Heterodera guangdongensis, Zhuo & Wang & Zhang & Liao, 2014

Zhuo, Kan, Wang, Honghong, Zhang, Hongling & Liao, Jinling, 2014, Heterodera guangdongensis n. sp. (Nematoda: Heteroderinae) from bamboo in Guangdong Province, China-a new cyst nematode in the Cyperi group, Zootaxa 3881 (5), pp. 488-500 : 489-498

Heterodera guangdongensis n. sp.

Figs. 1–3

= Heterodera sp. GD 1 in Zhuo et al. 2013

Measurements. See Table 2 .

Material examined. Type-material: Holotype cyst, three paratype vulval cones, one paratype male and five paratype J2s are deposited in the USDA Nematode Collection , Beltsville , Maryland ; three paratype vulval cones, one paratype male and five paratype J2s in the Canadian National Nematode Collection, Ottawa, Canada . Other specimens are available in the Laboratory of Plant Nematology , South China Agricultural University, Guangzhou, China.

Description. Cyst. Lemon-shaped with distinct neck and vulval cone, light brown to dark brown in color. Stylet and other internal structures indistinct. Cuticle with irregular zig-zag pattern, but without remnant subcrystalline layer. Vulval cone ambifenestrate. Underbridge weak. Bullae absent. Eggs packed in the body. Anus distinct.

White female. Body lemon-shaped, white in color, with prominent neck and vulval cone. Subcrystalline layer present. Head set off, with two annuli, the second annule larger and disc-shaped. Stylet long, with rounded knobs sloping slightly posteriorly. Dorsal pharyngeal gland duct opens 3–4 µm behind the basal knobs. Median bulb massive, with strongly developed valve plates. Excretory pore indistinct. Egg sac sometimes present, ranging from one-half to two times the size of the female, containing eggs.

Male. Body vermiform, slightly curved ventrally when heat-relaxed. Lateral field with four incisures, outer bands areolated, wider than central band. Annulus width at mid-body about 1.8 µm. Head hemispherical, slightly offset, with a distinct labial disc and four or five postlabial distinct annuli. Stylet developed, with knobs slightly projecting or flat anteriorly. Dorsal pharyngeal gland duct opens 4–6 µm behind the basal knobs. Median bulb oval, with strong valve plates. Nerve ring slightly posterior to median bulb. Excretory pore 108–152.5 µm from anterior end of the body. Hemizonid 112–140 µm from anterior end of the body. Spicules arcuate, with finely bifurcate tip. Gubernaculum straight and simple. Tail very short and bluntly rounded. Phasmids indistinct.

J2. Body vermiform, slightly ventrally curved when heat-relaxed. Lateral field with three incisures. Body annuli 1.5–1.7 µm at mid-body. Head offset, hemispherical, with a labial disc and three or four indistinct postlabial annuli. Stylet robust with knobs slightly projecting or flat anteriorly. Dorsal pharyngeal gland duct opens 5.8–7 µm behind the basal knobs. Median bulb ovoid and prominent. Pharyngeal glands well developed. Hemizonid situated slightly anterior to excretory pore. Genital primordium oval, 8.0 (7.7–8.3) µm long, 5.8 (5.7–6.0) µm wide, 129.8 (100.7–143.7) µm anterior to tail terminus. Anus distinct. Tail conoid, narrowly tapering to a fine rounded terminus, hyaline terminal section distinct, 24.6 (18.3–33.8) µm long, occupying 50.1 (43.0–57.1)% of tail length. Phasmids pore-like, 5–8 annuli posterior to anus.

