Morphological and molecular characterisation of Discotylenchus lorestanensis sp. n. (Nematoda: Tylenchidae) from Iran Author Mehrabian, Fatemeh Author Azizi, Kourosh Author Bazgir, Eadi Author Darvishnia, Mostafa text Zootaxa 2017 2017-07-06 4290 1 167 176 journal article 25101 10.11646/zootaxa.4290.1.10 ef5dd783-13c1-4c93-94bb-7450c208a901 1175-5326 829179 8DB33697-570D-4640-B1D6-EFB0DD759832 Description of Discotylenchus lorestanensis sp. n. ( FigureS 1 & 3 ; Table 1 ) Female. Body Straight or Slightly curved ventrally after heat fixation. Cuticle with fine tranSverSe annuli, 0.8–1 µm wide at mid-body. Lateral fieldS with two inciSureS, almoSt one-fifth aS wide aS body diameter. Lip region continuouS, with diStinct diSk, 2–3 µm high and 5–6 µm wide at baSe; diSk diameter 3–4 µm, cephalic framework poorly developed. Amphidial aperture longitudinal Slit. Stylet Short with rounded knobS, ConuS Slightly Shorter than the Shaft, 40.5–47.2 % of total Stylet length (figureS 1B and 2C, D). DorSal pharyngeal gland orifice located 1– 2 µm behind the Stylet knobS. ProcorpuS Slender, median bulb oval in Shape, 9–11 µm long, 6–7 µm wide, with weak valve plateS. Nerve ring located in anterior half of the iSthmuS. Excretory pore 67–71 µm from anterior end, hemizonid 2 to 3 annuli anterior to excretory pore. ISthmuS Slender, baSal pharyngeal bulb Saccate, offSet from inteStine (figureS 1A and 2A, B). Reproductive SyStem monoprodelphic, with outStretched ovary and Short poStuterine Sac (figureS 1D and 2G), oocyteS in Single row, Spermatheca lobed and offSet, filled with rounded Sperm ( Figure 1 K). Tail elongated, not filiform, tapering to a rounded tip (figure 1L and 2H–J). Male . Similar to female in general view. Body Straight or Slightly curved ventrally, lateral field with two inciSureS. TeStiS outStretched, Sperm in Single row. SpiculeS Slender, ventrally arcuate, burSa Short, 17–20 µm; gubernaculum Simple. ( FigureS 1 E, J and 2A–F). TABLE 1. Morphometric data for Discotylenchus lorestanensis sp. n., collected from the rhizosphere of oak ( Quercus brantii ) in Lorestan province, Iran. All measurements are in µm and in the form: mean ± SD (range).

Character\Source	Female			Male
	Holotype	Paratype	CV	Paratype
N	1	1 9	7
L	494	508 ± 31 (449–566)	5.8	483 ± 14 (437–508)
a	33.3	33.2 ± 2.6 (28.3–39.5)	7.6	37.8 ± 1.4 (31.7–39.8)
b	5.4	5.5 ± 0.2 (5.2–5.8)	3.7	5.0 ± 0.4 (4.4–5.4)
c	6.8	6.9 ± 0.4 (6.2–8.0)	6.2	6.5 ± 0.3 (6.2–7.0)
c'	8.2	8.0 ± 1.1 (5.8–9.2)	12.5	7.8 ± 0.3 (5.9–7.9)
V	65.6	67.9 ± 2.9 (63.3–71.1)	3.8	-
stylet	8	8.8 ± 0.3 (8–9)	4.0	8.2 ± 0.1 (8–9)
Conus	4	3.9 ± 0.4 (3–5)	10.5	3.4 ± 0.1 (3–4)
m (conus/stylet %)	41.5	43.7 ± 2.2 (40.5–47.2)	5.3	41.2 ± 0.9 (40.1–42.2)
DGO	0.9	1.1 ± 0.2 (0.9–1.5)	16.7	0.9 ± 0.2 (0.8–1.1)
Pharynx	90	93 ± 4 (86–98)	4.9	99 ± 6 (94–106)
Median bulb	40	41.7 ± 2.4 (38–45)	6.7	45.1 ± 3.1 (43–50)
MB	44.2	46.0 ± 2.6 (41.7–50.5)	4.7	45.7 ± 0.5 (45.2–46.5)
Excretory pore	75	76 ± 4 (67–81)	5.3	77 ± 6 (74–85)
Nerve ring	61	62 ± 3 (58–68)	4.6	63 ± 0.4 (62–63)
Hemizonid	71.7	73 ± 4 (66–78)	5.3	73 ± 5.5 (66–79)
Head-vulva	350	340 ± 16 (309–389)	5.9	-
Head-anus	421	439 ± 26 (383–469)	5.6	407 ± 11 (369–426)
Vulva-anus	71	84 ± 13 (65–100)	17	-
Tail length	73	73 ± 4 (66–78)	6.4	74 ± 6 (68–82)
