Morphological and molecular characterization of Zygotylenchus gansuensis n. sp. (Nematoda: Pratylenchinae) from China Author Wang, Honghong Author Zhuo, Kan Author Liao, Jinling text Zootaxa 2014 3821 4 465 475 journal article 45395 10.11646/zootaxa.3821.4.5 cc5d93b9-1827-4143-a0bb-faf612a7117e 1175-5326 231666 3D6C9C52-B778-4387-95FD-83945478EBF9 Zygotylenchus gansuensis sp. n. Figs. 1–2 Measurements . See Table 1 . Material examined. Type-material: Holotype female, three paratype females are deposited in the USDA Nematode Collection, Beltsville, Maryland; three paratype females in the Canadian National Nematode Collection, Ottawa, Canada . Other voucher specimens are available at the Laboratory of Plant Nematology, College of Resources and Environmental Sciences, South China Agricultural University, Guangzhou, China . Description. Female. Body straight to curved ventrally after heat relaxation. Body annuli 1.1 ± 0.1 (0.9–1.3) µm wide at mid-body. Lateral fields 6.7 ± 1.0 (5.0–9.5) µm wide, occupying one-third to two-fifths of the mid-body diameter, with five lines in vulval region and four lines in tail region, outer bands areolated in some specimens. Deirids absent. Labial region with three annuli, low and flattened, slightly offset from body, 2.7 ± 0.2 (2.6–2.9) µm high and 8.1 ± 0.4 (7.6–8.8) µm wide. Cephalic framework moderately developed. Stylet short, robust; basal knobs flattened or slightly concave anteriorly, 1.7 ± 0.1 (1.6–2.0) µm high and 3.8 ± 0.1 (3.6–4.0) µm wide. Stylet conus shorter than length of shaft including knobs. Orifice of dorsal pharyngeal gland 2.1 ± 0.2 (1.8–2.5) µm posterior to stylet base. Procorpus cylindrical. Median bulb large and muscular, oval, occupying half of corresponding body diameter, 13.7 ± 0.6 (12.9–14.9) × 10.4 ± 0.5 (9.3–11.1) µm. Isthmus short, nerve ring encircling isthmus just posterior to median bulb. Excretory pore slightly anterior to pharyngo-intestinal junction. Hemizonid ca 2 annuli long, 0–3 annuli anterior to excretory pore. Hemizonion not visible. Pharyngeal glands overlapping intestine ventrally or ventrolaterally. Vulval lips elevated distinctly. Vulva slightly posterior to middle of body. Ovaries paired, outstretched, oocytes in single row. Spermatheca indistinct; when observed in three specimens, empty and rounded. Tail subcylindrical with 25 ± 3 (21–31) annuli, tail terminus smooth and rounded, hyaline portion of tail terminus indistinct, no more than 2.5 µm in length. Phasmid minute, dot-like, situated at or near the middle of tail, 12 ± 3 (9–17) annuli posterior to anus. TABLE 1 Morphometrics of females of Zygotylenchus gansuensis n. sp. [All measurements are in Μm and in the form: mean ± standard deviation (range)].

Character	Holotype	Paratypes
n	20
L	544.6	522.0 ± 46.5 (453.5–609.5)
a	29.6	27.5 ± 1.7 (24.7–30.1)
b	7.0	6.8 ± 0.5 (5.9–7.6)
b'	4.3	3.8 ± 0.4 (3.3–4.6)
c	17.1	16.4 ± 1.4 (12.9–19.6)
c'	2.5	2.7 ± 0.2 (2.3–3.3)
V	57.0	56.4 ± 1.6 (54.0–60.8)
Stylet length	14.2	14.1 ± 0.5 (13.0–14.9)
Stylet shaft	7.2	7.3 ± 0.3 (6.9–7.9)
Stylet knob width	3.9	3.8 ± 0.1 (3.6–4.0)
Stylet knob height	1.7	1.7 ± 0.1 (1.6–2.0)
m	49.3	47.9 ± 0.9 (46.7–49.6)
Lip width	8.8	8.1 ± 0.4 (7.6–8.8)
Lip height	2.7	2.7 ± 0.2 (2.4–2.9)
DGO from stylet base	2.2	2.1 ± 0.2 (1.8–2.5)
Anterior end to:
center of metacorpus	49.7	47.7 ± 2.1 (43.9–51.6)
cardia	77.4	75.0 ± 3.9 (69.1–81.6)
end of pharyngeal gland lobe	126.0	133.4 ± 8.6 (116.3–145.0)
secretory/excretory pore	86.8	83.7 ± 5.3 (73.9–95.2)
Pharyngeal overlap	48.6	58.5 ± 8.1 (43.6–68.3)
Maximum body diam.	18.4	18.6 ± 1.6 (16.0–22.4)
Vulval body diam.	18.4	18.6 ± 1.6 (16.0–22.4)
Anal body diam.	12.6	11.6 ± 0.9 (10.1–13.2)