Diagnosis and relationships. Heterodera guangdongensis n. sp. belongs to the Cyperi group of Subbotin et al. (2010a). Cysts are characterized by a protruding, ambifenestrate vulval cone with small fenestrae (22.0– 33.8 µm in length), weak underbridge, short vulva-anus distance (28.9–35.9 µm) and a 31.1–41.0 µm long vulval slit, but without bullae; females are characterized by a 25.1–27.6 µm long stylet with rounded knobs sloping slightly posteriorly; males are characterized by a 21.5–23.0 µm long stylet with knobs slightly projecting or flat anteriorly, four incisures in lateral field, a 22.0–26.0 µm long spicule with bifurcate tip; J2s are characterized by a 19.3–21.3 long stylet with slightly projecting or anteriorly flattened knobs, three incisures in lateral field, a 41.7–61.3 µm long tail with finely rounded terminus and hyaline portion forming 50.1 (43.0–57.1)% of the tail length.

In its morphology, the new species is most closely related to four members of the H. cardiolata Kirjanova & Ivanova, 1969 complex in the Cyperi group, including H. cardiolata , H. graminis Stynes, 1971 , H. phragmitidis Kazachenko, 1986 and H. longicolla Golden & Dickerson, 1973 . The new species differs from H. cardiolata by the more oblate fenestrae (22–33.8 µm vs 44–70 µm in length), the shorter vulva-anus distance (28.9–35.9 µm vs 42–50 µm), the longer female stylet (25.1–27.6 µm vs 19–21.4 µm) and the J2 stylet knob shape (slightly projecting or anteriorly flattened vs prominently anteriorly directed). It differs from H. graminis by the longer female stylet (25.1–27.6 µm vs 19–24 µm), the smaller female DGO (3–4 µm vs 5–7 µm), the more oblate fenestrae (22.0– 33.8 µm vs 32–63 µm in length), the shorter vulva-anus distance (28.9–35.9 µm vs 40–50 µm), the J2 stylet knob shape (slightly projecting or anteriorly flattened vs prominent anteriorly concave), the male stylet knob shape (slightly projecting or flat anteriorly vs rounded) and the shorter spicules (22–26 µm vs 31–35 µm). From H. phragmitidis , it differs by the longer female stylet (25.1–27.6 µm vs 19.5–21 µm), the smaller female (223.6–398.5 × 207.9–367.9 µm vs 467–812 × 394–677 µm), the smaller cyst (266.5–415.1 × 207–361.2 µm vs 407–836 × 318.2–738 µm), the shorter spicules (22–26 µm vs 28.6–41.6 µm), the shorter gubernaculum (7.8–8 µm vs 11.7–14.3 µm), the male stylet knob shape (slightly projecting or flat anteriorly vs rounded) and the larger J2 DGO (5.8–7 µm vs 3.6–4.8 µm). It can be distinguished from H. longicolla by the longer female stylet (25.1–27.6 µm vs 19–21 µm), the more oblate fenestrae (22.0– 33.8 µm vs 37 µm in length), the longer male stylet (21.5–23 µm vs 17.9–20.2 µm), more incisures in the male lateral field (4 vs 3), more head annuli in the male (4–5 vs 3), the slightly shorter spicules (22–26 µm vs 25.2–28.6 µm), the shorter gubernaculum (7.8–8 µm vs 9–10.6 µm), the longer J2 stylet (19.3–21.3 µm vs 16.8–18.5 µm) and the larger J2 DGO (5.8–7 µm vs 3.4–6.1 µm).