Body width	15	15.2 ± 1.2 (13–17)	7.6	12.4 ± 0.8 (12–14)
Vulval body width	14	14.5 ± 0.8 (14–16)	5.9	-
Anal body width	9	9.5 ± 1.5 (9–13)	11.5	10.0 ± 0.6 (9–11)
Vagina (µm)	6	6.0 ± 0.7 (5–7)	12.6	-
Lateral field width	3	3.1 ± 0.3 (3–4)	11.2	2.5 ± 0.2 (2–3)
Lateral field/BW %	20.2	20.4 ± 2.1 (15.9–23.7)	10.5	19.5 ± 0.3 (19.2–20.0)
Annulus width	0.8	0.9 ± 0.1 (0.8–1.0)	9.5	0.9 ± 0.1 (0.8–1.0)
PUS	6	5.6 ± 0.5 (5–7)	11.2	-
PUS/BW	0.4	0.4 ± 0.6 (0.3–0.4)	8.5	-
Spicule	-	-	-	14.1 ± 0.9 (13–16)
Bursa	-	-	-	18.5 ± 1.1 (17–20)
Gubernaculum	-	-	-	4.3 ± 0.2 (4–5)

Diagnosis and Relationships. Discotylenchus SpecieS are divided into two groupS baSed on the number of lateral field inciSureS. ThoSe with two inciSureS include D. azadkashmirensis , D. discolabialis , and D. longicauda , while the Second group, with four inciSureS, includeS D. attenuatus , D. brevicaudatus , D. iranicus , and D. discretus . Discotylenchus lorestanensis Sp. n., belongS to the firSt group on the baSiS of itS two lateral field inciSureS, and iS further characterized by a Stylet length of 8–9 µm, tail 66–78 µm long and tapering to a rounded tip, offSet, lobed Spermatheca filled with rounded Sperm, and an oval median bulb. The new SpecieS can be diStinguiShed from D. azadkashmirensis by having a Shorter tail (66–78 vs 100–105 µm), Spermatheca Shape (lobed vs elongated), different baSal bulb Shape (Saccate to elongate vs elongate), longer body (449–566 vs 350–460 µm), higher c value (6.2–8.0 vs 3.5–4.6), lower c’ (5.8–9.2 vS 10.0–13.3), and higher V (63–71 vs 57–61). DifferenceS with D. longicauda include the Shorter tail (66–78 vs 140–188 µm), median bulb Shape (oval vs Spindle), baSal bulb Shape (Saccate vs elongated), Spermatheca Shape (round to ellipSoid vs elongated), and longer Stylet (8–9 vs 6.5–7.5 µm). It differS from D. discolabialis by the longer Stylet (8–9 vs 6–7 µm), median bulb Shape (oval vs Spindle), baSal bulb Shape (Saccate vs elongated), Spermatheca (filled vs empty), and in the preSence of maleS. The new SpecieS differS from memberS of the Second group, above, in Several featureS in addition to the number of lateral field inciSureS. In compariSon with D. attenuatus , it differS by having a longer Stylet (8–9 vs 6–7 µm), and in tail Shape (not filiform, tapering to a rounded tip vs filiform, with extremely narrow terminuS). It can be Separated from D. brevicaudatus by the longer Stylet (8–9 vs 6–8 µm), greater body length (449–566 vs 320–360 µm), tail length (66–78 vs 35–47 µm), and vulva poSition (63–71 vs 79–81%), aS well aS in the preSence of maleS. It can be diStinguiShed from D. discretus by having a longer Stylet (8–9 vs 7–8 µm), by tail Shape (elongated, not filiform tapering to rounded vs gradually tapering, filiform), and Shorter tail length (66–78 vs 80–115 µm). D. lorestanensis sp. n. , differS from Discopersicus iranicus Yaghoubi, Pourjam, Ortega , LiebanaS, Atighi & Pedram 2016 by itS longitudinal amphidial aperture on the lip region ( vs oblique amphidial aperture), aS well aS in having a Shorter Stylet (8–9 vs 12–15 µm) and body (449–566 vs 681–748 µm). FIGURE 1. Photomicrographs of Discotylenchus lorestanensis sp. n. Female (A–D, F–I, K, L) and male (E, J). A: Pharyngeal region; B: Head and stylet; C: Amphid; D: PUS; E: Male