Integrative taxonomy of the stunt nematodes of the genera Bitylenchus and Tylenchorhynchus (Nematoda, Telotylenchidae) with description of two new species and a molecular phylogeny Author Handoo, Zafar A. Author Palomares-Rius, Juan E. Author Cantalapiedra-Navarrete, Carolina Author Liébanas, Gracia Author Subbotin, Sergei A. Author Castillo, Pablo text Zoological Journal of the Linnean Society 2014 2014-10-31 172 2 231 264 http://doi.wiley.com/10.1111/zoj.12175 journal article 10.1111/zoj.12175 0024-4082 5314438 TYLENCHORHYNCHUS MEDITERRANEUS SP. NOV. ( FIGS 4–6 , TABLES 4–6 ) Holotype : Female extracted from soil samples collect- ed from the rhizosphere of grapevine ( Vitis vinifera ), in Jerez de la Frontera , Cádiz, Spain , by P. Castillo , mounted in pure glycerine, and deposited in the USDA Nematode collection, Beltsville, Maryland (collection number T- 640t ). Paratypes : Female paratypes extracted from soil samples collected from the rhizosphere of grapevine ( Vitis vinifera ), in Jerez de la Frontera , Cádiz, Spain , were deposited in the following nematode collections: the USDA Nematode collection, Beltsville, Maryland (collection numbers T-6249p to T-6264p); and the Nematode collection of the Department of Nematology , Landbouwhogeschool, Wageningen, the Netherlands (collection number VIDV-5) . Figure 1. Line drawings of Bitylenchus hispaniensis sp. nov. A, female pharyngeal region; B, vulval region showing part of gonads and spermatheca; C, female lip region; D, details of lip region showing oral disc ( en face view); E, F, male tails showing spicules and gubernaculum; G–J, female tails, with H and I showing intestinal fasciculi/sinuous canals present and extending back to tail, and J with areolated lateral fields and phasmid in the middle. Figure 2. Photomicrographs of Bitylenchus hispaniensis sp. nov. A, whole body of female and male; B, female pharyngeal region; C, female lip region; D, vulval region; E, female tails; F, male tail; G, lateral fields at mid-body. Scale bars: A = 50 μm, B, E = 10 μm; C, D, G = 5 μm; F = 20 μm. Etymology: The specific epithet refers to the geographical origin and is derived from the Latin mediterraneus = from the Mediterranean Basin. Description of female: Body ventrally arcuate after fixation, no longitudinal striae or ridges outside lateral fields. Body annuli distinct but fine, 1.0–1.5 μm wide around mid-body. Lateral fields originating at the level of the conus of the stylet and extending up to hyaline region of tail to tail terminus, with four incisures, areolated throughout the body. Lip region hemispherical, offset by a constriction, with five to seven annuli; lip sclerotization weakly developed. The en face view (SEM) observations revealed a labial disc flattened laterally, dorsally and