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Citation Information : Journal of Nematology. Volume 51, Pages 1-12, DOI: https://doi.org/10.21307/jofnem-2019-025
License : (CC-BY-4.0)
Published Online: 27-May-2019
A new species of the genus
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 (2011, 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.
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).
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.
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.
DNA was extracted from a single individual nematode, following Waeyenberge et al. (2000). The nematode was transferred to a 0.5 ml Eppendorf tube, containing 18 µl of Worm Lysis Buffer (50 mM KCL, 10 mM Tris pH 8.3, 2.5 mMMgCl2, 0.45% NP 40, and 0.45% Tween 20) and 2 µl of proteinase K (600 mg ml−1) (Thermo Scientific). The tubes were incubated at 65oC (1 hr) and then at 95oC (15 min). PCR and sequencing protocols are described in detail by De Ley et al. (1999). Primers for D2–D3 of 28S rDNA amplification were D2A (59-ACAAGTACCGTGGGGAAAGTTG-39) and D3B (59-TCGGAAGGAACCAGCTACTA-39) (Subbotin et al., 2006). Primers for ITS rDNA amplification were modified from Vrain et al. (1992): VRAIN 2F (59-CTTTGTACACACCGCCCGTCGCT-39) and VRAIN 2R (59-TTTCACTCGCCGTTACTAAGGGAATC-39).
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.
Geocenamus vietnamensis n. sp.
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 body surface; epyptigma is double; vaginal length is 30% of the body diameter, moderately sclerotized; spermatheca is rounded, with rounded sperm cells; ovaries are outstretched with a single row of oocytes. The tail is conical, annulated with hyaline, and the tail tip is pointed; 58 to 73 tail annuli are present on the ventral side (Figs. 1H, 2L, 3I,J). Phasmid is slightly enlarged, at about one-fourth of the tail length. The lateral field ends before the hyaline part of the tail tip.
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).
The alignment of D2–D3 of 28S rDNA sequences contained 31 sequences including the sequences from two outgroup taxa (Psilenchus spp. and Rotylenchulus macrosoma). The length of alignment was 602 bp. The D2–D3 of 28S rDNA sequence of Geocenamus vietnamensis n. sp. is 91 to 93% similar to those of Merlinius spp. (KP313847, KT213560, KJ585417), Scutylenchus spp. (KT213558, KP313854, KP313851, KJ585422), and Geocenamus sp. (KY560459). The variations between the D2–D3 of 28S rDNA sequence of G. vietnamensis n. sp. and other studied species were 10% (70 bp) compared to the sequence of Geocenamus sp. (KY560459), 10 to 13% (68-91 bp), 10 to 11% (68–78 bp) compared to the sequences of Merlinius spp., 10 to 12% (69–82 bp) compared to the sequences of Scutylenchus spp., 11 to 12% (77–79 bp) compared to the sequences of Nagelus spp., and 12 to 13% (81–91 bp) compared to the sequences of Pratylenchoides spp. The variations were 26 to 28% (186–196 bp) compared to two outgroup taxa (Psilenchus and Rotylenchulus).
The phylogenetic relationships between G. vietnamensis n. sp. and other species, based on the maximum likelihood method (GTR+G model), were presented in Figure 5. The D2–D3 of 28S rDNA sequence of Geocenamus vietnamensis n. sp. has a sister relationship with Geocenamus sp. (KY560459), Scutylenchus spp. (KT213558, KP313854, KP313851, KJ585422), and Merlinius spp. (KP313847, KT213560, KJ585417). Together they form a group with 98% bootstrap support.
The ITS rDNA sequence alignment contained 27 sequences including the sequences from Rotylenchulus spp. (outgroup taxon). The length of the alignment was 897 bp. The ITS rDNA sequence of Geocenamus vietnamensis n. sp. is 88 to 91% similar to those of Geocenamus sp (KY560464, KY560463), Scutylenchus sp (JQ069956), Helicotylenchus digonicus (GQ906353, GQ906351), and Pratylenchoides spp. (KY424249, KY468918). The sequence variations between the ITS rDNA sequence of G. vietnamensis n. sp. and other species varied from 6 to 10% (63–99 bp) compared to Geocenamus sp. (KY560464, KY560463), 6% (58 bp) compared to Scutylenchus sp. (JQ069956), 6% (61 bp) compared to Helicotylenchus digonicus (GQ906353, GQ906351), 85 to 86% compared to Pratylenchoides spp. (KY424249, KY468918), and 30 to 35% (264-318 bp) compared to outgroup taxa (KT003781, KT003785, KT003806).
The phylogenetic relationships between the ITS rDNA sequence of Geocenamus vietnamensis n. sp. and other species, based on maximum likelihood (TN92 + G model), are presented in Figure 6. The ITS rDNA sequence of G. vietnamensis n. sp. has a sister relationship with the sequences of Geocenamus sp. (KY560464, KY560463), Scutylenchus sp (JQ069956), Helicotylenchus digonicus (GQ906353, GQ906351), and Pratylenchoides spp. (KY424249, KY468918). They were placed together in a clade with maximal bootstrap support (100%).
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.
Among Geocenamus spp., G. vietnamensis n. sp. is most similar to G. boghiae (Choi and Geraert, 1993) in having a non-sclerotized head framework and the males without a bursa. They can be differentiated from all other Geocenamus spp. by these characteristics. However, this new species differs from G. boghiae in having a shorter stylet (24–28 vs 48–54 μ m and 22.9–25.5 vs 45–52 μ m in females and males, respectively), a shorter body length in males (631–754 vs 750–900 μ m), a shorter length of the anterior end to the end of pharynx (124–152 vs 162–185 μ m and 114–135 vs 134–167 μ m in females and males, respectively), a longer female tail (85–125 vs 52–69 μ m), a shorter spicule length (18.7–22 vs 22.35 μ m), a shorter gubernaculum length (5.2–7.8 vs 9–11 μ m), a more anterior vulva (47–52 vs 50–57), a smaller c value (7.3–10.2 vs 14.2–18.3 and 9.3–9.5 vs 10.9–15.5 in females and males, respectively), and a larger c value (4.3–6.5 vs 2–3.2 and 4.3–6.3 vs 2.7–3.8 in females and males, respectively).
One holotype female, one allotype male and 33 paratypes (18 females and 15 males) from a population were extracted from the rhizosphere of Casuarina equisetifolia (Australian pine tree) (Casuarinaceae) in Quang Nam Province, Vietnam (15°56′56′′ N, 108°30′36′′ E).
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.
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.