posterior end; F: Female tail; G: Entire body; H: Cross section of female showing lateral field; I: Lateral field; J: Bursa; K: spermatheca; L: tail terminus (all scale bars = 10 µm). (A: anus; B: bursa; L.L: lateral line; PUS: post-vulval uterine sac; S: spermatheca; V: vulva). FIGURE 2. Line drawings of Discotylenchus lorestanensis sp. n. female. A, B: Pharynx; C, D: Head and stylet; E: Entire body; F: Lateral field; G: PUS; H–J: tail; K: Cross section at mid-body. FIGURE 3. Line drawings of Discotylenchus lorestanensis sp. n. male. A: Entire body; B: Head and stylet; C: Pharynx; D, E: Tail and spicule; Head and stylet; F: Cross section at mid-body. Locality and habitat. The SpecimenS were collected from the rhizoSphere of oak treeS ( Quercus brantii ) in Sholabad region , Khoram-Abad county , LoreStan Province , weStern Iran (GPS coordinateS: N 33°26' 37'' , E 48° 12' 47'' ), in May, 2014. Type material. Holotype female (Slide number DL 200), 16 paratype femaleS, 6 paratype maleS and 2 juvenileS on Slide numberS DL 201–DL 208, kept in the nematode collection of the Department of Plant Protection , College of Agriculture , UniverSity of LoreStan , Iran ; 4 paratype femaleS, 2 paratype maleS and 1 juvenile on Slide numberS WT3698; WT3699; WT3700 and WT 3701 in the Nematode Collection of Plantenziektenkundige DienSt , Wageningen , The NetherlandS . Etymology. The Specific epithet iS derived from the LoreStan province of Iran , the region where the new SpecieS waS collected. Molecular characterization and phylogenetic relationships. For our molecular analySeS, we Sequenced two fragmentS of riboSomal DNA : 563 bp of LSU (28S D2–D3), and 1747 bp of SSU (18S) each from two Separate individualS. The newly obtained SequenceS were uSed for phylogenetic reconStructionS along with available SequenceS of memberS of Tylenchidae obtained from GenBank. We choSe a Sequence of Cephalenchus hexalineatus aS to Serveoutgroup taxon for each dataSet. In the SSU analySiS, our D. lorestanensis sp. n. SequenceS are tentatively placed aS SiSter taxa to Filenchus misellus ( AndráSSy, 1958 ) RaSki & Geraert, 1987 (differing by 16– 17 out of 1747 bp) and Filenchus chilensis RaSki & Geraert, 1987 (differing by 16 of 1747 bp). TheSe three SpecieS are placed in a well-Supported (0.95 pp) cluSter with Filenchus discrepans ( AndráSSy, 1954 ) RaSki & Geraert, 1987 and Filenchus longiurus ( Siddiqi & Lal, 1992 ) BrzeSki, 1997 , all SpecieS of Tylenchinae Örley, 1880 ( Geraert 2008 ) . The main clade of Boleodorinae Khan, 1964 ( Geraert 2008 ) in thiS phylogenetic tree includeS Basiria duplexa ( Hagemeyer & Allen, 1952 ) Geraert, 1968 , D. iranicus , Basiria Sp. and Neopsilenchus magnidens ( Thorne, 1949 ) Thorne & Malek, 1968 , placed together with Strong Support (1.00 pp). The relatively high number of autapomorphic characterS , aS well aS the maximal Support of the relevant branching pointS, SupportS the StatuS of D. lorestanensis sp. n. aS a diStinct SpecieS. BaSed on the type of amphidial aperture and the reSultS of our phylogenetic analySeS of SSU SequenceS, thiS new SpecieS belongS to the Subfamily Tylenchinae ( Geraert 2008 ) . AnalySiS of the LSU SequenceS (figure 4) ShowS that two individualS of D. lorestanensis sp. n. were identical.