Hemicycliophora subbotini, Maria & Cai & Qu & Castillo & Zheng, 2018

Maria, Munawar, Cai, Ruihang, Qu, Nan, Castillo, Pablo & Zheng, Jingwu, 2018, Morphological and molecular characterization of Hemicycliophora subbotini n. sp. (Tylenchida: Hemicycliophoridae) from China, Zootaxa 4433 (1), pp. 161-173 : 163-170

Hemicycliophora subbotini n. sp.

(See Figs. 1–5 )

Measurements. See Table 1.

Description. Female. Body straight or slightly ventrally arcuate after heat relaxation. Cuticular sheath detached from inner cuticle at pharyngeal, vulval and tail regions. Annuli rounded with ornamentation appearing as few breaks, irregularities and anastomosis. Lip region conoid, not set off from body contour containing 2–3 annuli and with bulging oral disc, sometimes protruding beyond first annulus. Oral disc narrow ovate, convex anteriorly, amphidial apertures covered by lateral plates, lying in the same plane as oral disc when observed en face. Stylet long and flexible. Stylet knobs posteriorly slopping, concave shaped with slight cavity. Dorsal pharyngeal gland opening ca 4.0–5.0 µm posterior to stylet knob base. Pharyngeal lumen looped in median pharyngeal bulb having medium sized refractive valvular apparatus. Isthmus narrow, short, encircled by nerve ring. Basal pharyngeal bulb distinct. Excretory pore one or two annuli posterior to the base of pharynx. Hemizonid and hemizonion not seen. Vulva lips slightly modified with 1–2 annuli long vulval sleeve. Vagina straight extending less than half of body diam. Spermatheca rounded to oblong, scarcely filled with rounded sperm cells. Oviduct very short, ovary single, prodelphic. Oocytes arranged in single file except for a short region of multiplication near anterior end. Anus small, indistinct, located at 15th–22nd annuli posterior to vulva. Tail almost cylindrical anteriorly, tapering gradually to a distal finely conoid terminus or slightly offset spike with a narrowly rounded terminus. Tail annuli distinct until terminus except terminal annuli become irregular.

Male. Not detected.

Diagnosis. Hemicycliophora subbotini n. sp. is characterized by the combination of three unique characters, tail morphology (elongated, conoid or slightly offset spike with narrowly rounded terminus), long stylet 118 (108– 125) µm and posteriorly located vulva 91 (90–93). Additionally, a lip region conoid, not set off with bulging oral disc, sometimes protruding beyond the first annulus. Oral disc narrow ovate, convex anteriorly, amphidial apertures covered by lateral plates, lying in the same plane as oral disc when observed en face. The excretory pore is located 1–2 annuli posterior to the base of pharyngeal bulb. Reproductive system prodelphic, vulval lips slightly modified with a small vulval sleeve, spermatheca rounded to oblong. Anus located 18 (15–22) annuli posterior to vulva.

Relationships. Chitambar & Subbotin (2014) presented a polytomous key to identify Hemicycliophora species. According to the polytomous key, new species is close to H. filicauda Doucet, 1982 , H. metleri Jenkins & Reed, 1964 , H. nucleata Loof, 1968 , H. similis Thorne, 1955 , H. vaccinii Reed & Jenkins, 1963 because new species share similar tail shape (elongate conoid, tapering gradually to a distal finely conoid terminus or slightly offset spike with a narrowly rounded terminus, and tail annuli are distinct until terminus except terminal annuli become irregular), cuticle ornamentation (consists of few breaks, irregularities and anastomosis) and general morphology (slightly ventrally arcuate, cuticle attached at lip region and the terminal end of body with spike-like projection) with these five species. However, together with above-mentioned characters, longer stylet length and posteriorly located vulva make this species unique among rest of Hemicycliophora species. It can be differentiated from H. filicauda by more body annuli, R= 275 (255–297) vs. 262 (246–274), longer stylet, 118 (108–125.) vs. 85 (80–90) µm, higher V, 91 (90–93) vs. 82.5 (82–83), higher number of annuli from anterior end to excretory pore, Rex, 60 (54–66) vs. 44 (43–460), lower Ran, 28 (24–25) vs. 59 (53–60), lower VL/VB, 2.5 (2.0–2.9) vs. 5.1 (4.7– 5.2) and shorter tail, 94 (76–111) vs. 142 (132–147) µm.

