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JOURNAL of NEMATOLOGY Molecular And JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2019-034 e2019-34 | Vol. 51 Molecular and morphological characterization of Paurodontella composticola n. sp. (Nematoda: Hexatylina, Sphaerulariidae) from Iran Mehrab Esmaeili1, Ramin Heydari1,*, Ahmad Kheiri1 and Weimin Ye2 1Department of Plant Protection, Abstract College of Agriculture and natural resources, University of Tehran, A new species of the genus Paurodontella, P. composticola n. sp., Karaj, Iran. collected from Nazar Abad City, Alborz Province, Iran, is described 2Nematode Assay Section, North and illustrated. The new species has a body length of 803–1053 μ m Carolina Department of Agriculture (females n = 10) and 620 and 739 μ m (males n = 2). The cuticle is and Consumer Services, Agronomic weakly annulated with four lateral lines. Cephalic region is annulated Division, Raleigh, NC, 27607, USA. and continuous with body contour. The stylet is 8.0 to 9.0 μ m long *E-mail: [email protected] with asymmetrical knobs. Esophageal basal bulb is present with a small posterior extension projecting into the intestine. Excretory pore This paper was edited by Zafar is situated at the level of esophageal basal bulb region. Post-uterine Ahmad Handoo. sac is 5 to 8 μ m long and uterus is without diverticulum. Tails of both Received for publication November sexes are similar, short and sub-cylindrical. Males have 24 to 25 μ m 20, 2018. long bursa leptoderan and spicules 24 or 25 µm long. A non-branch- ing oviduct is present to form a uterine diverticulum; the new spe- cies is closely related to five known species of the genus, namelyP . asymmetrica, P. balochistanica, P. densa, P. iranica and P. niger. It most closely resembles P. iranica, but differs from it morphological- ly by a shorter stem-like extension projecting into lumen of intestine and male with sub-cylindrical tail vs conoid. In addition to morpho- logical comparisons, the molecular phylogenetic analyses based on 733 bp of the partial sequence of 28S D2/D3 expansion segments of the large subunit rDNA gene (LSU) revealed this as a new species. Key words 28S D2/D3, Molecular phylogeny, Morphology, New species, Taxonomy. The genus Paurodontella (Husain and Khan, 1968) ca (Handoo et al., 2010), P. myceliophaga (Handoo et is a diverse and worldwide distributed Paurodontid al., 2010), P. iranica (Golhasan et al., 2016), P. persica taxon. To date, Paurodontella contains 14 nominal (Esmaeili et al., 2017a), P. parapitica (Esmaeili et al., species (Handoo et al., 2010; Esmaeili et al., 2016b; 2016b), and P. gilanica (Yaghoubi et al., 2018). Among Yaghoubi et al., 2018), including P. aberrans (Nanda- these, P. iranica, P. persica, P. gilanica and P. parapitica kumar and Khera, 1969; Sumenkova, 1975), P. apitica were described from Iran. (Thorne, 1941; Husain and Khan, 1968), P. asymme- Paurodontella is mainly characterized by the tricus (Tikyani and Khera, 1968; Sumenkova, 1975), presence of basal stylet knobs, the location of excre- P. densa (Thorne, 1941; Husain and Khan, 1968), tory pore near the nerve ring, the esophageal bulb P. minuta (Husain and Khan, 1968), P. niger (Thorne, being present with long/or short stem-like exten- 1941; Husain and Khan, 1968), P. sohaili (Maqbool, sion, the absence of post uterine sac (PUS) or pres- 1982), P. auriculata (Anderson, 1985), P. balochistani- ent only in rudimentary form, simple vulval lips and 1 © 2019 Authors. This is an Open Access article licensed under the Creative Commons CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ Molecular and morphological characterization of Paurodontella composticola n. sp. Table 1. Species used for the analysis of phylogenetic relationships and the accession numbers for LSU sequences deposited in GenBank Species. Accession Accession Authors Species Authors number number Abursanema KF885742 Yaghoubi et al. Howardulla sp. JX291131 Chizhov et al. iranicum (2014) (2012) Anguina sp. KU317615 unpublished data Litylenchus GU727547 Zhao et al. (2012) coprosma Anguina tritici DQ328723, Subbotin et al. Nothotylenchus KT149799 Esmaeili et al. KC818620 (2006) persicus (2015) Anguinata KY067443 unpublished data Nothotylenchus KX549319 Esmaeili et al. phoneixae (2017c) Cephalenchus KU723245 Pereira and Baldwin Nothotylenchus MH243749 unpublished data sp. (2016) sp. Cephalenchus KX685166 Pereira et al. (2017) Paurodontella MH427864 Present study cephalodiscus composticolla n. sp. Cephalenchus KX462033 Pereira et al. (2017) Paurodontella MF543010 Yaghoubi et al. daisuce gilanica (2018) Cephalenchus KU723245 Pereira and Baldwin Paurodontella KP642168 Golhasan et al. nemoralis (2016) iranica (2016) Deladenus AY633444 Ye et al. (2007) Paurodontella KU522237 Esmaeili et al. siricidicola parapitica (2016b) Deladenus sp. JX104332 