Research Article |
Corresponding author: Hui Xie ( xiehui@scau.edu.cn ) Academic editor: Steven Nadler
© 2018 Wen-Jia Wu, Lu Yu, Hui Xie, Chun-Ling Xu, Jiao Yu, Dong-Wei Wang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Wu W-J, Yu L, Xie H, Xu C-L, Yu J, Wang D-W (2018) Description and molecular analysis of Tylencholaimus helanensis sp. n. from China (Dorylaimida, Tylencholaimidea). ZooKeys 792: 1-14. https://doi.org/10.3897/zookeys.792.27255
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A new species, Tylencholaimus helanensis sp. n., extracted from the rhizosphere soil of unidentified grasses from Helan Mountain, Inner Mongolia, China was identified. The new species is characterized by having a body length of 0.93–1.07 mm with the lip region approximately one-quarter of the body diameter at the posterior end of the neck region wide; female didelphic-amphidelphic; pars proximalis vaginae violin-shaped. Males were not found. SEM observations of the new species were made and a phylogenetic analysis of both the 18S rDNA and the D2-D3 region of 28S rDNA is presented.
China, morphology, new species, phylogenetic analysis, taxonomy, Tylencholaimus
The genus Tylencholaimus de Man, 1876 is common in most soils all over the world and contains more than 50 valid species. It is mainly characterized by having small body, cap-shaped lip region, weak odontostyle and knobbed odontophore. The types of female genital system and tail of the genus are various (amphidelphic, monoprodelphic, or mono-opisthodelphic for female genital system, hemispherical to elongate-conical for tail) (
With detailed examinations based on light microscopy, SEM observations and phylogenetic analysis of 18S rDNA and the D2–D3 region of 28S rDNA, one nematode population from Inner Mongolia, China, was identified to be a new member of Tylencholaimus and is described as Tylencholaimus helanensis sp. n.
Soil samples were collected from the rhizosphere soil of unidentified grasses from Helan Mountain, Alxa Left Banner, Alxa League, Inner Mongolia, China. Nematode populations were extracted from the soil samples by using the modified Baermann funnel method (
A single nematode was picked into 10 μL mixed solution (distilled water: 2×buffer for KOD FX = 1:1) and cut using a sterilized needle. 1 μL 20 μg/mL proteinase K was added and then reacted at 65 °C for 1 h and 95 °C for 15 min to release the genomic DNA. PCR reactions were performed in a 10 μL reaction mixture containing 5 μL of 2×buffer for KOD FX, 0.3 μL of each primer (10 μM) , 2 μL of dNTPs (200 μM), 1 μL of DNA, 1.2 μL of distilled water and 0.2 μL of KOD FX polymerase (1 U/ μL). Two overlapping fragments of the 18S rDNA were amplified using two primer sets, 988F (5'–CTCAAAGATTAAGCCATGC–3’) and 1912R (5'–TTTACGGTCAGAACTAGGG–3’) for the first fragment, and 1813F (5'–CTGCGTGAGAGGTGAAAT–3’) and 2646R (5'–GCTACCTTGTTACGACTTTT–3’) for the second one (
The sequences of the new species were compared with sequences in GenBank using BLAST. The sequences of other dorylaimid species and the outgroup taxa were chosen according to previous studies (
Seven females from Qinghai Province; 38°40.311’N, 105°50.905’E; 22 August 2014; collected by Dong-Wei Wang, Wen-Jia Wu, Lu Yu, and Hui Xie. Female holotype (M51.B.a) and six female paratype specimens (slide numbers: M51.A.a, b, c, d, e and M51.B.b) are deposited in the Lab of Plant Nematology/Research Center of Nematodes of Plant Quarantine, South China Agricultural University, Guangzhou, Guangdong 510642, China.
Tylencholaimus helanensis sp. n. Female: A, B entire body C anterior region showing pharynx D, E amphidial fovea F, G posterior region H, I anterior region showing odontostyle and odontophore Jvulva in ventral view K, L vulva in lateral view M, N genital system O SEM of the lip region. Scale bars: 200 μm (A, B); 20 μm (C, F, G, M, N); 10 μm (D, E, H–L); 2 μm (O).