From H. metleri , differs by the shape of lip region (conoid with narrow ovate oral disc vs. truncate with rectangular oral disc), slenderer body, a = 21.7 (18.9–23.7) vs. 28.3 (23–38), lower R, 275 (255–297) vs. 414 (385– 2455), shorter stylet, 118 (108–125) vs. 143 (122–158) µm, higher V, 91 (90–93) vs. 85 (83–87), and lower RVan, 15–22 vs. 24–36.

From H. nucleata differs by shorter body length 1057 (896–1218) vs. 1450 (1240–1550) µm, lower R, 275 (255–297) vs. 347 (320–373), slenderer body, a = 21.7 (18.9–23.7) vs. 33 (30–38), lower c' =2.4 (2.1–3.1) vs. 4.4 (3.3–5.5), shorter stylet, 118 (108–125) vs. 138 (130–150) µm, higher V, 91 (90–93) vs. 82 (79–85), lower RV, 46 (42–53) vs. 83 (62–92), lower Ran, 28 (24–25) vs. 57 (48–64).

From H. similis (after Subbotin et al., 2014) differs by longer stylet length, 118 (108–125) vs. 100.3 (91–108) µm, higher V, 91 (90–93) vs. 79.8 (78–81), lower RV, 46 (42–53) vs. 64.4 (57–70), lower Ran, 28 (24–25) vs. 42.8 (36–51), lower VL/VB, 2.5 (2.0–2.9) vs. 5.9 (4.8–7.6).

From H. vaccinii differs by slenderer body, a = 21.7 (18.9–23.7) vs. 30.1 (25.6–35.3), longer stylet, 118 (108– 125) vs. 102 (95–112) µm, higher V, 91 (90–93) vs. 76.7 (74.7–80.4), lower c' =11.3 (10.4–12.6) vs. 15.6 (14.9– 16.7), lower RVan (15–22 vs. 30–35), longer tail (after Subbotin et al., 2014) 94 (76–111) vs. 116.5 (113–121) µm, lower R, 275 (255–297) vs. 304.5 (273–339), lower RV, 46 (42–53) vs. 73.3 (70–76), lower Ran, 28 (24–25) vs. 49.6 (46–52).

Type host and locality. The type specimens were extracted from the rhizosphere of camphor tree ( Cinnamomum camphora (L.) Presl), Hu-shan Garden, Zhejiang Province, P. R. China, on October 2, 2017. The geographical position of the sampling site is 120°11"60' E; 30°21"47' N.

Etymology. The species is named in honor of Dr. Sergei A. Subbotin for his extraordinary contribution to our knowledge of nematode taxonomy and systematics.

Type-material. Holotype female, 10 female paratypes (slide numbers ZJU-19-01-ZJU-19-06) deposited in the nematode collection, Zhejiang University, Hangzhou, China. Two female paratypes deposited in the Nematode collection in Institute for Sustainable Agriculture, CSIC, Córdoba, Spain. Two females (slide number T-6986p) at USDA nematode collection, Beltsville, MD, USA.

Polytomous key code. According to the polytomous key of Chitambar and Subbotin (2014), the new species has the following specific alphanumeric codes: A4, B2, C1, D21, E2, F12, G3, H3, I2, J2, K4, L3, M2, N2, O2, P1, Q3, R6, S2, T2, U2, V2, W1, X1.

Molecular characterization and phylogenetic relationships. The sequenced fragments of D2–D3 of 28S rRNA, ITS, and the partial 18S rRNA were ca 729–732 bp, 797–799 bp, and 1795–1798 bp, respectively. Sequences from H. subbotini n. sp. matched well with the Hemicycliophora spp. sequences deposited in GenBank all of them is clearly different. Three new D2–D3 of 28S rRNA gene sequences of H. subbotini n. sp. ( MG701275 - MG701277 ) were obtained in the present study showing 99% similarity among them (1–2 bp, 1–2 indels). These sequences showed a 92–93% identity (differed in 43–53 bp) with several Hemicycliophora spp. such as H. epicharoides ( KF430512 , KF430513 ), Hemicycliophora sp. 13 ( KF430507 ), Hemicycliophora sp. 9 ( KF430511 , AY780973 , KF430515 ), H. signata ( MG019824 ), Hemicycliophora sp. 2 ( KF430516 , KF430517 ), and H. obtusa ( KF430521 ).

