Females and juveniles were killed and fixed by addition of 80°C FG 4:1 fixative (Southey, 1986). Nematodes were fixed for at least 24 hr and, then, processed according to the Seinhorst method (Cid Del Prado Vera and Subbotin, 2012). For light microscopic observations, specimens were mounted on Cobb slides and sealed with a paraffin ring and glycerin (Cobb, 1917). Nematodes were observed, measured and photographed with the aid of a compound microscope (BX53, Olympus) equipped with microscope digital camera (DP73, Olympus). For SEM study, nematodes were dehydrated through an ethanol series ranging from 20% through 100%, with specimens left at each step for about 1 day (Cid Del Prado Vera et al., 2012). Subsequently, the specimens were critical point-dried and mounted on studs in suitable position arrangements. Nematodes were coated with gold palladium (SC7620; Polaron) and observed under a scanning electron microscope (Jeol JSM-6390) at 10 kV.
For molecular analysis, DNA was extracted with DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA). Two rRNA fragments, i.e. the LSU D2–D3 and ITS regions, were amplified. Primers for D2–D3 segments amplification were D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′). Primers for ITS amplification were TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′). Detailed protocols of polymerase chain reaction (PCR) are as follows: after an initial 5-min denaturation step at 94°C, a 40-cycle amplification (94°C for 1 min, 56°C (D2–D3 segments) to 58°C (ITS region) for 1 min, and 72°C for 2 min) was conducted. The final extension step continued for 10 min at 72°C. In order to confirm the successful amplification of DNA by PCR, electrophoresis was performed using 0.5 × TAE buffer on 1% agarose gel. The PCR product was subsequently purified with a PCR Purification Kit (Qiagen Inc.). The amplicons were cloned in pGEM-T Easy Vector System (Promega), and the resultant plasmid DNA was isolated with a Plasmid Midiprep System (Promega). Both strands of the PCR amplicons were cycle-sequenced with an ABI PRISM BigDye Terminator version 1.1 Cycle Sequencing Kit and electrophoresed in each direction on an ABI Prism ABI 377 Genetic Analyzer (PE Applied Biosystems). The newly obtained sequences were submitted to the GenBank database under accession numbers MN244153 and MN244164.
For phylogenetic analyses, the sequences of
Systematics
Morphometric measurements of
Paratype | ||||
---|---|---|---|---|
Stage | Character | Holotype | Range (min.–max.) | Mean ± SD |
|
||||
|
10 | |||
L (including neck) | 462.7 | 363.1–544.6 | 471.5 ± 53.8 | |
L (excluding neck) | 380.3 | 430.6–255.5 | 367.8 ± 51.4 | |
W (width) | 286.5 | 166.6–329.1 | 259.3 ± 46.2 | |
Neck | 82.5 | 77.2–164.8 | 103.6 ± 26.9 | |
L (inc. neck)/W ratio | 1.6 | 1.6–2.2 | 1.8 ± 0.2 | |
Stylet length | 30.7 | 28.2–31.9 | 30.1 ± 1.0 | |
Anterior end to median bulb | 65.0 | 53.9–93.7 | 70.3 ± 14.7 | |
Length of medium bulb | 28.3 | 22.6–31.9 | 25.9 ± 2.9 | |
Width of medium bulb | 25.2 | 20.6–29.9 | 24.3 ± 3.2 | |
Vulva-nus distance | 46.9 | 35.8–51.6 | 44.7 ± 4.3 | |
Vulval slit length | – | 51.9–54.1 | 52.6 ± 1.3 | |
Excretory pore from anterior end | 80.1 | 72.6–124.5 | 92.4 ± 17.5 | |
|
||||
|
20 | |||
L (body length) | 469.1 | 424.2–525.9 | 482.8 ± 28.5 | |
|
22.7 | 18.5–22.7 | 20.1 ± 1.4 | |
|
3.9 | 3.4–4.4 | 3.9 ± 0.3 | |
|
2.7 | 2.2–3.0 | 2.7 ± 0.2 | |
|
7.2 | 6.2–7.4 | 7.0 ± 0.3 | |
|
4.4 | 3.3–5.0 | 4.2 ± 0.4 | |
Stylet length | 29.8 | 26.8–31.3 | 29.3 ± 1.2 | |
Tail length | 64.8 | 59.6–76.7 | 69.4 ± 4.4 | |
Max. body diam. | 20.6 | 20.3–28.3 | 24.1 ± 2.5 | |
Hyaline length | 39.0 | 31.7–47.8 | 38.4 ± 3.9 | |
Excretory pore from anterior end | 111.93 | 101.43–132.66 | 118.6 ± 8.9 | |
|
||||
|
10 | |||
L | 92.8–144.2 | 114.1 ± 15.1 | ||
W | 31.4–39.7 | 36.1 ± 2.8 | ||
L/W ratio | 2.9–3.9 | 3.2 ± 0.3 |
All measurements are in µm.
Measurements Table 1.