The new species is also similar to other members of the Cyperi group, including H. cyperi Golden, Rau & Cobb, 1962 , H. elachista Ohshima, 1974 and H. oryzicola Rao & Jayaprakash, 1978 . It can be distinguished from H. cyperi by the longer female stylet (25.1–27.6 µm vs 22–22.4 µm), the smaller female DGO (3–4 µm vs 4.5–5.6 µm), the shorter female (223.6–398.5 µm vs 459–663 µm), the shorter cyst (266.5–415.1 µm vs 459–742 µm), the shorter males (624.0– 861.3 µm vs 874–1159 µm), the shorter spicules (22–26 µm vs 28–30.8 µm), the shorter gubernaculum (7.8–8 µm vs 8–10 µm), the male stylet knob shape (slightly projecting or flat anteriorly vs rounded), the shorter J2 (318.1–409.5 µm vs 414–532 µm), the slightly larger J2 DGO (5.8–7 µm vs 3.8–6.1 µm) and the slightly shorter J2 tail (41.7–61.3 µm vs 56–79 µm); from H. elachista by the longer female stylet (25.1–27.6 µm vs 19–23 µm), the absence of bullae (vs presence), the absence of subcrystalline in cyst stage (vs presence), the longer male stylet (21.5–23 µm vs 20–21 µm), the shorter spicules (22–26 µm vs 26–29 µm), the shorter gubernaculum (7.8–8 µm vs 8–13 µm) and the male stylet knob shape (slightly projecting or flat anteriorly vs sloping posteriorly); from H. oryzicola by the shorter female (223.6–398.5 µm vs 414–520 µm), the longer female stylet (25.1–27.6 µm vs 18–20 µm), the absence of bullae (vs presence), the shorter males (624–861.3 µm vs 896–980 µm) and the J2 stylet knob shape (slightly projecting or anteriorly flattened vs rounded).

Compared with the four Heterodera species from bamboo, i.e. H. bamboosi , H. koreana , H. hainanensis and H. fengi , H. guangdongensis n. sp. can be easily distinguished from the first three species by the presence of fenestrae (vs afenestrate). In addition, the new species further differs from H. bamboosi by the presence of a prominent vulval cone (vs absence of vulval cone) and more incisures in the male lateral field (4 vs 3); from H. koreana and H. hainanensis by more J2 lip annuli (3–4 vs 2). H. guangdongensis n. sp. differs from H. fengi by the longer female stylet (25.1–27.6 µm vs 23–24 µm), the smaller fenestrae (22.0–33.8 × 27.8–35.5 µm vs 40–65 × 45–62.5µm), the shorter vulval slit (31.1–41 µm vs 40–60 µm), the shorter male stylet (21.5–23 µm vs 24.5–26.3 µm), the male stylet knob shape (slightly projecting or flat anteriorly vs rounded, sloping posteriorly), the shorter spicules (22–26 µm vs 27.5–31.3 µm), the shorter J2 (318.1–409.5 µm vs 440–520 µm), the shorter J2 stylet (19.3–21.3 µm vs 22–24 µm), the larger J2 DGO (5.8–7 µm vs 4.5–5.3 µm), the shorter J2 tail (41.7–61.3 µm vs 62.5–77 µm) and the shorter J2 hyaline tail portion (18.3–33.8 µm vs 35–45 µm).

Type-locality and habitat: Heterodera guangdongensis n. sp. was originally collected from roots and soils around roots of bamboo ( Phyllostachys pubescens ) in Guangzhou City (latitude N23°9'23'', longitude E113°21'40''), Guangdong Province, China in 2009. The soil type is sandy loam and the local climate is subtropical .

Other locality: Other than the type locality, H. guangdongensis n. sp. was also collected from roots and soils around roots of P. pubescens in Nanning City (latitude N22°52'44'', longitude E108°25'3'') and Hechi City (latitude N24°33'29'', longitude E107°46'30''), Guangxi Province, China in 2011. The soil type is sandy loam and the local climate is also subtropical.

Etymology: The specific name refers to the type locality, Guangdong Province.

Molecular profiles and phylogenetic status. Three rDNA amplicons of 784-bp LSU D2D3 were sequenced, obtained from single J2s collected from Guangzhou, Hechi and Nanning, respectively. GenBank accession numbers of the three sequences are JX081320 , KM224882 and JX081321 . The identities among these three D2D3 sequences of H. guangdongensis n. sp. were 99.6–99.7%. A BlastN search of H. guangdongensis n. sp. on the LSU D2D3 showed high-scoring matches with some Heterodera species , the highest being with two sequences of H. elachista (GenBank accession number HM560842 and HM560843 ). The identities of these two sequences and the three D2D3 sequences from the new species ranged from 96.8% to 97.2%.

