Nematology 17 (2015) 27-52 brill.com/nemy

Morphological and molecular characterisation of pseudorobustus (Steiner, 1914) Golden, 1956 and related species (: ) with a phylogeny of the genus ∗ Sergei A. SUBBOTIN 1,2, ,NicolaVOVLAS 3, Gregor W. YEATES†, Johannes HALLMANN 4, Sebastian KIEWNICK 5,VladimirN.CHIZHOV 2,RosaH.MANZANILLA-LÓPEZ 6, Renato N. INSERRA 7 and Pablo CASTILLO 8 1 Plant Pest Diagnostic Center, California Department of Food and Agriculture, 3294 Meadowview Road, Sacramento, CA 95832, USA 2 Center of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, Leninskii Prospect 33, Moscow 117071, Russia 3 Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (C.N.R.), U.O.S. di Bari, Via G. Amendola 122D, 70126 Bari, Italy 4 Julius Kühn-Institut, Bundesforschungsinstitut für Kulturpflanzen Institut für Epidemiologie und Pathogendiagnostik Toppheideweg 88, 48161 Münster, Germany 5 Agroscope, Institute for Plant Production Sciences, IPS, Schloss 1, P.O. Box, 8820 Wädenswil, Switzerland 6 Department of AgroEcology, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK 7 Florida Department of Agriculture and Consumer Services, DPI, Nematology Section, P.O. Box 147100, Gainesville, FL 32614-7100, USA 8 Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avenida Menéndez Pidal s/n, Apdo. 4084, 14080-Córdoba, Spain Received: 17 September 2014; revised: 28 October 2014 Accepted for publication: 29 October 2014; available online: 2 December 2014

Summary – Morphological identification of spiral of the genus Helicotylenchus is a difficult task because most characters used for their diagnosis vary within species. In this paper we provide morphological and molecular characterisations of several spiral nematodes, H. broadbalkiensis, H. digonicus, H. dihystera, H. microlobus, H. paxilli and H. pseudorobustus, collected in different geographical areas of USA, Switzerland, Italy, New Zealand, Spain, UK, South Korea and Russia. We suggest that H. microlobus and H. pseudorobustus are valid species separated from each other morphologically and molecularly. Seven species with distinct molecular characteristics are also distinguished, but are not ascribed morphologically to any specific taxon because of the low number of specimens available. Phylogenetic relationships of H. pseudorobustus with other Helicotylenchus species are given as inferred from the analyses of 154 sequences of the D2-D3 of 28S rRNA gene and 37 sequences of ITS rRNA gene. Keywords – 28S rRNA gene, Helicotylenchus broadbalkiensis, Helicotylenchus depressus, Helicotylenchus digonicus, Helicotylenchus microlobus, Helicotylenchus paxilli, ITS rRNA gene, morphology, morphometrics, spiral nematodes, .

Spiral nematodes of the genus Helicotylenchus com- H. multicinctus (Cobb, 1893) Golden, 1956 and H. digo- prise numerous species of worldwide distribution occur- nicus Perry, 1959 (Marais, 2001). Helicotylenchus pseu- ring in a variety of habitats. Among the spiral nema- dorobustus is widely distributed in both temperate and todes, H. pseudorobustus (Steiner, 1914) Golden, 1956 is tropical regions and is a root feeder with ecto- or semi- one of the most commonly reported species in the liter- endoparasitic habits. It parasitises many crops and is a ature along with H. dihystera (Cobb, 1893) Sher, 1961, contributing factor to growth suppression of crops con-