unpublished data Paurodontella KP000034 Esmaeili et al. persica (2017a) Deladenus sp. JX104322 Morris et al. (2013) Paurodontoides MG836264 Esmaeili et al. sp. (2018) Ditylenchus KX426052, Wang et al. (2017) Rubzovinema KF155283 Koshel et al. (2014) arachis KX426054 sp. Ditylenchus EU400626 Wang et al. (2017) Rubzovinema KF373736 Koshel et al. (2014) destructor sp. Ditylenchus FJ707363, Douda et al. (2013) Sphaerularia cf. AB733664, Ye et al. (2007) dipsaci KT806479, bombi AB733665, JF327763, DQ328726 JF327765, Ditylenchus gigas HQ219217, Vovlas et al. (2011) Sphaerularia AB300596 Ye et al. (2007) KC310734 vespae Ditylenchus KF612015 Vovlas et al. (2016) Sphaerularioidea KR920361 unpublished data oncogenus Ditylenchus KX463285 Esmaeili et al. Subanguina JN885540 Yao et al. (2012) persicus (2016a) moxae Ditylenchus KX400577 Esmaeili et al. Subanguina JN885539 Yao et al. (2012) stenurus (2017b) radicicola Ditylenchus MG551886 Madani and Tenuta Subanguina sp. KT205566, unpublished data weischeri (2017) KT205560, KX776479 Continued 2 JOURNAL OF NEMATOLOGY Fergusobia sp. AY633446 Ye et al. (2007) Travassosinema KX844645 Morffe and claudiae Hasegawa (2017) Tylenchida sp. FJ661086 unpublished data Travassosinema LC214839 unpublished data sp. Tylenchomorpha LC147027 unpublished data – – – sp. bursa not being leptoderan (Siddiqi, 2000). Accord- Material and methods ing to the classification by Siddiqi (2000), Pauro- dontella belongs to the subfamily Paurodontinae Sampling, extraction, mounting, and (Thorne, 1941), family Paurodontidae (Thorne, 1941), drawing superfamily Sphaerularioidea (Lubbock, 1861), suborder Hexatylina (Siddiqi, 1980) in the order of Specimens of Paurodontella composticola n. sp. Tylenchida (Thorne, 1949). Siddiqi (2000) consid- were obtained from compost collected in Nazar Abad ered the family Paurodontidae a junior synonym of City, Alborz Province, Iran in August 2017. To obtain Sphaerulariidae, since the genera included in these a cleaner suspension of nematodes, the tray of ex- families are morphologically similar and have similar traction was employed (Whitehead and Hemming, life cycles. However, there is not enough available 1965). Specimens for light microscopy were killed by molecular data to support these statements. Biolog- gentle heat, fixed in a solution of 4% formaldehyde ically, in Paurodontidae, a fungus-feeding generation + 2% glycerol and transferred to anhydrous glycer- is well known and nothing is known about entomo- in, according to De Grisse (1969), and mounted on parasitic forms, whereas in Sphaerulariidae, an en- permanent slides. Specimens were examined using tomoparasitic form is present. Some other nema- an Olympus BH-2 (Japan) compound microscope tologists (Chizhov, 2004; Andrássy, 2007; Handoo at magnifications up to 1,000× magnification. Meas- et al., 2010) followed Siddiqi’s opinion considering urements were carried out using a drawing tube at- Paurodontidae as a synonym of Sphaerulariidae. In tached to a Nikon E200 light microscope (Japan). All this study, Siddiqi’s (2000) scheme was followed. measurements were expressed in micrometers (μ m) A history of the taxonomic studies on mem- and photographs of nematodes were taken by a digi- bers of the superfamily Sphaerularioidea and a list tal camera attached with the same microscope. of species occurring in Iran were given by Esmaeili et al. (2016b). In order to study the species diversi- DNA extraction, PCR, and sequencing ty of this superfamily in Iran, we conducted several samplings in cultivated and natural areas of Iran dur- Nematode DNA was extracted from single live indi- ing August 2017 and, as a result, a population of a viduals. Single nematode specimen was transferred Paurodontella species has been found in compost to an Eppendorf tube containing 16 µl ddH2O, 2 µl 10× from a few places where mushrooms failed to grow. PCR buffer and 2 µl proteinase K (600 µg/ml) (Pro- This population morphologically resembled a group mega, Benelux, The Netherlands) and crushed for of Paurodontella species by having a short stylet 2 min with a micro-homogeniser, Vibro Mixer (Zürich, with asymmetrical knobs at the base, the subventral Switzerland). The tubes were incubated at 65°C for knobs larger than dorsal, a short stem-like extension 1 hr, then at 95°C for 10 min. In total, 1 µl of extracted projecting and extended immediately behind the DNA was transferred to an Eppendorf tube containing base of esophageal basal bulb, short PUS and four 2.5 µl 10× NH4 reaction buffer, 0.75 µl MgCl2 (50 mM), incisures in the lateral fields. These traits prompt- 0.25 µl dNTPs mixture (10 mM each), 0.75 µl of each ed us to perform much detailed morphological and primer (10 mM), 0.2 µl BIOTAQ DNA Polymerase (BIO- molecular study to compare with all previously
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