Three ITS-rDNA sequences, two 1161 bp and one 1163 bp, were obtained, respectively, from the same J2 DNA templates mentioned above. GenBank accession numbers of these three sequences are JX081324 , KM224880 and KM224881 . The identities among these three ITS sequences of H. guangdongensis n. sp. were 99.7%. A BlastN search of H. guangdongensis n. sp. on the ITS also indicated high-scoring matches with some Heterodera species , but the identities were less than 90%. The highest match was also two sequences of H. elachista (GenBank accession number JN201917 and JN201914) with 87.5% to 88% identities.

The molecular phylogenetic relationships of H. guangdongensis n. sp. reconstructed by our analyses are presented in Figures 4 and 5. A phylogenetic tree based on LSU D2D3 from a multiple alignment of 676 total characters was constructed ( Figure 4 ). The average nucleotide composition is as follows: 16.98% A, 22.45% C, 34.34% G and 26.23% T. When Cryphodera brinkmani Karssen & van Aelst, 1999 was used as the outgroup taxon, all Heterodera species form a monophyletic group with strong support [100% posterior probability (PP)]. H. guangdongensis n. sp. is positioned in a 100%-supported clade with several species in the Cyperi group, such as H. elachista , H. fengi and H. oryzicola .

A phylogenetic tree based on ITS-rRNA was generated from a multiple alignment of 1174 total characters ( Figure 5 ). The average nucleotide composition is as follows: 17.6% A, 23.15% C, 30. 25% G and 29.01% T. When using H. circeae Subbotin & Sturhan, 2004 as an outgroup taxon, H. guangdongensis n. sp. is sister to H. elachista in the Cyperi group, with high support (94% PP), and they were placed in a moderately supported clade (82% PP) with other species including H. oryzicola , H. cyperi and H. mothi belonging to the Cyperi group. This clade is closely related to another member in the Cyperi group, H. fengi , with 100% support.

The RFLP-ITS-rRNA profile of H. guangdongensis n. sp. is presented in Figure 6 and Table 3 , and differs from profiles of other known Heterodera species , as seen in Subbotin et al. (2010a), especially from profiles of those species in the Cyperi group, including H. cardiolata , H. cyperi , H. elachista , H. fengi , H. mothi Khan & Husain, 1965 and H. oryzicola , obtained with the same PCR primers and the same set of restriction enzymes, as summarized by Subbotin et al. (2010b) and Wang et al. (2013).

Figures

FIGURE 1. Heterodera guangdongensis n. sp. A: Cysts; B: Anterior region of white female; C: Vulval cone; D: Pharyngeal region of male; E: Anterior region of male; F: Lateral field of male; G: Tail of male; H: Pharyngeal region of J2; I: Anterior region of J2; J: Lateral field of J2; K: Tail of J2.

FIGURE 2. Cysts and females of Heterodera guangdongensis n. sp. under light microscope. A: Entire cysts; B: En face view of vulval cone; C: Same as B, deeper level, with underbridge; D: Female; E: Female with egg sac; F–G: Anterior region of white females; H: Posterior end of white female. (Scale bar: A = 200 µm; D–E = 100 µm; B–C, F–H = 20 µm.)

FIGURE 3. Males and J2s of Heterodera guangdongensis n. sp. under light microscope. A: Entire male; B: Entire J2; C: Pharyngeal region of J2; D: Anterior region of J2; E: Lateral field of J2; F: Tail of J2; G: Pharyngeal region of male; H: Anterior region of male; I: Lateral field of male; J: Tail of male. (Scale bar: A–B = 100 µm; C, F–H, J= 20 µm; D–E, I = 10 µm.)