The body is globose, varies in size (513.4–778.3 µm). Cuticle is annulated on entire body (Fig. 3A). Female is pearly white when young and becoming yellowish. Cuticle does not transform to a cyst and is partially covered by some tissue materials (Fig. 1B). Head region is not set off, labial disk is distinct and neck is distinct, usually bent laterally. Stylet is 30.1 µm long. Median bulb is nearly rounded to oval, with distinct valve plates situated centrally (Fig. 3D). Except for the stylet and median bulb, most internal structures are difficult to observe in adult females. Excretory pore is located posterior to metacorpus. Vulva is located sub-terminal to terminal, near the anus. Vulval lips are pronounced with protrusion. Vulva-anus region is slightly concave to flat (Fig. 3B). Mature females contain less than 100 eggs in various stages of development. Perineal patterns are difficult to prepare flat due to a prominent protuberance. Perineal pattern is ovoid with fine, smooth and continuous striae. Vulva is surrounded by circles of striae (Fig. 3C).
Not found.
The body is cylindrical, tapering posteriorly, straight or slightly ventrally curved after fixation (Fig. 4A). The lip region is offset, a labial disc with four annuli (Fig. 5A). Stylet is long, cone is straight, and shaft is cylindrical. Stylet knobs are well developed with distinct flat or droop to posterior. Body annuli is 1.5–1.6 µm in width at mid-body and three incisures are visible in lateral position (Fig. 4E). Tail is long conoid, gradually tapering to a sharply pointing terminus. Hyaline terminal section is 38.4 (31.7–47.8) µm long, occupying 49.4 to 62.4% of tail length. Particularly, nearly 65% of J2 specimens of the present population exhibited a distinct constriction around middle of the hyaline region (Fig. 4C).
Comparative morphometrics (in µm) of female in species of
|
|
|
|
|
|
|
|
||
---|---|---|---|---|---|---|---|---|---|
Present study | Zhuo et al. (2014) | Colbran (1966) | Wouts (1973) | Bajaj et al. (1989) | Karssen and Van Aelst (1999) | Wouts (1973) | Wouts (1973) | Wouts (1973) | |
Body length (L) | 471.5 ± 53.8 (363.1–544.6) | 460.9 ± 53.1 (362.9–526.8) | 400–636 | 353 (286–428) | 311 ± 21 (272–353) | 450 ± 52 (384–544) | 393 (300–473) | 341 (261–423) | 331 (218–455) |
Body diameter | 259.3 ± 46.2 (166.6–329.1) | 240.1 ± 51.6 (172.4–314.0) | 201–274 | 210 (147–290) | 205.5 ± 51 (112–288) | 238 ± 35 (160–339) | 276 (217–335) | 226 (148–305) | 184 (116–369) |
Number of lip annulus | – | – | – | 1 | 2–3 | – | 2 | 2 more or less | 2 more or less |
Vulval slit length | 52.6 ± 1.3 (51.9–54.1) | 51.3 ± 6.8 (42.8–58.3) | 45 (44–49) | – | 35 | 51 ± 3.8 (44–60) | – | – | – |
Anus-vulva profile | Flat to concave | Distinctly concave | Flat to concave | Concave | Distinctly concave | Flat to concave | Flat | Flat | Flat |
Data referred to Colbran (1966), Wouts (1973), Bajaj et al. (1989), Karssen and Van Aelst (1999), Siddiqi (2000), Sturhan (2010), Zhuo et al. (2014). –, not available.
Comparative morphometrics (in µm) of second-stage juveniles (J2) in species of
|
|
|
|
|
|
|
|
||
---|---|---|---|---|---|---|---|---|---|
Present study | Zhuo et al. (2014) | Colbran (1966) | Wouts (1973) | Bajaj et al. (1989) | Karssen and Van Aelst (1999) | Wouts (1973) | Wouts (1973) | Wouts (1973) | |
Body length (L) | 482.8 ± 28.5 (424.2–525.9) | 435.2 ± 25.4 (388.8–474.5) | 379–461 | 428 ± 6 | 383 ± 19 (353–424) | 393 ± 23 (450–541) | 525 ± 5 | 450 ± 4 | 457 ± 4 |
|
20.1 ± 1.4 (18.5–22.7) | 22.6 ± 1.5 (19.4–25.9) | 22.0–26.0 | 24.8 ± 0.6 | 22 ± 1.5 (18–26) | 23.2 ± 1.3 (20.3–24.6) | 27.2 ± 0.5 | 24.5 ± 0.3 | 25.3 ± 0.4 |
|
3.9 ± 0.3 (3.4–4.4) | 3.9 ± 0.2 (3.7–4.4) | 3.5-4.8 | 3.7 ± 0.06 | 3.84 ± 0.38 (3.2–5.0) | – | 3.8 ± 0.06 | 3.7 ± 0.05 | 3.7 ± 0.04 |
