Description of Geocenamus vietnamensis sp. n. (Nematoda: Merliniidae) from Vietnam

Abstract A new species of the genus Geocenamus was isolated from soil and root samples from the rhizosphere of Casuarina equisetifolia (Casuarinaceae) in Quang Nam province, Vietnam. This species is characterized by a round-to-hexagonal labial disc, the presence of a labial region, which is continuous or slightly offset from the body with six sectors, lateral sectors of first labial annulus being smaller than the submedian sectors, the presence of six to seven labial annules; the absence of deirids; stylet length 24 to 28 μm long, body length 776 to 979 μm long; lateral field with six to eight lateral lines, without areolation at mid-body and with areolation in outer bands at the tail region and a pointed tail terminus. Geocenamus vietnamensis n. sp. most closely resembles G. boghiae in having a non-sclerotized head framework and lacking a bursa in the males. It can be clearly distinguished from all other species of the genus Geocenamus by these characteristics. The combination of morphology, morphometric features, and phylogenetic trees, based on D2–D3 of 28S and ITS rDNA sequences, showed that this new species can be clearly separated from all other sequenced species. This record is the first for Geocenamus in Vietnam.

The genus Geocenamus was proposed by Thorne and Malek (1968), with G. tenuidens (Thorne and Malek, 1968) as its type species. After that Fortuner and Luc (1987) and Maggenti et al. (1988) placed the genus Geocenamus in the subfamily Belonolaiminae (Whitehead, 1959). The classifications presented by Siddiqi (2000) distinguished the following five genera within the subfamily Merliniinae: Geocenamus, Nagelus, Merlinius (Siddiqi, 1970), Scutylenchus, and Amplimerlinius (Siddiqi, 1976). However, Geraert (2011) retained only the genera Amplimerlinius, Geocenamus, and Nagelus in subfamily Merliniinae of the family Dolichodoridae. Remarkably, the main morphological characters that distinguish Scutylenchus from other genera in the subfamily Merliniinae are the longitudinal striation of the cuticle and the absence of deirids (Siddiqi, 2000). Deirids are also absent in Geocenamus species, but they are obviously present in all other members of the family Merliniidae. Handoo et al. (2007) considered the number of lateral lines as one of the most important characters in identifying these genera that are ranging from three to six lines.
Based on the observations from two studies, Sturhan (2011Sturhan ( , 2012 concluded that the absence of longitudinal cuticular striae along the entire body appears to be the only essential character that distinguishes Geocenamus from Scutylenchus. However, this character has not been considered in classifications of Tylenchorhynchus and Rotylenchus species (Sturhan, 2012). Therefore, Sturhan (2012) agreed with Brzeski (1991) in synonymizing Scutylenchus with the "senior" genus Geocenamus, but he considered Merlinius as a valid genus.
Following the classification of Sturhan (2012), this study provides morphological and molecular characterizations of a new species of the genus Geocenamus in Vietnam.

Nematode population sampling
Soil and root samples were collected from the rhizosphere of Casuarina equisetifolia (Casuarinaceae) in Quang Nam province in May 2017. Nematodes were extracted from the soil and the roots by the method described by Nguyen (2003).

Light microscopy
For morphometric and morphological examination, the extracted nematodes were killed by heat, fixed in a TAF solution, processed to anhydrous glycerol, following Seinhorst (1959), and mounted on permanent glass slides. Nematodes on permanent slides were observed through a Carl Zeiss Axio Lab. A1 light microscope. Measurements and pictures were taken using the ZEN lite software on ZEISS Axiocam ERc5s digital camera. Raw photographs were edited with Adobe Illustrator CS 3.

Scanning electron microscopy (SEM)
After examination and identification, good specimens were selected for SEM imaging, following the protocol of Abolafia (2015). The nematodes were hydrated in distilled water, dehydrated in a graded ethanol and acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin Scanning Electron Microscope.

Phylogenetic analyses
The BLAST homology search program was used to search for closely related species on GenBank. The sequence data set was aligned with the ClustalW software (Thompson et al., 1994). Sequence alignments were manually edited using ChromasPro software (ChromasPro 1.7.4; Technelysium Pty Ltd, Tewantin, QLD, Australia). The sequence data set was analyzed using the maximum likelihood (ML) method in the MEGA 6 program (Tamura et al., 2013). The best fit model for DNA evolution was obtained using the Modeltest in MEGA 6. The model TN93 + G was chosen for D2-D3 of 28S rDNA data set and model GTR + G was chosen for ITS rDNA data set; 1,000 bootstrap replications were executed. Outgroup taxa were chosen according to the results of previously published data (Subbotin et al., 2006;Ghaderi et al., 2014). The trees were visualized in FigTree v1.4.0.