† Passed away in August 2012. ∗ Corresponding author, e-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2015 DOI 10.1163/15685411-00002850 S.A. Subbotin et al. comitantly infected by other pathogens or adversely af- i) integrative morphological, morphometric and molecu- fected by nutrient or water imbalance (Fortuner, 1984, lar characteristics of H. pseudorobustus from the type and 1985; Ma et al., 1994; Davis et al., 2004). other localities; ii) morphological and molecular features Since Sher (1966) redescribed H. pseudorobustus from of other species closely related to H. pseudorobustus; and topotypes, many populations of this species have been in- iii) phylogenetic relationships of H. pseudorobustus with correctly identified in various countries (Fortuner et al., other Helicotylenchus species using the D2-D3 of 28S 1981, 1984). Identification of H. pseudorobustus popu- rRNA and ITS of rRNA gene sequences. lations and related species is a difficult, if not impossi- ble, process, because most characters used for their di- Materials and methods agnosis appear to vary within species (Fortuner, 1984). Their discrimination is complicated by their phenotypic POPULATIONS plasticity (intraspecific variability and minor interspecific Nematode populations used in this study were collected differences) leading to potential mis-identification. As a from geographically diverse locations (Table 1). Three result, taxonomic difficulties often arise from under- or Helicotylenchus species were obtained from the type lo- over-estimation of intraspecific variability of certain mor- calities, viz., H. pseudorobustus, H. paxilli Yuen, 1964 phological characters that are currently used for species and H. depressus Yeates, 1967. Additional information diagnosis. The existence in Helicotylenchus and other ge- about the collection sites of H. pseudorobustus popula- nera of plant-parasitic nematodes of species complexes, tions not reported in Table 1 include the GPS coordi- which are morphologically almost indistinguishable but nates of these sites within a radius of 2 km in the Altmatt may be phylogenetically distant from one another, has region, Switzerland, which were: i) 47°0743.9N lati- been revealed by molecular studies and complicates the tude, 08°4158.0E longitude; ii) 47°0756.7N latitude, identification of these nematodes (Oliveira et al., 2006; 08°4131.4E longitude; and iii) 47°0741.4N latitude, Gutiérrez-Gutiérrez et al., 2010; Cantalapiedra-Navarrete 08°4224.0E longitude. The H. pseudorobustus speci- et al., 2013). In recent years, sequences of nuclear ribo- mens from the type locality were used for both morpholo- somal RNA gene (rRNA) have been used for molecular gical and molecular analyses. The topotype specimens of characterisation and reconstruction of phylogenetic rela- H. paxilli and H. depressus were used only for molecular tionships within the Hoplolaimidae and especially within study after verifying that their morphology was congruent Helicotylenchus (Subbotin et al., 2007, 2011; Bae et al., with that of the original description. Samples of H. dihys- 2009; Holterman et al., 2009). tera from Florida (CD359) and H. microlobus from China An attempt to discriminate populations of putative H. (718) and California (CD599) were included for integra- pseudorobustus from a large number of populations of tive morphological and molecular comparisons. related species was made by Subbotin et al. (2011) who analysed sequences of the D2-D3 expansion segments of MORPHOLOGICAL STUDY 28S rRNA gene from 54 Helicotylenchus isolates. Nine Nematode specimens from the soil samples were ex- highly or moderately supported major clades were dis- tracted using the centrifugal-flotation method (Coolen, tinguished by these authors within the genus, including a 1979). Specimens were fixed in hot TAF (no more than well-supported clade I containing populations tentatively 70°C) or 4% formaldehyde + 1% propionic acid. Adult identified as H. pseudorobustus. However, the populations specimens of each sample were processed to glycerin of this species were not unambiguously identified and (Seinhorst, 1962, 1966) and mounted on glass slides for were assembled in groups of four different types: ‘A’, ‘B’, species identification. Nematode specimens were exam- ‘C’ and ‘D’, suggesting that the populations in this com- ined, measured and photographed in two laboratories plex were representatives of different species. The unre- (IAS-CSIC, Spain and CDFA, USA) using compound mi- solved characterisation of H. pseudorobustus populations croscopes Zeiss III compound microscope or Olympus in that study emphasised the need to obtain morpholo- BX51, respectively, equipped with a Nomarski differen- gical and molecular data of topotype specimens of H. tial interference contrast. Slides with topotype specimens pseudorobustus from Switzerland to be used in diagnos- were deposited in the nematode collections of IAS-CSIC, tic works for comparison with those of other populations Spain, and the Julius Kühn-Institut, Germany. of this species from distant geographical areas. In order to Species delimitation of some Helicotylenchus in this reach this objective a study was conducted to determine: and the previous study (Subbotin et al., 2011) was under-

