTGAGGGAAA GTTG-3′) and the D3B (5′-TCGGAAGGAACCAGCT ACTA-3′) primers (De Ley et al., 1999;Nunn, 1992). PCR cycle conditions were as follows: one denaturation cycle of 94°C for 15 min., followed by 35 cycles of 94°C for 45 sec; annealing cycle of 55°C for 45 sec; extension cycle of 72°C for 45 sec, and finally one extension cycle of 72°C for 5 min. After DNA amplification, 5 μ l of product was loaded on a 1% agarose gel in 0.5% Tris-acetate-EDTA (40 mM Tris, 20 mM glacial acetic acid and 2 mM EDTA; pH = 8) to verify the amplification using an electrophoresis system (Labnet Gel XL Ultra V-2, Progen Scientific, London, UK). The bands were stained with RedSafe (20,000x) previously added to the agarose gel solution. The sequencing reactions of the PCR products were performed at Sistemas Genómicos (Paterna, Valencia, Spain) according the Sanger et al. (1977) method. The rDNA sequences obtained for Spinocephalus tessellatus n. gen., n. sp. were submitted to the GenBank database.

Phylogenetic analyses
For phylogenetic relationships, analysis was based on 18S and 28S rDNA. The newly obtained sequences were manually edited using BioEdit 7.2.6 (Hall, 1999) and aligned with other 28S rRNA gene sequences available in GenBank using ClustalW (Thompson et al., 1994) alignment tool implemented in the MEGA7 (Kumar et al., 2016). Alignments ends were trimmed using MEGA7. The best-fit model of nucleotide substitution used for the phylogenetic analysis was statistically selected using jModelTest 2.1.10 (Darriba et al., 2012). A phylogenetic trees were generated with the Bayesian inference method using MrBayes 3.2.6 (Ronquist et al., 2012). Drilocephalobus sp. (AY284680) for the 18S tree and Deficephalobus desenderi De Ley and Coomans, 1990 (GU062820) for the 28S tree was chosen as the outgroup. The analysis under GTR + I + G model was initiated with a random starting tree and run with the Markov Chain Monte Carlo (MCMC) (Larget and Simon, 1999) for 1 × 10 6 generations. The tree was visualized and saved with FigTree 1.4.4 (Rambaut, 2018).

Diagnosis
Cephalobidae, Cephalobinae. Cuticle with tessellations, lateral field with two tessellated wings. Lip region with six triangular lips bearing two conoidelongate processes at primary axils and one thorn-like process (labial probolae?) at secondary axils bearing a small rounded protuberance fused at oral plate with triradiate symmetry, oral opening surrounded by a hexagonal margin, amphids large and clearly rounded to slightly oval, stoma cephaloboid with cheilostom bearing minute and rounded rhabdia. Pharynx cephaloboid with corpus subcylindrical and isthmus unusually very long. Nerve ring and excre tory pore at isthmus level. Female monodelphic-prodelphic with spermatheca well developed and post-vulval uterine sac poorly developed. Female tail conoid with rounded terminus. Male monorchic with spicules paired and symmetrical and gubernaculum well-developed. Male tail conical and ventrally curved with rounded terminus.