Description Female
Their body is ventrally curved after fixation, tapered at both ends. The lateral field is present with 6 to 8 lines without areolation at mid-body, about 6.0 µm wide (Figs. 1F, 2K, 3D). Body annuli are distinct, 1.2 ± 0.1 µm wide, and they are divided into blocks. The longitudinal striation is conspicuous with 18 to 20 lines at the ventral side near vulva (Fig. 3D). The labial region bears 6 to 7 annuli without longitudinal striae, not offset or very slightly offset by a shallow depression. A projected, round-to-hexagonal oral disc is present. First labial annulus is divided into six sectors; the lateral sectors are much smaller than the other four. Lateral sectors of first annulus are present with oval amphidial apertures (Fig. 3C). The head framework is not sclerotized. A well-developed stylet is present with elongated conical part and sloping and rounded knobs (Figs. 1C,E, 2B,E). The dorsal gland orifice is located 2.6 to 3.1 µm posterior to stylet knobs. An oval median bulb and a saccate terminal bulb, not overlapping intestine, are present. Secretory-excretory pore is located at the level of the nerve ring, 86 to 102 µm from the anterior end; hemizonid is located anterior to the secretory-excretory pore; deirid is absent. Vulva is a transverse slit, sunken or flush to

Male
Males are similar to females but they are more ventrally arcuate (Figs. 1B, 2A). The male reproductive apparatus is present with slightly bent spicules and a curved gubernaculum; cloacal lips are protuberant. Two posterior hypoptygmata are well developed, with equal lengths (Fig. 4I). Bursa is absent. Phasmid is located 22 to 28 µm posterior to the cloacal aperture. The tail is conical, annulated with hyaline, and the tail tip is pointed (Figs. 1I, 2I, 4F,G,H).

Diagnosis
Geocenamus vietnamensis n. sp. is characterized by a long stylet, a slightly offset labial region, a non-sclerotized head framework, a labial region bearing 6 to 7 annuli without striation, body annuli divided into blocks, a conical and pointed tail with annulated, hyaline tip, and the absence of a bursa in the males.

Etymology
The specific epithet is derived from Vietnam, the country where the species was found.

Type material
Holotype and paratypes were deposited in the Nematode Collection of the Institute of Ecology and Biological Resources (IEBR), Vietnamese Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi, Vietnam, and 10 female paratypes were deposited in the nematode collection of the Zoology Museum, Ghent University, K. L. Ledeganckstraat 35, Ghent, Belgium. The D2-D3 and ITS sequences were deposited in GenBank with accession numbers MH191361 and MH191362, respectively.

Discussion
In terms of morphology, this new species possesses a relatively long stylet compared to other plant-parasitic nematode groups, a slightly offset labial region bearing 6 to 7 annuli without striation, a non-sclerotized head framework, a body cuticle with 26 to 28 longitudinal striae (excluding lateral lines), 6 to 8 lateral lines at lateral field, a conical and pointed tail with annulated tip, and the absence of a bursa in the male. These characteristics clearly indicate that this species belongs to the subfamily Merliniinae. According to Siddiqi (2000) and Sturhan (2011), this species should belong to the genus Scutylenchus due to the appearance of tessellated cuticle and the absence of deirids. However, by considering the inconsistency in the appearance of longitudinal striae in Rotylenchus and Tylenchorhynchus, Sturhan (2012) agreed with Brzeski (1991) to synonymize Scutylenchus with Geocenamus. Interestingly, although the classification of Geraert (2011) is in agreement with Sturhan (2012), he considered the absence of radial grooves on the labial region as the key feature to define the genus Nagelus in the subfamily Merliniinae. It is a bit confusing in classifying our species due to the absence of radial grooves on the labial region, but this species can be clearly excluded from Nagelus because of the absence of deirids. In conclusion, we placed this new species in the genus Geocenamus according to the most recent classification.
In both D2-D3 of 28S and ITS rDNA trees, our new species always shows a very close relationship with species of Scutylenchus and Geocenamus, and a more distant relationship with Nagelus. In this study, the sister relationship of the genera Merlinius, Scutylenchus, and Geocenamus in the ITS rDNA tree strongly supports the recent classification in the subfamily Merliniinae. However, the number of D2-D3 of 28S and ITS rDNA sequences in GenBank for Merliniinae, as well as the genus Geocenamus, are very limited. More molecular data are needed to further resolve relationships in Merliniinae.