|
7.0 ± 0.3 (6.2–7.4) | 7.8 ± 0.5 (6.4–8.6) | 7.9–9.8 | 8.3 ± 0.15 | 8.4 ± 1.2 (7–13) | 7.1 ± 0.4 (6.4–7.8) | 7.7 ± 0.07 | 8.2 ± 0.08 | 8.5 ± 0.08 |
Number of lip annuli | 4 | 3 | 3 | 3 | 3 | 4 | 5 | 4 | 4 |
Stylet length | 29.3 ± 1.2 (26.8–31.3) | 29.1 ± 0.8 (28.0–31.0) | – a | 32.9 ± 0.3 (31–35) | 26.5 ± 1 (24–29) | 33.3 ± 1.0 (31.6–35.4) | 38.7 ± 0.4 | 33.9 ± 0.2 | 34.4 ± 0.2 |
Stylet knobs | Flat posteriorly | Project distinctly anteriorly | Concave | Flat or pointing slightly anteriorly | Flat to concave | Concave | Pointing distinctly anteriorly | Flat anteriorly | Pointing slightly anteriorly |
DGO | 4.6 ± 0.9 (3.3–6.1) | 5.4 ± 0.7 (4.5–6.5) | 4.3–5.0 | 5.5 ± 0.17 | 3–4 | 5.8 ± 0.6 (4.4–7.0) | 6.0 ± 0.26 | 5.6 ± 0.17 | 6.1 ± 0.28 |
Number of lateral line | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 (occasionally 4)b | 3 |
Tail length | 69.4 ± 4.4 (59.6–76.7) | 56.0 ± 3.2 (52.0–65.0) | – | 46.5 ± 6 (27–54) | 46.5 ± 6 (27–54) | 69.7 ± 4.4 (61.3–76.5) | 68.9 ± 0.1 | 55.3 ± 0.8 | 53.7 ± 0.8 |
Hyaline portion of tail | 38.4 ± 3.9 (31.7–47.8) | 28.3 ± 2.3 (24.5–35.0) | – | 21.5 ± 2.5 (18–26) | 21.5 ± 2.5 (18–26) | 37.2 ± 3.6 (30.3–42.3) | 36.0 ± 0.1 | 30.1 ± 0.7 | 30.6 ± 0.5 |
Tail terminus | Sharp point with constriction at the middle of hyaline | Point with mucro-like tip | Buntly rounded | Narrow rounded | Narrow rounded | Point with mucro-like tip | Narrow rounded | Narrow rounded | Narrow rounded |
DGO, distance of dorsal esophageal gland orifice to base of stylet; Data referred to Colbran (1966), Wouts (1973), Bajaj et al. (1989), Karssen and Van Aelst (1999), Siddiqi (2000), Sturhan (2010), Zhuo et al. (2014). –, not available; a Colbran (1966) reported stylet length for J2 of C. eucalypti was 26–35.9 mm. Wouts (1973) mentioned that the minimum 26 mm belonged to J2 of Heterodera. Bajaj et al. (1989) and Karssen and Van Aelst (1999) accepted the point of Wouts (1973). Thus, the value of C. eucalypti described by Colbran (1966) is omitted here; b Wouts (1973) described four incisures in the field of C. nothophagi J2. Sturhan (2010) re-examined paratype J2 of C. nothophagi, which revealed three incisures in the field, with the inner incisure occasionally diverging into two. Thus, the point of Sturhan (2010) is accepted here.
The sequenced LSU D2–D3 segments and ITS region are 792 and 1,028 bp, respectively. A BLASTn search of
The molecular phylogenetic relationships of the new species are shown in Figures 6 and 7. Figure 6 represents a phylogenetic tree based on LSU D2–D3 segments. The average nucleotide composition is as follows: 22.34% A, 22.47% C, 29.58% G and 25.61% T. Using
Holotype (female and J2): isolated from roots from type locality and habitat. Slides T-721t and T-722t deposited in the United States Department of Agriculture Nematode Collection (USDANC), Beltsville, Maryland. Paratype (female and J2): same data and repository as holotype. Slides T-7228p and T-7229p.
The specific epithet refers to the Gaya dynasty before 400 AD in ancient history of Korea. The species habitat is the geographical capital region of Gaya. Gaya was a confederacy of territorial polities nobility in the Nakdong river basin of southern Korea.
Key to the species of Cryphodera, based on second-stage juveniles:
1. — Three lip annules (lateral)2
— Four to five lip annules (lateral)4
2. — Stylet
— Stylet > 29 μm3
3. — Tail of J2 with narrow rounded
— Tail of J2 with point, mucro-like tip
4. — Four lip annules5
— Five lip annules
5. — Three lateral lines near mid-body6
— Four lateral lines near mid-body
6. — Tail length < 60 μm, tail tip narrow rounded
— Tail length
7. — Tail of J2 with mucro-like tip; stylet = 33.3 μm, DGO = 5.8
— Tail of J2 with constriction at the middle of hyaline; style L = 29.8,DGO = 4.6