FIGURE 4. The 10001st Bayesian tree inferred from LSU D2D3 under model GTR+I+G (lnL=4670.1465; AIC=9360.293; freqA=0.1698; freqC=0.2245; freqG=0.3434; freqT=0.2623; R(a)=0.3607; R(b)=3.4605; R(c)=1.5668; R(d)=0.2269; R(e)=5.5144; R(f)=1; Pinvar=0.41; Shape=0.6903). Posterior probability values exceeding 50% are given on appropriate clades. *identified as H. cynodontis by Subbotin et al. (2006), the species is considered as a synonym of H. cardiolata by Subbotin et al. (2010a).

FIGURE 5. The 10001st Bayesian tree inferred from ITS-rRNA under model TVM+I+G (lnL=12448.9229; AIC=24915.8457; freqA=0.176; freqC=0.2315; freqG=0.3025; freqT=0.2901; R(a)=0.9087; R(b)=3.144; R(c)=1.5713; R(d)=0.581; R(e)=3.144; R(f)=1; Pinvar=0.186; Shape=1.5228). Posterior probability values exceeding 50% are given on appropriate clades. * identified as H. cynodontis by Subbotin et al. (2001), the species is considered as a synonym of H. cardiolata by Subbotin et al. (2010a).

FIGURE 6. RFLP-ITS-rRNA pattern of Heterodera guangdongensis n. sp. Lanes: M = 100 bp DNA ladder (NEB); 1 = ITSrRNA amplification product; 2 = AluI; 3 = AvaI; 4 = Bsh1236I (BstuI); 5 =BsuRI (HaeIII); 6 = CfoI (HhaI); 7 = MvaI; 8 = RsaI (AfaI).

Tables

TABLE 2. Morphometrics of Heterodera guangdongensis n. sp. [All measurements are in µm and in the form: mean ± standard deviation (range)].

Stage	Character	Holotype	Paratype
Cyst
	n		21
	L (excluding neck)	303.1	345.9±38.3 (266.5–415.1)
	W	275.8	292.9±46.7 (207.0–361.2)
	Neck	109.1	107.3±10.7 (94.0–120.9)
	L (excluding neck)/Diam. ratio	1.1	1.2±0.1 (1.0–1.5)
Vulval plate
	n		19
	Fenestra length	25.1	29.4±3.2 (22.0–33.8)
	Fenestra width	28.0	30.6±2.2 (27.8–35.5)
	Vulval slit length	35.4	35.6±2.5 (31.1–41.0)
	Vulval-anal distance	35.2	31.6±2.1 (28.9–35.9)
	Underbridge	74.1	75.5±6.5 (69.7–85.1)
Female
	n		13
	L (excluding neck)		323.0±53.9 (223.6–398.5)
	W		291.5±44.6 (207.9–367.9)
	Neck		103.5±10.0 (93.6–121.3)
	L (excluding neck)/Diam. ratio		1.1 ±0.1 (1.0–1.3)
	Stylet length		26.5±0.9 (25.1–27.6)
	Stylet shaft plus knobs		12.6±1.1 (11.9–14.2)
	DGO		3.7±0.5 (3.0–4.0)
	Anterior end to median bulb		71.6±3.8 (64.9–75.6)
	Length of median bulb		30.9±1.6 (29.5–33.3)
	Width of median bulb		27.5±3.9 (23.8–32.8)
J2
	n		25
	L		355.0±26.2 (318.1–409.5)
	a		22.1±1.0 (20.8–24.5)
	b		3.8±0.1 (3.7–4.0)
	b'		2.4±0.1 (2.2–2.5)
	c		7.7±0.3 (7.1–8.1)
	c'		4.2±0.2 (3.9–4.6)
	Stylet length		20.2±0.6 (19.3–21.3)
	Lip height		3.4±0.3 (3.0–4.0)
	Lip diam.		7.7±0.3 (7.3–8.5)
	DGO		6.3±0.5 (5.8–7.0)
	Anterior end to excretory pore		84.1±8 (74.6–100.0)
	Anterior end to median bulb		60.7±5.8(52.4–72.0)