28 Nematology Helicotylenchus pseudorobustus and genus phylogeny (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2011) et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Subbotin P. Castillo C. Blomquist, J. Stack Subbotin G. Yeates Subbotin Subbotin Subbotin Subbotin Subbotin Subbotin S.A. Subbotin, Subbotin Subbotin References or sources HM014259 KM506832, KM506833 KM506827- KM506831 HM014250 KM506849 HM014275 HM014272 HM014267 HM014269 HM014270 M014246 HM014252 HM014277 HM014274 28S rRNA GenBank accession number KM506858 KM506853- KM506855 ITS rRNA D2-D3 of J94round KM506857, CD423 – HM014247 Subbotin 14623 – HM014271, 14619 – HM014270, CD1072 CD603 – HM014245 Subbotin 1463014621 – – HM014273 Subbotin HM014265, 14622 – HM014266, 14620 – HM014268, codes CD556 – HM014300 Subbotin sp. CD359 – HM014258, Ficus Schefflera Amorpha Ceratonia siliqua Amorpha Ficus benjamina Solanum Koeleria Panicum virgatum Koeleria Panicum virgatum – CD706 – KM506848, Grassesarboricola CD864, canescens pyramidata pyramidata canescens mauritianum USA, Florida, Lake Worth USA, Nebraska, Lincoln USA, Florida, Ft. Lauderdale USA, Nebraska, Lincoln USA, Florida, Ft. Pierce USA, Hawaii, Kawai Grasses CA152 – HM014243, USA, Hawaii, KawaiUSA, Georgia, Athens Grasses – CA157 14627 – – HM014248 Subbotin HM014249, USA, Florida, Goulds Bromeliads CD508 – HM014242, South Africa, Magaliesberg (type locality) Washington Marlatt park USA, Kansas, Manhattan USA, Nebraska, Lincoln USA, Kansas, Manhattan USA, Nebraska, Lincoln LocationUSA, California, Death Valley Grasses Plant hosts Sample CD363 KM506856 HM014276, populations and species obtained for the present study and used for the phylogenetic analysis. – Spain, Jerez de la Frontera – New Zealand, Birdling Flat – USA, Kansas, Manhattan, spI-5 type B type C type D Previous identification spI-9 Helicotylenchus H. dihystera H. dihystera H. depressus H. digonicus H. dihystera H. dihystera H. digonicusH. digonicus Helicotylenchus H. platyurus H. digonicus H. digonicus H. digonicus H. pseudorobustus H. dihystera H. dihystera H. dihystera H. dihystera H. broadbalkiensis H. digonicus H. leiocephalus H. digonicus H. pseudorobustus H. dihysteraH. dihystera H. dihystera H. dihystera Table 1. Identification based on the result of the present study H. brevis H. brevis H. dihystera H. dihystera H. broadbalkiensis Helicotylenchus

Vol. 17(1), 2015 29 S.A. Subbotin et al. (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2011) (2006) (unpubl.) (unpubl.) (2009) et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Subbotin (unpubl.) S.A. Subbotin Subbotin Subbotin Subbotin Subbotin References or sources Subbotin Subbotin Subbotin HM014244, HM014254 KM506840 HM014262 HM014253 HM014254 HM014262 HM014260 28S rRNA HM014296 HM014298 GenBank accession number KM506884, KM506886 ITS rRNA D2-D3 of – – KF443217 Xiao – – KF486503 Xiao 14625 – HM014293, CD1391 – KM506834 S.A. Subbotin codes 14631 – HM014295, sp. CD616 KM506885 HM014261, sp. CD616 – HM014261, sp. CD385 – HM014256, sp. – FJ440620 – Rahman Musa Musa paradisiaca Neoregelia Musa paradisiaca Schizachyrium Neoregelia Psidium Vitis vinifera Grasses CD1215 – KM506836 S.A. Subbotin scoparium Republic of ChinaMalaysia – DQ309585 – Cheng USA, Kansas, Manhattan USA, Hawaii, Maui – CD617 KM506883, USA, Florida, Homestead Bromeliads CD600 – HM014257 Subbotin USA, Hawaii, KawaiUSA, Georgia, MidvilleChina, Fujia China, Zhang Zhou Grasses – CA150 GAC177 – – HM14255 Subbotin FJ485651 Bae USA, Florida, Apopka USA, Georgia, Athens – 14626 – HM014251, USA, Hawaii, Maui – CD617 – HM014244, USA, Kansas, Manhattan Wasco county, Atwater USA, Florida, Apopka South Africa, Nelspruit Location Plant hosts Sample – USA, North Carolina – CD1431 – KM506837 W. Ye ––––– USA, Florida, Homestead USA, Florida, Homestead Bromeliads USA, Texas (plant shipment) USA, California, Kern county, Bromeliads – USA, California, CD703 Merced CD894 – – CD1067 KM506839, – KM506838 S.A. Subbotin KM506835 S.A. Subbotin Previous identification (Continued.) H. dihysteraH. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. labiodiscinus H. labiodiscinus H. dihystera H. dihystera H. dihysteraH. dihysteraH. dihysteraH. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera H. dihystera Table 1. Identification based on the result of the present study H. dihystera H. dihystera H. labiodiscinus H. labiodiscinus