Female. Body robust and cylindrical, tapering towards the anterior end. Habitus variable, almost straight or slightly twisted after fixation. Cuticle two layers, 1.0–2.0 μm thick in anterior region, 1.5–2.5 μm at mid-body, and 2.5–3.5 μm on tail; outer layer with fine transverse striations, the inner one loose and often shrunken after fixation. Lateral chord occupying about one-third of the body diameter at mid-body, lateral pores indistinct. Lip region cap-shaped, offset from the body by a constriction, 2.4–2.8 times as wide as high or 25% in average of the body diameter at posterior end of the neck region wide. Lips not amalgamated, the outer part of each lip not distinct from the inner one. Labial and cephalic papillae distinct but not interfering with the contour. Amphidial foveae cup-shaped, opening at the level of the constriction, apertures 0.4 times on average as wide as the lip region. Odontostyle straight with a distinct lumen, 8–9.5 μm long, 0.9–1.0 times as long as the lip region width, its aperture about one-third of its length. Odontophore rod-like with small basal knobs, 9–11 μm long, 1–1.3 times as long as the odontostyle. Guiding ring single. Nerve ring situated at 35–42% of the neck length. Anterior part of pharynx slender and expanded gradually, basal expansion occupying 39–43% of the total neck length. Pharyngeal gland nuclei locations (
Fore measurements see Table
Morphometrics of Tylencholaimus helanensis sp. n. and the females of its six close species. Measurements for Tylencholaimus helanensis sp. n. are in the form: mean ± s.d. (range), for other six species are in the form of range, and all in μm (except for ‘L’ in mm).
Character | Tylencholaimus helanensis sp. n. | T. teres | T. congestus | T. cosmos | T. crassus | T. paracrassus | T. sinensis | |
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Holotype | Paratypes | (1–4)* | (4, 5)* | (6, 7)* | (4, 5)* | (4)* | (8)* | |
n | 1♀ | 6♀♀ | >20♀♀ | 5♀♀ | 15♀♀ | 28♀♀ | 5♀♀ | 2♀♀ |
L | 1.00 | 1.00 ± 0.06 (0.93–1.07) | 0.8–1.06 | 0.72–0.83 | 0.57–0.9 | 0.68–0.92 | 0.9–1.1 | 0.76–0.82 |
a | 26.3 | 25.6 ± 1.0 (24.8–27.5) | 20–35.8 | 29.2–33 | 25.7–35 | 20–24 | 24–31 | 27–28.5 |
b | 4.4 | 4.4 ± 0.4 (3.7–4.9) | 4.0–5.1 | 3.2–4.2 | 3.7–5 | 3.2–4.7 | 4.0–4.8 | 3.6–4.3 |
c | 54.5 | 51.3 ± 5.8 (46.2–60.7) | 55.0–67.3 | 40–46 | 34.8–39.1 | 28–39 | 35–37 | 45–45.8 |
c’ | 0.6 | 0.7 ± 0.1 (0.6–0.8) | 0.7–1.0 | 1 | 1.0–1.3 | 1 | 1.1–1.9 | 0.8–0.85 |
V | 55.1 | 55.3 ± 1.2 (53.3–56.5) | 51–66 | 60–62 | 57–63.4 | 52–57 | 52–58.5 | 57–57.5 |
G1 | 18.2 | 14.0 ± 3.2 (8.6–17.3) | 18.4–27.9 | 17.3 | 9–24 | – | – | 18.5–19.5 |
G2 | 14.2 | 14.3 ± 1.8 (11.7–16.3) | 13.9–21.5 | 17.0 | 10–17 | – | – | 16.5–18.5 |
Lip region diameter | 10 | 10 ± 0.2 (9.5–10) | 8–9.5 | 8 | 7–8 | 10.5–12 | 11.5–13 | 8 |