Anterior genital tract length	113.4	96.6 ± 13 (73.7–114.8)
OV1	20.8	18.9 ± 1.8 (16.2–22.8)
Posterior genital tract length	114.8	102.2 ± 17.1 (81.3–141.5)
OV2	21.1	20.0 ± 2.9 (15.4–24.6)
Tail length	31.8	31.3 ± 2.8 (27.0–35.9)
Number of tail annuli	23	25 ± 3 (21–31)
Vulva to anus distance	203.4	192.5 ± 24.7 (157.0–249.7)
Phasmid from tail terminus	18.6	16.4 ± 1.1 (14.0–18.6)
E.P. (%)	15.9	16.4 ± 0.9 (14.6–18.1)

FIGURE 1. Female of Zygotylenchus gansuensis n. sp. A: Entire body; B: Anterior region; C–D: Lip region; E: Lateral lines in prevulval region; F: Lateral lines in vulval region; G: Ovaries; H–K: Tail. FIGURE 2. Light micrographs of female of Zygotylenchus gansuensis n. sp. . A: Entire body; B–C: Anterior region; D, F: ovaries (arrow shows spermatheca); E: Lip region; G: Lateral lines in prevulval region; H: Lateral lines in vulval region; I–L: Lateral view of tail (arrow shows phasmid); M: Ventral view of tail (arrows show phasmids). ( Scale bars: A = 50 µm; B–D, F, I–M = 200 µm; E, G–H = 10 µm ). Male . Not found. Diagnosis and relationships. Zygotylenchus gansuensis sp. n. is characterized by the following morphological features: low and flattened labial region with three annuli, stylet 14.1 ± 0.5 (13.0–14.9) µm long, absence of deirids, five lateral lines in the vulval region, ventrally or ventrolaterally pharyngeal glands overlap 58.5 ± 8.1 (43.6–68.3) µm long, V = 56.4 ± 1.6 (54.0–60.8), indistinct spermatheca, subcylindrical tail with smooth and rounded terminus, and absence of males. Z . gansuensis sp. n. is most similar to Z . taomasinae (de Guiran, 1964 ) Braun & Loof, 1966 in morphology, but it can be differentiated from Z . taomasinae by the shorter tail (27.0–35.9 vs 40.4–44.5 µm), fewer tail annuli (21–31 vs 30–45), lower c’ value (2.3–3.3 vs 3.7–4.5), shorter distance from median bulb valve to anterior end (43.9–51.6 vs 50–58 µm), stylet conus shorter than length of shaft plus knobs, m = 46.7–49.6 ( vs conus longer than shaft plus knobs, m = 50–55), tail terminus shape (rounded vs narrowly rounded), shorter hyaline portion of tail terminus (no more than 2.5 vs 6 µm) and males absent (vs. present). The new species can be readily distinguished from the other two species of the genus, Z . guevarai (de Guiran, 1964 ) Braun & Loof, 1966 and Z . natalensis van den Berg & Tiedt, 2003 . It differs from Z . natalensis in tail shape (subcylindrical vs conical), tail terminus shape (rounded vs irregularly annulated terminus always with a ventral projection), vulval lips elevated ( vs not prominent), males absent (vs. present), lower ‘a’ value (24.7–30.1 vs 29.5–40.5 µm), position of vulva (V = 54–60.8 vs 58–65), and shorter hyaline portion of tail terminus (no more than 2.5 vs 2–6 µm). From Z. guevarai by spermatheca shape (indistinct in most specimens, rounded and empty in three specimens vs rounded, offset and filled with round sperms), lip region shape (flattened, slightly offset vs rounded, very slightly flattened), shorter stylet (13.0–14.9 vs 14.7–20.0 µm), more lateral lines in vulval region (five vs four) and males absent (vs. present). Type-locality and habitat: Soil samples were collected from the rhizosphere of jujube ( Zizyphus jujuba Mill. ) in Lanzhou City ( 36°7'30" N , 103°41'47" W ), Gansu province, China in July, 2013. Etymology: The species name is given to show the type locality of Gansu province. Molecular profiles and phylogenetic status. Three 890-bp partial SSU sequences from three single females (GenBank accession numbers KJ129767 , KJ129768 and KJ129766 ) were sequenced. Intraspecific variation of the partial SSU for Z . gansuensis n. sp. was 0.1–0.3% (1–3 bp). A BlastN search of Z. gansuensis n. sp. on the partial SSU GenBank database