30 Nematology Helicotylenchus pseudorobustus and genus phylogeny (2007) (2007) (2007) (2011) (2011) (2011) (unpubl.) (2009) et al. et al. et al. et al. et al. et al. et al. et al. N. Vovlas S.A. Subbotin Subbotin Subbotin Subbotin References or sources Subbotin S.A. Subbotin KM506805 KM506799, KM506802 KM506793 N. Vovlas KM506797, DQ328749 HM014305 H01464264 HM014297 28S rRNA KM506798 GenBank accession number KM506860 KM506866 KM506864 ITS rRNA D2-D3 of J94typ KM506862 KM506806 P. Castillo ST20/J238 – KM506795 P. Castillo Bari –CD989Palag KM506861 KM506804Locubin KM506803, G. Tylka KM506865, KM506867 KM506796 P. Castillo CD599 – H01464263, codes 14629 – HM014294, europaea sp. CD1559 – KM506800 V.N. Chizhov Ceratonia siliqua Olea europaea Olea europaea Andropogon subsp. Acer GrassesZea mays CD743 KM506859, Zea mays Vitis vinifera Populus nigra Unknown tree CD740 KM506863, Turfgrass ILC171 – FJ485649 Bae gerardii – CD1560 – KM506801 R.V. Khusainov China, Linfen – – GQ906356 – Zheng Fairmount Park Ames USA, Kansas, Manhattan province, Palagiano de Locubín Fairmount Park USA, Illinois, University of Illinois South Africa, Lambani Grasses CD613 – HM014304, USA, California, Fresno – CA4 – DQ328748 Subbotin USA, California, Fresno Italy, AnconaChina, Beijing – – 727 718 – – DQ328750 Subbotin DQ328747, Location Plant hosts Sample Anapa –– USA, California, Riverside, USA, Iowa, Story county, – Italy, Bari – Spain, Jerez de la Frontera ––––– Italy, Apulia region, Tarento Spain, Jaén province, Castillo Spain, Jaén province, Andújar USA, Arizona USA, California, Riverside, Grasses CD1327 – KM506794 S.A. Subbotin type B type B type B type B type B – Russia, Saratov region, Engels Previous identification – Russia, Krasnodar Territory, (Continued.) H. microlobus H. microlobus H. microlobus H. microlobus H. microlobus H. microlobus H. microlobus H. microlobus H. microlobus H. microlobus H. crenacauda H. microlobus H. pseudorobustus H. microlobus H. pseudorobustus H. microlobusH. microlobus H. pseudorobustus H. microlobus H. pseudorobustus H. pseudorobustus H. martini H. martini Table 1. Identification based on the result of the present study H. labiodiscinus H. labiodiscinus H. microlobus H. microlobus

Vol. 17(1), 2015 31 S.A. Subbotin et al. (2011) (2006) (2011) (2007) (unpubl.) et al. et al. et al. et al. (2011) (2011) et al. G. Yeates, Subbotin et al. Manzanilla-López R.N. Inserra J. Hallmann, S. Kiewnick G. Yeates, Subbotin et al. Subbotin Subbotin References or sources S.A. Subbotin KM506819, HM014278, KM506809, KM506822 KM506824 R.N. Inserra KM506825, KM506826 KM506811, KM506815, KM506817 HM014279, HM014280 HM014291 DQ328746 28S rRNA KM506820 GenBank accession number – KF443214 Xiao KM506875 KM506878, KM506879 KM506871 KM506873 KM506880- KM506882 KM506877s ITS rRNA D2-D3 of CD423 – HM014292 Subbotin CH256 KM506876, N453; CD428, CD704 CD694 KM506872, CD794; CD2, CD795; CH4, CD796 codes , sp. CD511 – HM014290, sp. – – DQ328745, Ficus benjamina Musa paradisiaca Musa Paspalum Lolium perenne Musa Grasses CD785 KM506874, Lolium perenne Trifolium repens Unknown plant CD1538 KM506868 – R.H. Grasses CD1227 KM506870 KM506823 S.A. Subbotin vaginatun Moss/swamp CH1, Grasses CD881 – KM506816 S.A. Subbotin – CD914 – KM506813 S.A. Subbotin USA, Florida, Ft. Pierce China, Fujia South Africa, Lambani Germany, Münster, BBA – 590 – DQ328751 Subbotin county Sudan New Zealand, MAF farm, Kaitoke Research, Broadbalk Wilderness (type locality) county, Friant locality) New Zealand, Rotorua Location Plant hosts Sample Tomales county – USA, California, Mendocino type A – Switzerland, Altmatt (type – UK, Harpenden, Rothamsted – USA, California, Fresno type A –– USA, Florida, Hague USA, Florida, Osteen – CD1066 KM506869, type A – USA, California, Marin county, – USA, Washington, Clallam – Germany, Münnerstadt – CD975 – KM506814 J. Hallmann Previous identification (Continued.) H. pseudorobustus H. pseudorobustus H. pseudorobustus H. pseudorobustus H. pseudorobustus H. paxilli H. paxilli H. pseudorobustus H. paxilli H. paxilli H. pseudorobustus H. pseudorobustus H. multicinctusH. multicinctus H. multicinctus H. multicinctus H. multicinctus H. multicinctus Table 1. Identification based on the result of the present study H. multicinctus H. multicinctus H. pseudorobustus H. pseudorobustus H. pseudorobustus