Relationships
The new genus Spinocephalus n. gen. resembles, morphologically, other genera of the superfamily Cephaloboidea that have cuticle divided in blocks and arranged into longitudinal crests such as Acromoldavicus Nesterov, 1970, Penjatinema Heyns and Swart, 1998, and Stegelleta Thorne, 1938 According to observations of SEM studies (Baldwin et al., 2001;Karegar et al., 1997Karegar et al., , 1998Susulovsky et al., 2001), the new genus Spinocephalus is distinguished from Acromoldavicus by having lips divided into plates with one long, acute process at primary axils and one large thorn-like process curved toward the oral opening at secondary axils (vs expanded lips with acute tips at primary axils and rounded at secondary axils), oral opening surrounded by a triacute margin with tips directed toward the primary axils and bearing three small rounded protuberance, one dorsal and two subdorsal (vs surrounded by three triangular labial probolae). Likewise, the new genus is different from Penjatinema (Heyns and Swart, 1998;Holovachov et al., 2009) by the lip region morphology (vs lips with fimbriated margin and oral opening surrounded by three long labial probolae with dendriform distal part). Finally, Spinocephalus n. gen. can be differentiate from Stegelleta (SEM by Holovachov, 2012, 2014; also by the lip region morphology (vs fused lips in pairs with smooth margin or having short acute tip at primary axils and oral opening surrounded by three bifurcate labial probolae with smooth prongs. Molecularly, only Acromoldavicus presents closer relationships with Spinocephalus n. gen.
On the other hand, the morphology of the lip region resembles Chilodellus Boström and Holovachov, 2012 with irregular lips and unusually large amphids. However, labial probolae are very different, having bifurcate distal halves with convergent prongs in Chilodellus. Unfortunately, molecular studies are not available to confirm this relationship.

Etymology
The generic name refers to the presence of acute processes (Latin spina = thorn) on the lip region (Latin cephalus from Greek kephale = head).

Description Adults
Stout to moderately slender nematodes, body length 0.55-0.67 mm. Habitus slightly ventrally curved in females and J-shaped in males after fixation. Cuticle deeply tessellated, having deep transversal and longitudinal incisures dividing the cuticle in blocks, the first annuli with wider than long blocks, later quadrangular and posteriorly longer than wide until the tail end. Lateral field with three longitudinal incisures or two narrow and tessellated wings, occupying 26-31% of mid-body diam., extending to tail end. Anterior body end narrower with lip region continuous with adjacent body, having six lips, slightly triangular, the lateral ones larger, and bearing six smaller labial and four larger cephalic sensilla; primary axils deep, V-shaped, bearing two conoid-elongate guard processes originating from each lip; secondary axils

Female
Reproductive system cephaloboid, monodelphicprodelphic having a globular sac-like spermatheca, in dextral position to intestine; ovary short, lacking flexures, with oocytes in one row; oviduct very short, areolate; spermatheca well-developed, as width as corresponding body diam., sometimes with sperm, this with 3-4 µm long; uterus about three times as long as corresponding body diam., distally tubular with thick walls and proximally swollen with thin walls; post-vulval uterine sac poorly developed, 0.8-1.0 times body diam., proximally swollen with thin walls and distally narrower lacking lumen; vagina short, extending inward 22-28% of body diam.; vulva very reduced, oval. Rectum 0.8-1.3 times anal body diam., with three small gland-like cells distinguishable around intestine-rectum junction. Tail conoid with small rounded terminus, with 9-12 annuli at ventral side. Phasmids located at 43-47% of tail length.

Male
General morphology similar to female. Reproductive system monorchic, dextral in position, with testis reflexed ventral anteriorly. Spicules paired and symmetrical, with rounded, ventral bent manubrium, conoid calamus and slightly ventrally curved lamina with very small dorsal hump, poorly developed ventral wing and acute tip. Gubernaculum well developed, ventrally curved, 0.5-6.0 times the spicule length, with thin manubrium and corpus, and crura well developed. Three small gland-like cells distinguishable around beginning of cloaca. Genital papillae one pair pre-cloacal, one pair ad-cloacal and five pairs post-cloacal arranged as follows: two in middle tail region (one lateral located at lateral field level and one subventral), and three pairs near tail terminus (one subdorsal, one lateral, and one subventral). Tail conical and ventrally curved with small rounded terminus. Phasmids located at 43-55% of tail length, slightly posterior to genital papillae GP3.

Type locality and habitat
The specimens were collected at sandy soil in the rhizosphere of Tamarix passerinoides Delile ex Desv. in Shush (ancient Persian city of Susa, GPS coordinates: 32°17.28′N, 48°25.07′E), Khuzestan province, Iran.

Type material
Six females (holotype and paratypes) and five males (paratypes) deposited in the nematode collection of the Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Spain. One female and one male (paratypes) deposited in the nematode collection of the Department of Plant Protection, College of Agriculture, University of Zanjan, Zanjan, Iran.