Three new ITS sequences of H. subbotini n. sp. ( MG701272 - MG701274 ) were obtained in the present study (identical among them), showing 87–88% identity (79–87 bp) with H. californica ( KF430576 ) and H. raskii ( KF430577 ). Three near-full-length 18S rRNA sequences of H. subbotini n. sp. ( MG701278 - MG701278 ) (identical among them), showed a high similarity with other three Hemicycliophora spp. being 97–98% similar (49–52 bp) to H. thienemanni ( AY284629 , AY284628 , EU3066341) and H. thornei ( KJ636437 ). Phylogenetic relationships among Hemicycliophora species inferred from analyses of D2–D3 expansion segments of 28S and ITS sequences using BI are given in Figures 4 and 5 .

The D2–D3 expansion segments of 28S rRNA tree ( Fig. 4 ), based on a multiple edited alignment of 80 sequences and 667 total characters, revealed nine major well-to low supported clades (PP = 1.00, PP ≤0.70, respectively), I, II, III, IV, Va, Vb, VI, VII, and VIII as numbered by Subbotin et al. (2014) and Van den Berg et al. (2018). Hemicycliophora subbotini n. sp. appeared as a separate basal clade within species of clade I, II, and III ( Fig. 4 ). For ITS rRNA tree ( Fig. 5 ), the 50% majority-rule BI tree from a multiple sequence alignment of 91 sequences and 723 characters also showed seven (I to VIb) major well to moderately supported clades (PP = 1.00, PP =0.87, respectively). Hemicycliophora subbotini n. sp., H. californica Brzeski 1974 and H. raskii Brzeski 1974 clustered together in a moderately supported clade Vb (PP = 0.93) ( Fig. 5 ). No phylogenetic tree based on the nearfull 18S rRNA was carried out in this research since only three species for this gene have been sequenced.

Figures

FIGURE 1. Line drawings of Hemicycliophora subbotini n. sp. Female. A: Pharyngeal region; B–D: Various lip regions; E–G: En face view of lip region; H: SEM Vulval region; I: SEM Lateral field; J: Reproductive system; K, L: Vulval region; M, N: Stylet basal knobs; M: SEM Posterior region showing vulva and anus; P–R: Female tails. (Scale bars: A = 20 µm, B–D, E–G, K–N, P–R = 10 µm, H, I = 30 µm, O = 50 µm.)

FIGURE 2. Light photomicrographs of Hemicycliophora subbotini n. sp. Female. A: Entire body; B, C Pharyngeal region, arrows showing position of base of pharyngeal bulb and excretory pore; D: Posterior region, arrows showing position of vulva and anus; E, F: Stylet basal knobs; G, H: Vulval regions; I: Lateral field; J–L: Female tails arrows showing position of anus. (Scale bars: A, D = 40 µm, B, C = 20 µm, E–L= 10 µm.)

FIGURE 3. Scanning electron micrographs of Hemicycliophora subbotini n. sp. female. A: Entire body; B–D: En face view of lip region; E: Vulval region; F, G: Posterior region, arrows showing position of vulva (v) and anus (a); H: Female tail arrow showing the position of anus I: Lateral field. (Scale bars: A = 250 µm; B–D: 5 µm; E = 15 µm; F, G, I: 25 µm; H: 20 µm.)

FIGURE 4. Phylogenetic relationships of Hemicycliophora subbotini n. sp. Bayesian 50% majority rule consensus tree as inferred from D2 and D3 expansion segments of 28S rRNA sequence alignment under a transitional of invariable sites with invariable sites and a gamma-shaped distribution model (TIM3 + I + G). Posterior probabilities greater than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. Scale bar = expected changes per site.

FIGURE 5. Phylogenetic relationships of Hemicycliophora subbotini n. sp. Bayesian 50% majority rule consensus tree as inferred from ITS rRNA sequence alignment under a transitional of invariable sites with invariable sites and a gamma-shaped distribution model (TIM2 + I + G). Posterior probabilities greater than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. Scale bar = expected changes per site.