32 Nematology Helicotylenchus pseudorobustus and genus phylogeny (2011) (2011) (2011) (2011) (2011) (2007) (2005) (unpubl.) (2009) (2009) et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Subbotin Subbotin Subbotin Subbotin Subbotin References or sources HM014288 HM014286 HM014282 HM014239 DQ328761 28S rRNA GenBank accession number ITS rRNA D2-D3 of – – AB602602 Pang CA175 – HM014287, CA172 – HM014285, 14628 – HM014281, codes FLC180 – FJ485648 Bae sp. CD601 – HM014289 Subbotin sp. CD1558 – KM506807 V.N. Chizhov Cynodon dactylon Stenotaphrum Vigna unguiculata Vigna unguiculata Andropogon bladhi Agave Grasses CD1234 – KM506810 S.A. Subbotin Grasses CD1242 – KM506818 S.A. Subbotin Grasses CD1250 – KM506808 S.A. Subbotin Grasses CD1276 – KM506812 S.A. Subbotin Grasses CD1291 – KM506821 S.A. Subbotin Acer secundatum USA, Florida, Ft. Lauderdale Burkina Faso, Farako-Ba USA, CaliforniaBurkina Faso, Leguema – 4I22G4 – DQ077794 De Ley USA, Kansas, Manhattan Obispo county, Paso Robles Location Plant hosts Sample county, Santa Nella county, Salinas Novato county, El Cajon Myschkin USA, Florida, Goulds sp. USA, Florida, Ft. Lauderdale type B South Africa, Lambani Grasses CD620 – HM014238, type A Italy, Ancona – AI36, AI13 – DQ328759- type A USA, Arkansas – KrC210 – FJ485650 Bae Helicotylenchus spIII-1 Helicotylenchus spIII-1 Helicotylenchus spI-10 Helicotylenchus Helicotylenchus spIII-1 H. vulgaris – USA, California, San Luis – USA, California, Merced – USA, California, Monterey Helicotylenchus spI-8 – USA, California, Marin county, – USA, California, San Diego –H. vulgaris Russia, Jaroslavl region, H. vulgaris Previous identification Helicotylenchus spIII-2 – USA, Florida (shipment) Soil CD901 – KM506846 S.A. Subbotin type B type A type A (Continued.) spIII-1 spIII-1 spI-10 spI-11 spIII-1 spI-8 spIII-2 spIII-3 Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus H. vulgaris Table 1. Identification based on the result of the present study H. pseudorobustus H. pseudorobustus H. pseudorobustus H. pseudorobustus H. pseudorobustus H. pseudorobustus H. vulgaris Helicotylenchus Helicotylenchus H. vulgaris