ventrally, resulting in a squarish pattern, margins of labial disc interrupted laterally by amphidial openings ( Fig. 6B, C ). Oral aperture slitlike, orientated dorsoventrally with three papillae on each side parallel to long axis ( Fig. 6B, C ). Stylet moderately strong, shaft and conus equally long; knobs rounded and posteriorly directed. DGO about 2.5 to 3 μm behind stylet base. Median pharyngeal bulb oblong to rounded, 16–18 μm long, basal bulb 55–60 μm long, pyriform or extending back over the intestine. Hemizonid usually just four to five annuli anterior to excretory pore, one to three annuli wide. Vulva a transverse slit slightly posterior to the middle of the body and distinctly protruding, most of the specimens had loose cuticle just above the vulval area. Epiptygma absent. Reproductive system amphidelphic, didelphic; anteri- or and posterior ovaries equally developed. Spermatheca rounded, filled with rounded spermatozoa. Tail conoid to cylindrical straight, with 42–81 annuli, tail terminus rounded, annulated. Phasmids located slightly anterior to middle of the tail. Postanal extension of intestine absent. Figure 3. Scanning electron microscope photographs of Bitylenchus hispaniensis sp. nov. A, female lip region; B, en face view showing oral (oa) and amphidial (am) apertures; C, lateral fields at mid-body; D, E, female tails showing anus (a) and phasmid (ph); F, male tail showing spicules (sp). Scale bars: A, C = 10 μm; B = 5 μm; D–F = 20 μm. Description of the male: Abundant, morphologically similar to female except for sexual characters, and body in posterior region usually more curved than in female. Gubernaculum well developed, half of the spicule length. Tail terminus pointed. Diagnosis: Tylenchorhynchus mediterraneus sp. nov. is characterized by a long body (850–1040 μm), a lip region hemispherical, offset by a constriction, with five to seven annuli, stylet moderately strong, 19.0–22.0 μm long with laterally to posteriorly directed rounded knobs, lateral fields with four incisures, areolated throughout the body, most of the specimens had loose cuticle just above the vulval area, tail conoid to cylindrical, straight, with 42–81 annuli, tail terminus rounded, annulated, phasmids located slightly anterior to middle of the tail, and postanal extension of intestine absent. Specific D 2– D3, ITS , and 18S rRNA sequences were deposited in GenBank with accession numbers KJ461553 – KJ461558 , KJ461584 – KJ461587 , and KJ461613 – KJ461616 , respectively . Table 2. Morphometrics of paratypes and several populations of Bitylenchus hispaniensis sp. nov. from southern Spain*

Córdoba, Córdoba, Spain, wild olive			Castro del Río, Córdoba, Spain, olive		Albanchez, Jaén, Spain, olive		Bonares, Huelva, Spain olive,
Locality