Lip region height | 4 | 4 ± 0.2 (3.5–4.0) | 4 | 3.5 | 2–3 | 5–5.5 | – | 3 |
Amphid aperture | 4 | 4 ± 0.2 (3.5–4) | 3–4 | 4 | 2 | – | – | 4–5 |
Odontostyle length | 9 | 9 ± 0.4 (8–9.5) | 5–6 | 7–8 | 7–8 | 8.5–9.0 | 10–11.5 | 7 |
Odontophore length | 10 | 10 ± 0.7 (9–11) | 8–9 | 8–9 | 9–14 | 9–11 | – | 8 |
Guiding ring from anterior end | 6 | 5.8 ± 0.4 (5.5–6.5) | 5–6 | – | 4–5.5 | – | – | 4–5 |
Nerve ring from anterior end | 94 | 86 ± 6.6 (78–93) | 72–90 | 80 | 63–71 | – | – | 73–83 |
Pharyngeal length | 230 | 222 ± 8.5 (216–237) | 202–244 | 193–220 | 146–207 | 211–222 | 213–249 | 191–208 |
Expanded part of pharynx | 98 | 91.0 ± 6.4 (87–102) | 81–110 | 76 | 61–87 | 90 | 90–106 | 67–75 |
Cardia length | 12 | 11 ± 1.2 (9–12) | 6–12 | 6 | 5–7 | – | – | 5–8 |
Body diameter at neck base | 34 | 36 ± 4.1 (31.5–41.5) | 24–31.5 | 26 | 19–24 | – | – | – |
Body diameter at mid-body | 38 | 39 ± 2.7 (36–43) | 26–34.5 | 28 | 19–26 | – | – | – |
Body diameter at anus | 28 | 25 ± 1.3 (24–27) | 19–22 | 18 | 15–21 | 28 | 21–25 | – |
Anterior genital branch | 182 | 142 ± 38.2 (80–185) | 172–265 | 141 | 58–95 | – | – | 142–164 |
Posterior genital branch | 142 | 144 ± 16.3 (117–161) | 134–219 | 139 | 68–87 | – | – | 125–155 |
Vaginal depth | 14.5 | 19.0 ± 1.0 (18–20) | 11–16.5 | 13 | 8–11 | – | – | – |
Vulva from anterior end | 552 | 559 ± 34.8 (513–607) | 553–658 | 492 | 345–420 | – | – | 440–474 |
Prerectum length | 68.5 | 85 ± 12.9 (71–100) | 69–200 | 94 | 22–70 | – | 47–66 | 100–105 |
Rectum length | 26 | 25 ± 2.7 (22–28) | 17–23 | 18 | 13–25 | – | – | 18–20 |
Tail length | 18 | 19 ± 1.5 (16.5–21) | 14–19 | 18 | 16–22 | 28 | 30–38 | 17–18 |
The sequences of 18S rDNA and D2-D3 region of 28S rDNA of Tylencholaimus helanensis sp. n. were obtained. The inter-individual variabilities of the 18S rDNA sequences and the 28S rDNA sequences are one gap and two base pair differences, respectively. Two sequences for each of the genes were deposited in GenBank (accession numbers: KU992903 (1746 bp long) and KU992904 (1747 bp long) for 18S rDNA, KU992905 and KU992906 (both 840 bp long) for D2-D3 region of 28S rDNA). The BLAST search for the 18S rDNA showed the highest similarity (94% and 95%) to the sequence of an unidentified species of Tylencholaimus (AJ966510). For the D2-D3 region of 28S rDNA, both sequences showed the highest similarity (79%) to the sequences of Xiphinema brevicollum Lordello & Da Costa, 1961 (AY580057). In the 18S rDNA phylogenetic reconstructions (Fig.
Phylogenetic relationships of Tylencholaimus helanensis sp. n. and other Dorylaimida species for the D2-D3 region of 28S rDNA. Bayesian inference strict consensus tree is acquired under GTR+I+G model. Posterior probabilities higher than 50% are presented. Newly obtained sequences presented in bold.