Vol. 17(1), 2015 33 S.A. Subbotin et al. (2007) (2007) (2007) (2007) (2011) (2011) (2011) (2011) (2011) et al. et al. et al. et al. et al. et al. et al. et al. et al. S.A. Subbotin R. Inserra Subbotin Subbotin References or sources KM506851 KM506845 HM014241 HM014303 28S rRNA GenBank accession number ITS rRNA D2-D3 of CD951 – KM506792 S.A. Subbotin CD382 – HM014240, codes sp. CD761 – KM506844, sp. CD505 – HM014302, Vitis vinifera Glycine max Juglans Potato CD567 – KM506850, Grasses CD1052 – KM506852 S.A. Subbotin Calathea Grasses– CD1051 – CD1013 KM506841 S.A. Subbotin – KM506843 S.A. Subbotin Italy – 723 – DQ328758 Subbotin Russia, Moscow region – RU21 – DQ328755 Subbotin Belgium, Ghent – LG43 – DQ328754 Subbotin USAcounty, Brawley – CD347 – HM014301 Subbotin Strawberry canyon USA, California, Winters South Africa, Koedoeskop LocationUSA, Hawaii, KawaiUSA, Hawaii, Kawai Plant hosts GrassesWinter garden Grasses Sample CA157 CA150 – – HM014284 Subbotin HM014283 Subbotin Strawberry canyon UCR campus sp. Russia, Moscow region – – – DQ328734 Subbotin H. digonicus type A – USA, California, Berkley, Helicotylenchus spIX-4 Helicotylenchus spIX-3 –Helicotylenchus spVII-1 – South Korea USA, California, Imperial – CD1383 – KM506847 S.A. Subbotin –Helicotylenchus USA, California, Woodland –Helicotylenchus spIX-1 USA, California, Berkley, H. digonicus type C Previous identification Helicotylenchus spIV Helicotylenchus spIV – USA, Florida, Gainesville, –– USA, California USA, California, Riverside, – CD874 – KM506842 S.A. Subbotin (Continued.) spIX-6 spX-1 spIX-4 spIX-3 spVII-1 spV-2 spVII-2 spIX-1 spIX-2 spVII-3 spIX-1 spV-1 spIV-1 spIV-1 spIV-2 spX-1 spX-1 Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Table 1. Identification based on the result of the present study Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus Helicotylenchus

34 Nematology Helicotylenchus pseudorobustus and genus phylogeny taken using an integrated approach that considered mor- analysis. The topologies were used to generate a 50% phological and morphometric evaluation combined with majority rule consensus tree. Posterior probabilities (PP) molecular based phylogenetic inference (tree-based meth- are given on appropriate clades. Sequence analyses of ods) and sequence analyses (genetic distance methods) alignments were performed with PAUP∗ 4b10 (Swofford, (Sites & Marshall, 2004). Clade numbers on a phyloge- 2003). Pairwise divergences between taxa were computed netic tree were used for specific coding of unidentified as absolute distance values and as percentage mean dis- species. tance values based on whole alignment, with adjustment for missing data. DNA EXTRACTION,PCRAND SEQUENCING For molecular analyses, DNA was extracted from sev- Results eral specimens of each population using the proteinase K protocol (Tanha Maafi et al., 2003). DNA extraction, PCR Within the pool of spiral nematodes studied, seven valid and cloning protocols were as described by Tanha Maafi Helicotylenchus species were distinguished by integrating et al. (2003). The forward D2A (5-ACA AGT ACC GTG   the results of their morphological and molecular analyses. AGG GAA AGT TG-3 ) and the reverse D3B (5 -TCG The remaining populations were considered representa- GAA GGA ACC AGC TAC TA-3) primers (Subbotin  tives of seven undetermined taxa since these populations et al., 2007) and the forward TW81 (5 -GTT TCC GTA did not contain sufficient number of specimens for their GGT GAA CCT GC-3) and the reserve AB28 (5-ATA  morphological identification and determination of their TGC TTA AGT TCA GCG GGT-3 ) (Tanha Maafi et al., taxonomic status. The valid identified species were: H. 2003) were used for amplification of the fragment of D2- broadbalkiensis, H. depressus, H. digonicus, H. dihystera, D3 regions of the 28S rRNA and the ITS of rRNA genes, H. microlobus, H. paxilli and H. pseudorobustus. These respectively. PCR products of some samples were puri- samples included topotype populations of H. pseudoro- fied using QIAquick (Qiagen) gel extraction kits and then bustus, H. paxilli and H. depressus and non-type popu- cloned using pGEM-T Vector System II kit (Promega). lations from distant geographical areas fitting the char- Sequences were obtained directly from PCR products or acteristics of each of these species. The undetermined from one or more clones. The newly obtained sequences species were indicated as: Helicotylenchus spIII-3, Heli- were submitted to the GenBank database under acces- cotylenchus spIV-2, Helicotylenchus spV-2, Helicotylen- sion numbers (KM506792-KM506886) as indicated in chus spVII-2, Helicotylenchus spVII-3, Helicotylenchus Table 1. spIX-1 and Helicotylenchus spX-1. Brief morphological descriptions with illustrations PHYLOGENETIC ANALYSIS (Figs 1-8) and morphometric values (Tables 2-5) are given The newly obtained sequences for D2-D3 of 28S rRNA and ITS rRNA were aligned using ClustalX 1.83 (Thomp- son et al., 1997) with default parameters with their corre- sponding published gene sequences (De Ley et al., 2005; Chen et al., 2006; Subbotin et al., 2007, 2011; Zhao et al., 2010; Pang et al., unpubl.). Outgroups for each dataset were selected as proposed in the previous study (Sub- botin et al., 2011). Sequence datasets were analysed with Bayesian inference (BI) using MrBayes 3.1.2 (Huelsen- beck & Ronquist, 2001) under the GTR + G + I model. BI analysis for each gene was initiated with a random starting tree and was run with four chains for 1 × 106 generations. The Markov chains were sampled at inter- vals of 100 generations. Two runs were performed for each analysis. The log-likelihood values of the sample points stabilised after approximately 1000 generations. After discarding burn-in samples and evaluating conver- Fig. 1. Entire view of Helicotylenchus pseudorobustus (CD1250, gence, the remaining samples were retained for further CA, USA). (Scale bar = 20 μm.)