Differential diagnosis
The body length of the new species range from 0.55 -0.67 mm long in females and 0.58-0.65 mm long in males. The cuticle shows deep transversal and longitudinal incisures which divides the cuticle in blocks, or tessellated, and the lateral fields with two tessellated longitudinal wings or three longitudinal incisures. The lip region has six triangular lips appearing the primary axils deeper with V-shaped bearing two conoid-elongate guard processes originating from each lip while the secondary axils are deeper with U-shaped bearing one thorn-like process (labial probolae?) observed in lateral view and bearing a small rounded protuberance fused at an oral plate having triradiate symmetry which develops a more acute margin toward each primary axil. The oral opening is hexagonal The amphids are large and rounded to slightly oval. The stoma is cephaloboid with cheilostom bearing minute and rounded rhabdia. The pharynx is cephaloboid with subcylindrical corpus and very long isthmus being 1.4-1.7 times corpus length. The nerve ring surrounds the isthmus and the excretory pore appears at 65-73% of neck length at isthmus level. The female reproductive system is monodelphic-prodelphic with spermatheca as long as the body diam. and post-vulval uterine sac 0.8-1.0 times body diameter. The female tail is conoid with small rounded terminus. The male reproductive system is monorchic having spicules 24-26 µm long and gubernaculum 11-14 µm long. The male tail is conical and ventrally curved ending in a small, rounded terminus.

Etymology
The specific name refers to the presence of cuticular blocks or tessellation (Latin tessella = mosaic pavers). Zoobank code urn:lsid:zoobank.org:pub:53D4EE5A-A6B4-4A91-9896-88A5A6134D6C Figure 6: Bayesian inference tree showing the phylogenetic position of Spinocephalus tessellatus n. gen., n. sp. and its related taxa based on sequences of the 28S rDNA region. Bayesian posterior probabilities (%) are given for each clade. Scale bar shows the number of substitutions per site.

Relationships
The morphological analysis of Spinocephalus tessellatus n. gen., n. sp. shows the presence of an unusual lip pattern which structure is not similar to other genera belonging to the superfamily Cephaloboidea. The presence of a stoma with small rhabdia and sac-like spermatheca clearly indicates its relationship with other species of the infraorder Cephalobomorpha.
However, the absence of clear labial probolae and the unusual structure of the lips make difficult to know its position at family level. Molecular analysis based on 18S rDNA (Fig. 5) does not resolve well the phylogenetic relationships of Spinocephalus n. gen. because few genera of the superfamily Cephaloboidea are sequenced currently, appearing the new genus grouped to some species of the genus Acrobeles von Linstow, 1877 although they do not maintain closemorphological similarities. On the other hand, the 28S rDNA phylogenetic tree (Fig. 6) shows that the new genus is related with species of the genera Nothacrobeles and Acromoldavicus, both containing species with tessellated cuticle such as N. cancellatus (see Karegar et al., 1997Karegar et al., , 1998 and Acromoldavicus species (Fig. 7). With respect to the lip region, the lip pattern of Spinocephalus n. gen. (Fig. 8A) resembles slightly to that in Acromoldavicus (Fig. 8B). Thus, the ventral triangular process of Acromoldavicus could be homologous to the polygonal plate appearing in Spinocephalus n. gen., while the elongate process visible at the primary axils could be homologous to the acute tip of each lip present in Acromoldavicus. However, the lip pattern observed in the Nothacrobeles species, with dentate lips, is very different. Other species with an irregular lip pattern is Chilodellus eremus Boström and Holovachov, 2012 (Fig. 8C). This species has very large amphid openings, similar to Spinocephalus tessellatus n. gen., n. sp., and lips with long, acute processes, the lateral ones more reduced, which have a large amphid opening. However, the labial probolae have very different morphology with bifurcate distal part with pinnate outer margin.
According to this, Spinocephalus tessellatus n. gen., n. sp. is tentatively located in the family Cephalobidae, subfamily Cephalobinae instead of the family Elaphonematidae, subfamily Kirjanoviinae.