Genus
During the present study, a population of
Several twigs collected from declining Chinese pine (
DNA samples were prepared according to Li et al. (2008). Four sets of primers (synthesized by Majorbio, Shanghai, China) were used in the PCR analyses to amplify the near full length 18 S, full length ITS region and D2-D3 expansion segments of the 28 S ribosomal RNA genes (rDNA). The near full length 18 S region was amplified as two partially overlapping fragments; for the first fragment, 988 F (5-CTC AAA GAT TAA GCC ATG C-3) and 1912R (5-TTT ACG GTC AGA ACT AGG G-3) were used and for the second fragment 1813F (5-CTG CGT GAG AGG TGA AAT-3) and 2646 R (5-GCT ACC TTG TTA CGA CTT TT-3) (Holterman et al., 2006). The full length ITS region was amplified with the forward primer TW81 (5’-GTT TCC GTA GGT GAA CCT GC-3’) and the reverse primer AB28 (5’-ATA TGC TTA AGT TCA GCG GGT-3’) (Joyce et al., 1994). The 28 S D2-D3 region was amplified with the forward primer D2A (5’-ACA AGT ACC GTG AGG GAA AGT TG-3’) and the reverse primer D3B (5’-TCG GAA GGA ACC AGC TAC TA-3’) (De Ley et al., 1999). PCR conditions were as described by Li et al. (2008) and Ye et al. (2007). PCR products were separated on 1.5% agarose gels and visualized by staining with ethidium bromide. PCR products of sufficiently high quality were sent for sequencing by Invitrogen, Shanghai, China.
The newly generated near full length 18 S and 28 S D2-D3 rDNA sequences of
Measurements of the new species are given in Table 1.
Morphometrics data for
Female | Male | ||
---|---|---|---|
Character | Holotype | Paratypes | Paratypes |
n | – | 6 | 5 |
L | 914 | 968±46.1 (914-1064) | 876±69.1 (750-956) |
a | 42.1 | 41.5±1.9 (38.6-44.8) | 46.2±2.6 (42.4-50) |
b | 11.2 | 11.7±0.6 (11.2-12.7) | 10.5±0.6 (9.4-11.1) |
b’ | 4.3 | 4.8±0.4 (4.3-5.6) | 4.6±0.3 (4.1-4.9) |
c | 25.3 | 28±3.1 (25-32.9) | 19.9±1.4 (17.9-22.3) |
c’ | 2.6 | 2.6±0.3 (2.1-2.9) | 2.7±0.2 (2.4-2.9) |
V or T | 70.5 | 69.1±0.9 (67.7-70.7) | 70.8±4.4 (63.4-76.5) |
Lip region height | 2.3 | 2.4±0.1 (2.2-2.6) | 2.7±0.1 (2.6-2.8) |
Lip region width | 6.9 | 7.1±0.3 (6.8-7.4) | 7.1±0.6 (6.4-7.9) |
Stylet length | 12.2 | 12.3±0.3 (11.8-12.6) | 12.3±0.6 (11.1-12.9) |
Body diam. | 21.7 | 23.4±1.7 (21.6-26.4) | 19±1.1 (17.7-20.9) |
Median bulb width | 18.9 | 13.2±0.7 (12.4-14) | 12±0.7 (11-12.8) |
Median bulb length | 12.6 | 18.5±0.5 (17.6-18.9) | 17.4±0.8 (16.2-18) |
Median bulb length/diam. ratio | 1.5 | 1.4±0.1 (1.3-1.5) | 1.5±0.1 (1.4-1.5) |
Excretory pore from anterior end | 92.2 | 96.3±4.7 (88-104) | 83.2±2.5 (80.1-87) |
Ovary length or testis | 430 | 454±24 (420-480) | 622.6±76.6 (476-690) |
Post-uterine sac | 121 | 130.3±6.1 (119-138) | – |
Vulva to anus distance | 270 | 264.2±20.9 (233-300) | – |
Post-uterine sac length/vulva to anus (%) | 44.8 | 49.8±5.5 (39.7-55.8) | – |
Anal (cloacal) body diameter | 14.1 | 13.3±0.7 (12-14.1) | 16.3±1.0 (15.3-18.2) |
Tail length | 36.1 | 34.9±3.2 (29-38.6) | 44.1±1.8 (41.8-46.4) |
Spicule (curved median line) | – | – | 14.0±0.6 (13.2-15) |
Spicule (Chord) | – | – | 15.6±0.9 (14.1-16.6) |
Body is slender, cylindrical, and J-shaped when heat killed. Cuticle has fine annulations. Lateral field has four incisures. Lip region is convex in lateral view, offset, more than twice as broad as high. Stylet is 12.3 (11.8-12.6) μm long, divided into two parts with small basal swellings and conus occupying
Body is slender, cylindrical, and slightly ventrally arcuate when heat-relaxed. Cuticle and anterior body region are similar to female. Testis, outstretched, located on the left side of intestine. Anterior part of testis contains developing spermatocytes in a single row and gradually develops into two rows. Cloacal lips are non-protruded. Spicules are paired, condylus is broad squarish to rounded shaped with triangular rostrum, capitulum is straight to slightly bent in some individuals, and lamina/calomus is complex and smoothly curved to distal end. Distal ends of spicule have rounded terminus. Two pairs of papillae are present: one pair is subventral precloacal papillae (P2) located at the same level of cloacal opening, and the other pair is subventral postcloacal papillae (P3) located at mid of the tail. Tail is conoid ventrally curved with several tubercles. Bursa is absent.
The type material was isolated from declining Chinese pine,
Holotype female, four male and four female paratypes (slide numbers BJ1-1 to BJ1-3) were deposited in the nematode collection of Ningbo Entry-Exit Inspection and Quarantine Bureau, China. One paratype male and two paratype females (slide numbers T551) were deposited in the Canadian National Collection of Nematodes, Ottawa, ON, Canada.
The species epithet is formed from the country of origin.
The
The new species has a vulval flap and four lateral lines, whereas none of the other
The new species can be differentiated from
The new species was molecularly characterized using near full length 18 S, full length of ITS region and D2-D3 expansion segments of 28 S rDNA sequences which were deposited in GenBank under the following accession numbers: MN401302 (18 S, 1,675 bp), MN 401304 (ITS, 820 bp) and MN401303 (28 S, 726 bp). The 18 S data set had 1,874 characters and the 28 S data set had 976 characters after alignment and post-editing. Phylogenetic relationships among the isolates for each data set were assessed using Bayesian inference, with
The 18 S phylogenetic tree (Fig. 3) demonstrated
The phylogenetic tree of 28 S D2-D3 (Fig. 4) is similar to the 18 S tree and
Only the
The discovery of
The new species is the first