Vol. 17(1), 2015 35 S.A. Subbotin et al.

Fig. 2. Anterior region of Helicotylenchus spp. A-E: H. pseudorobustus (CD1250, California, USA); F-J: H. microlobus (F: CD599, California, USA; G-J: CD989, Iowa, USA); K, L: H. broadbalkiensis (CD363, California, USA); M-O: H. digonicus (Kansas, USA). (Scale bar = 10 μm.) for H. pseudorobustus and the closely related species H. unidentified species: Helicotylenchus spIV-2 (Fig. 6C, D), microlobus and H. paxilli. Additional descriptions are pro- Helicotylenchus spVII-2 (Figs 3F-H; 6B) and Helicoty- vided for H. digonicus and H. broadbalkiensis. Micropho- tographs of H. dihystera (Figs 3I-O; 6E-J) and three lenchus spX-1 (Figs 3E; 6A), are also included.

36 Nematology Helicotylenchus pseudorobustus and genus phylogeny

Fig. 3. Anterior region of Helicotylenchus spp. A: H. pseudorobustus (CD795, New Zealand); B: H. pseudorobustus (CD1276, California, USA); C: H. paxilli (CD694, Florida, USA); D: H. broadbalkiensis (J94round, Spain); E: Helicotylenchus spX-1 (CD1051, California, USA); F-H: Helicotylenchus spVII-2 (CD567, California, USA); I: H. dihystera (CD359, Florida, USA); J: H. dihystera (CD1067, Texas, USA); K-O: H. dihystera (CD617, Hawaii, USA). (Scale bar = 10 μm.)

Vol. 17(1), 2015 37 S.A. Subbotin et al.

Fig. 4. Posterior region of Helicotylenchus spp. A, B: H. pseudorobustus (CD795, Switzerland); C, D: H. pseudorobustus (CD704, New Zealand); E: H. pseudorobustus (CD1276, California, USA); F-J: H. pseudorobustus (CD1250, CA, USA); K: H. microlobus (CD599, California, USA); L-O: H. microlobus (CD989, Iowa, USA); P, Q: H. microlobus (Locubin, Spain); R: H. microlobus (718, China); S, T: H. microlobus (CD740, California, USA). (Scale bar = 10 μm.)