host-plant	Holotype	Paratype females	Paratype males	Females	Males	Females	Males	Females	Males
n	7	10	5	5	10	6	6	3
L	675.0	662 ± 38.4 (612–732)	640 ± 60.9 (548–760)	658 ± 65.8 (582–717)	682 ± 31.1 (645–727)	711 ± 39.9 (657–765)	693 ± 67.7 (585–790)	714 ± 45.5 (660–765)	691 ± 57.8 (627–740)
a	30.6	30.4 ± 1.7 (27.8–32.5)	31.0 ± 3.9 (27.0–39.1)	31.3 ± 4.1 (27.6–35.9)	33.2 ± 2.1 (31.5–36.3)	32.0 ± 1.0 (30.9–34.0)	34.0 ± 2.8 (30.2–38.5)	32.5 ± 1.1 (30.9–34.0)	30.2 ± 1.5 (28.5–31.3)
b	5.6	6.0 ± 0.4 (5.6–6.8)	6.1 ± 0.7 (5.4–7.8)	6.0 ± 0.8 (4.9–6.8)	6.0 ± 0.5 (5.6–6.9)	6.2 ± 0.4 (5.7–6.9)	5.9 ± 0.7 (4.9–6.9)	6.3 ± 0.4 (5.7–6.8)	6.1 ± 0.4 (5.8–6.6)
c	15.0	14.2 ± 1.7 (11.8–16.8)	17.7 ± 1.4 (15.2–20.7)	14.8 ± 0.8 (13.8–15.8)	17.3 ± 2.1 (15.6–20.2)	15.0 ± 0.6 (14.2–15.9)	18.7 ± 1.4 (16.6–20.0)	15.2 ± 0.6 (14.4–15.9)	14.5 ± 1.3 (13.1–15.7)
c′	2.7	2.9 ± 0.3 (2.5–3.3)	2.9 ± 0.3 (2.4–3.3)	2.7 ± 0.2 (2.5–3.0)	3.1 ± 0.3 (2.7–3.4)	2.9 ± 0.2 (2.5–3.3)	2.9 ± 0.2 (2.7–3.2)	2.9 ± 0.2 (2.7–3.2)	2.8 ± 0.1 (2.7–2.8)
V or T %	54.0	54.6 ± 1.5 (52.5–57.0)	–	56.0 ± 1.0 (55.0–57.0)	–	55.0 ± 1.8 (53.0–57.5)	–	55.4 ± 1.7 (53.0–57.5)	–
Stylet length	17.5	16.1 ± 0.5 (15.5–17.0)	15.9 ± 0.8 (15.0–18.0)	18.0 ± 0.4 (17.5–18.5)	17.0 ± 1.0 (16.0–18.0)	17.8 ± 0.3 (17.5–18.0)	17.6 ± 0.6 (17.0–18.5)	17.7 ± 0.3 (17.5–18.0)	17.5 ± 0.5 (17.0–18.0)
Maximum body	22.0	21.8 ± 1.0 (20.5–23.5)	20.7 ± 1.3 (18.0–23.0)	21.2 ± 2.2 (20.0–25.0)	20.6 ± 1.5 (19.0–23.0)	22.6 ± 1.2 (20.0–24.0)	20.4 ± 1.5 (18.0–22.0)	22.0 ± 1.2 (20.0–23.0)	22.8 ± 1.0 (22.0–24.0)
width
Pharynx length	120.0	110.0 ± 6.5	105.4 ± 8.2	110.2 ± 9.0	114.0 ± 5.5	114.6 ± 6.5	117.0 ± 4.5	114.0 ± 6.4	113.0 ± 8.5
(100.0–120.0)	(90.0–120.0)	(103.0–120.0)	(105.0–120.0)	(105.0–122.0)	(110.0–122.0)	(105.0–122.0)	(105.0–122.0)
Anterior end to	88.0	89.7 ± 3.9 (85.0–95.0)	–	97.0 ± 5.7 (90.0–103.0)	97.0 ± 4.5 (90.0–100.0)	102.3 ± 7.5 (92.0–120.0)	98.2 ± 3.8 (92.0–102.0)	103.7 ± 5.8 (98.0–114.0)	96.3 ± 7.4 (88.0–102.0)
excretory pore
Anal body width	17.0	16.1 ± 1.4 (13.5–17.5)	12.8 ± 0.8 (12.0–15.0)	16.5 ± 2.8 (13.5–20.0)	12.9 ± 0.2 (12.6–13.0)	16.6 ± 1.8 (14.0–20.0)	12.8 ± 1.0 (12.0–14.5)	16.3 ± 1.5 (14.0–18.0)	17.3 ± 1.2 (16.0–18.0)
Tail length	45.0	47.4 ± 6.3 (37.5–57.0)	36.6 ± 5.6 (30.0–50.0)	44.6 ± 5.5 (38.0–50.0)	39.8 ± 4.1 (35.0–44.0)	47.4 ± 3.2 (43.0–52.0)	37.1 ± 3.3 (32.5–40.0)	47.0 ± 2.8 (44.0–51.0)	47.7 ± 2.5 (45.0–50.0)
Tail annuli	54	55.1 ± 8.1 (40–66)	–	48.8 ± 5.9 (40–56)	–	52.1 ± 6.6 (45–63)	–	51.5 ± 5.0 (46–58)	52.3 ± 5.1 (48–58)
Spicule	–	–	25.3 ± 1.8 (23.0–30.0)	–	25.2 ± 1.5 (23.0–27.0)	–	25.3 ± 0.8 (24.0–26.0)	–	25.0 ± 1.0 (24.0–26.0)
Gubernaculum	–	–	11.4 ± 1.6 (10.0–15.0)	–	11.3 ± 0.6 (11.0–12.0)	–	12.5 ± 0.9 (11.0–13.5)	–	12.3 ± 0.6 (12.0–13.0)