revealed relatively high-scoring matches with Z. guevarai and P . bolivianus (GenBank accession numbers AF442189 and KC875390 , respectively). Interspecific sequence variation was 5.8–6.0% between the new species and Z . guevarai , and 5.0–5.3% between the new species and P . bolivianus . Figure 3 represents a phylogenetic tree based on partial SSU from a multiple alignment of 926 total characters with 605 constant characters (65.3%). The average nucleotide composition is as follows: 27.04% A, 21.76% C, 25.54 % G and 25.66% T. In this tree, Z . gansuensis n. sp. and Z . guevarai do not form a monophyletic clade. Z . gansuensis n. sp. forms a highly supported (PP = 100) clade with P . bolivianus , and this clade is sister to Z . guevarai (PP = 76). One 775-bp and two 774-bp LSU D2D3 sequences (GenBank accession numbers KJ129770 , KJ129771 and KJ129769 ) from the same females as mentioned above were also sequenced. Intraspecific variation of the D2-D3 LSU segment for Z . gansuensis n. sp. was 0–0.5% (0–4 bp). A BlastN search of Z . gansuensis n. sp. on the GenBank D2-D3 database returned the highest matches with Pratylenchus sp. DP-2010 and P . bhattii (GenBank accession numbers HM469436 and JN244270 , respectively). The new species differed in D2D3 sequences from Pratylenchus sp. DP-2010 and P . bhattii by 11.3–11.6% and 11.3–11.7%, respectively. Compared to congeneric sequences from Z . guevarai (GenBank accession numbers FJ717823 [ Palomares-Rius et al . 2010 ], JQ917439 and JX261956 [ Majd Taheri et al. 2013 ]), interspecific sequence variation ranges from 14.4% to 19.8%. Figure 4 represents a phylogenetic tree based on LSU D2D3 from a multiple alignment of 704 total characters with 288 constant characters (40.7 %). The average nucleotide composition is as follows: 16.93 % A, 24.18%C, 34.58% G and 24.31 % T. In this tree, Z . gansuensis n. sp. and Z . guevarai do not form a monophyletic clade, as in the tree inferred from SSU. Z . gansuensis n. sp. occupies a basal position within a relatively highly supported (PP = 85) clade together with P . zeae , P . bhattii , P . delattrei and Pratylenchus sp. DP-2010. Two 991-bp and one 992-bp ITS sequences from the same females as above were sequenced (GenBank accession numbers KJ129773 , KJ129774 and KJ129772 ). Intraspecific variation of the corresponding gene for Z . gansuensis n. sp. was 0–1.5% (0–15 bp). In comparison with sequences from GenBank, ITS sequences of Z . gansuensis n. sp. show very great divergence. Interspecific sequence variations were 27.1–27.3% between the new species and P . brachyurus ( KC538863 ), the most similar sequence in GenBank. Comparison of ITS sequences of FIGURE 3 . The 10001st Bayesian consensus tree inferred from partial SSU under the TrN+I+G model (lnL = 5547.4624; AIC = 11108.9248; freqA = 0.2704; freqC = 0.2176; freqG = 0.2554; freqT = 0.2566; R(a) = 1; R(b) = 2.0149; R(c) = 1; R(d) = 1; R(e) = 4.5776; R(f) = 1; Pinvar = 0.424; Shape = 0.6176). Posterior probability values exceeding 50% are given on appropriate clades. FIGURE 4 . The 10001st Bayesian consensus tree inferred from LSU D2D3 under the GTR+I+G model (lnL = 8138.25; AIC = 16296.5; freqA = 0.1693; freqC = 0.2418; freqG = 0.3458; freqT = 0.2431; R(a) = 1.1441; R(b) = 4.311; R(c) = 2.4485; R(d) = 0.4018; R(e) = 6.4698; R(f) = 1; Pinvar = 0.304; Shape = 0.8623). Posterior probability values exceeding 50% are given on appropriate clades. the new species with the available ITS sequence of Z . guevarai ( FJ717817 , Palomares-Rius et al. 2010 ) at interspecific level showed divergence between 47.0–47.4%. A phylogenetic analysis was not conducted because of limited availability of comparable sequences.