Rhizosphere soil of unidentified grasses from Helan Mountain, Alxa Left Banner, Alxa League, Inner Mongolia, China.
The new species is named after the mountain Helan, which is a famous mountain with a wealth of human history including rock paintings, architecture, vineyards, and a national park.
Tylencholaimus helanensis sp. n. is characterized by having a body length of 0.93–1.07 mm; body tapering towards the anterior end; lip region offset from the body by a constriction and 25% in average of the body diameter at posterior end of the neck region wide; amphid aperture 0.4 times in average as wide as the lip region; odontostyle 8–9.5 µm long and 0.85–1.0 times as long as the lip region width; odontophore 1–1.3 times as long as the odontostyle; basal expansion of pharynx 39–43% of the total neck length; female genital system didelphic-amphidelphic; vulva transverse; prerectum 2.4–4.2 times and rectum 0.9–1.2 times the body diameter at anus long; tail hemispheroid with blunt rounded to flat terminus; males not found.
Tylencholaimus helanensis sp. n. is close to T. congestus Loof & Jairajpuri, 1968, T. cosmos (Dhanam & Jairajpuri, 1999) Peña-Santiago, 2008, T. crassus Loof & Jairajpuri, 1968, T. paracrassus Monteiro, 1970, T. sinensis Li, Baniyamuddin, Ahmad & Wu, 2008 and T. teres Thorne, 1939 in having a body length about 1 mm or less, female genital system didelphic-amphidelphic, odontostyle less than 10 μm and ‘V’ value less than 62 in average, but can be differentiated by having panduriform pars proximalis vaginae. In addition, the new species differs from T. congestus (
In addition to the above characteristics used to differentiate the new species from its conspecifics, the pars proximalis vaginae of the new species should be noticed. Among the known didelphic species of Tylencholaimus, a cylindrical, spindle, convex, or pyriform pars proximalis vaginae has been described or illustrated. The violin-shaped structure in Tylencholaimus helanensis sp. n. is described here for the first time. This enriches the diversity of the pars proximalis vaginae and makes this characteristic more valuable for identification. In fact it is so distinctive that in the 18S rDNA and 28S rDNA Bayesian trees, Tylencholaimus helanensis sp. n. forms a monophyletic clade with 100% support. In the 18S rDNA tree, Tylencholaimus helanensis sp. n. is sister to a clade including T. teres and T. proximus. As mentioned previously, Tylencholaimus helanensis sp. n. is close to T. teres in morphology, but differs from the latter by several morphological characteristics such as a wider amphid aperture, a shorter prerectum, longer odontostyle and tail, and the fragments of their 18S rDNA sequences in common showed ten nucleotide differences. The new species does not otherwise show close relationships to T. teres in the 28S rDNA Bayesian trees, while the other close relative inferred from the 18S rDNA Bayesian tree, T. proximus, has a prodelphic genital system that is different to the didelphic-amphidelphic genital system of Tylencholaimus helanensis sp. n., and thus can be easily differentiated from the new species morphologically.
The sequences of Tylencholaimus species were not all grouped together in one clade in both the 18S rDNA and 28S rDNA Bayesian trees, suggesting that Tylencholaimus is not monophyletic. The deeper evolutionary relationships among Tylencholaimus currently cannot be further clarified due to because the few molecular data available for Tylencholaimus, especially 28S rDNA sequences, available on GenBank. For example, the relationship of the new species and T. proximus inferred from the 18S rDNA Bayesian tree was close, but this relationship cannot be confirmed because the 28S rDNA sequence of T. proximus is unavailable. Thus, the detailed relationships of Tylencholaimus species cannot be further resolved until more molecular data of Tylencholaimus are obtained.
This work was supported by a Special Project of Scientific and Technological Basis of the Ministry of Science and Technology of the People’s Republic of China to Hui Xie (Grant no. 2006FY120100).