*Measurements are in μm and in the form: mean ± SD (range). L, body length; a, body length/maximum body width; b, body length/pharyngeal length; c, body length/tail length; c′, tail length/body width at anus; V, (distance from anterior end to vulva/body length) × 100; T (distance from cloacal aperture to anterior end of testis/body length) × 100. Table 3. Morphometrics of several populations of Bitylenchus hispaniensis sp. nov. from southern Spain*

El Saucejo, Sevilla, Spain, olive		Montilla, Córdoba, Spain, grapevine		Génave, Jaén, Spain, olive
Locality host-plant	Females	Males	Females	Males	Females	Males
n	6	2	2	2	2	2
L	716 ± 44.4 (633–760)	683 ± 31.8 (660–705)	676 ± 26.9 (657–695)	683 ± 75.7 (630–737)	687 ± 24.7 (670–705)	701 ± 50.9 (585–790)
a	31.6 ± 0.2 (31.3–32.0)	32.2 ± 1.2 (31.3–33.0)	31.9 ± 1.4 (30.9–32.9)	31.4 ± 0.1 (31.4–31.5)	32.4 ± 1.5 (31.3–33.5)	30.5 ± 1.3 (29.6–31.4)
b	6.4 ± 0.4 (5.8–6.9)	6.0 ± 0.4 (5.8–6.3)	5.7 ± 0.0 (5.7–5.7)	6.1 ± 0.1 (6.0–6.1)	6.4 ± 0.3 (6.2–6.6)	6.0 ± 0.4 (5.8–6.3)
c	14.7 ± 0.6 (14.2–15.7)	15.2 ± 0.7 (14.7–15.7)	15.5 ± 0.4 (15.3–15.8)	14.1 ± 0.1 (14.0–14.2)	14.9 ± 0.2 (14.7–15.0)	14.0 ± 0.2 (13.9–14.2)
c′	2.8 ± 0.2 (2.5–3.0)	2.9 ± 0.1 (2.8–3.0)	2.8 ± 0.1 (2.8–2.9)	3.0 ± 0.0 (3.0–3.0)	3.1 ± 0.2 (2.9–3.3)	2.9 ± 0.1 (2.8–3.0)
V or T %	54.9 ± 1.5 (53.0–57.5)	–	55.5 ± 2.1 (54.0–57.0)	–	56.5 ± 0.7 (56.0–57.0)	–
Stylet length	17.8 ± 0.3 (17.5–18.0)	17.0 ± 1.4 (16.0–18.0)	17.8 ± 0.4 (17.5–18.0)	17.0 ± 1.4 (16.0–18.0)	17.0 ± 0.7 (16.5–17.5)	17.3 ± 0.4 (17.0–17.5)
Maximum body width	22.7 ± 1.4 (20.0–24.0)	21.3 ± 1.8 (20.0–22.5)	21.3 ± 1.8 (20.0–22.5)	21.8 ± 2.5 (20.0–23.5)	21.3 ± 1.8 (20.0–22.5)	23.0 ± 0.7 (22.5–23.5)
Pharynx length	112.8 ± 9.7 (98.0–122.0)	113.5 ± 12.0 (105.0–122.0)	118.5 ± 4.9 (115.0–122.0)	112.5 ± 10.6 (105.0–120.0)	108.0 ± 8.5 (102.0–114.0)	116.0 ± 1.4 (115.0–117.0)
Anterior end to	99.5 ± 8.1 (87.0–107.0)	94.0 ± 2.8 (92.0–96.0)	100.0 ± 7.1 (95.0–105.0)	102.5 ± 3.5 (100.0–105.0)	90.5 ± 2.1 (89.0–92.0)	101.5 ± 2.1 (100.0–103.0)
excretory pore
Anal body width	17.3 ± 1.7 (15.0–20.0)	15.5 ± 0.7 (15.0–16.0)	15.5 ± 0.7 (15.0–16.0)	16.3 ± 1.8 (15.0–17.5)	15.0 ± 1.4 (14.0–16.0)	17.3 ± 0.4 (17.0–17.5)
Tail length	48.7 ± 3.3 (44.0–52.0)	46.0 ± 1.4 (45.0–47.0)	43.5 ± 0.7 (43.0–44.0)	48.5 ± 4.9 (45.0–52.0)	46.3 ± 1.1 (45.5–47.0)	50.0 ± 2.8 (48.0–52.0)
Tail annuli	54.8 ± 6.0 (48–63)	–	46.0 ± 1.4 (45–47)	–	51.5 ± 6.4 (47–56)	–
Spicule	–	25.0 ± 1.4 (24.0–26.0)	–	24.5 ± 0.7 (24.0–25.0)	–	25.5 ± 0.7 (25.0–26.0)
Gubernaculum	–	13.0 ± 1.4 (12.0–14.0)	–	12.5 ± 0.7 (12.0–13.0)	–	13.5 ± 0.7 (13.0–14.0)

*Measurements are in μm and in the form: mean ± SD (range). L, body length; a, body length/maximum body width; b, body length/pharyngeal length; c, body length/tail length; c′, tail length/body width at anus; V, (distance from anterior end to vulva/body length) × 100; T (distance from cloacal aperture to anterior end of testis/body length) × 100.