Helicotylenchus pseudorobustus (Steiner, 1914) Female Golden, 1956 Body shape usually spiral to closed C-shape when (Figs1;2A-E;3A,B;4A-J) heat-relaxed. Lip region hemispherical with four or five annuli. Stylet well developed, knobs flattened in 98% of MEASUREMENTS specimens, rounded or indented anteriorly in remaining See Table 2. 2%. Excretory pore at about level of pharyngo-intestinal

38 Nematology Helicotylenchus pseudorobustus and genus phylogeny

Fig. 5. Posterior region of Helicotylenchus spp. A, B: H. broadbalkiensis (CD363, California, USA), C-E: H. broadbalkiensis (J94round, Spain); F-J: H. digonicus (CD864, Kansas, USA); K-M: H. paxilli (CD1538, UK); N, O: H. paxilli (CD694, Florida, USA); P-T: H. paxilli (CD1066, FL, USA). (Scale bar = 10 μm.) junction. Spermatheca empty, offset. Lateral field with proportion. Tail longer than anal body diam., dorsally areolation in both pharyngeal and tail regions. Pharyngeal convex-conoid, usually distinctly annulated and bearing gland overlapping intestine ventrally. Anal body diam. 5-10 ventral annuli, with pronounced ventral projection usually 15 μm. Phasmids 5-10 annuli anterior to anus usually rounded terminally and lacking a mucro. level. Junction of inner two incisures of lateral field on tail end variably shaped. The U- and M-shaped Male patterns (Fortuner et al., 1984) were observed in equal Not found.

Vol. 17(1), 2015 39 S.A. Subbotin et al.

Fig. 6. Posterior region of Helicotylenchus spp. A: Helicotylenchus spX-1 (CD1051, California, USA); B: Helicotylenchus spVII-2 (CD567, California, USA); C, D: Helicotylenchus spIV-2 (CD761, Florida, USA); E: H. dihystera (CD1067, Texas, USA); F-J: H. dihystera (CD617, Hawaii, USA). (Scale bar = 10 μm.)

Fig. 7. Helicotylenchus paxilli (CD694, Florida, USA). A: Entire body; B: Lip region; C-I: Tail variations; J: Lateral field at tail region. (Scale bar: B-J = 10 μm.)

40 Nematology Helicotylenchus pseudorobustus and genus phylogeny

Fig. 8. Helicotylenchus microlobus (J94typ, Spain). A: Entire body; B, C: Lip region; D-F: Tail variations; G: Lateral field at tail region. (Scale bar: B-G = 10 μm.)

REMARKS Helicotylenchus microlobus Perry in Perry, Darling & Thorne, 1959 (Figs 2F-J; 4K-T; 8) The congruency of the molecular features of the popu- lations studied with those of the topotype populations MEASUREMENTS from Switzerland validates their identification as repre- sentatives of H. pseudorobustus. Morphometrics of H. See Tables 3, 4. pseudorobustus populations from New Zealand and USA Female were similar to those of topotype specimens from Switzer- Body in spiral shape when heat-relaxed. Lip region land and other populations from the same locality in hemispherical with 4-5 annuli. Stylet knobs indented New Zealand as published previously by Yeates & Wouts anteriorly in California population (CD 599) and rounded (1992). The mean tail length of the topotype specimens in Iowa population (CD 989). Lateral field not areolated was greater (17.9 μm) than that (15.9 μm) reported by on tail. Pharyngeal gland overlapping intestine ventrally Fortuner et al. (1984), confirming the morphological vari- or ventrolaterally. Inner two incisures of lateral field ability observed by these authors in topotype specimens usually fusing distally for ca two annuli. Cumulative of this species. means of anal body diam. ranging from 12.7 to 14.4 μm

Vol. 17(1), 2015 41 S.A. Subbotin et al. s.d. ± 2 (8-14) 0.7 (2.5-5.0) 1.4 (9.0-12.5) 0.4 (5.1-6.3) 0.1 (0.9-1.2) 0.7 (6.0-7.5) 4.6 (135-150) 5.5 (105-122) 4.4 (145-160) 0.5 (11.0-12.5) 0.7 (25.0-26.0) 1.5 (16.0-20.0) 2.2 (56.4-62.5) 3.6 (38.0-46.9) 1.9 (16.0-22.5) 3.5 (77.5-87.5) 1.8 (27.1-31.8) 2.0 (58.4-64.6) 0.04 (3.5-4.0) 1.7 (26.0-31.0) ± 58.5 (712-895) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± m and in the form: mean μ 0.8 (4-6) – 1.7 (10-15) 11 0.4 (2.5-3.5) 3.8 0.3 (6.0-7.0) – 0.2 (3.0-4.0) 3.7 0.4 (5.2-6.4) 5.7 0.1 (1.0-1.3) 1.1 0.3 (5.5-6.5) 7.1 7.1 (122-144) 141 3.0 (112-122) 116 8.4 (137-162) 153 0.6 (11.0-12.5) 11.5 0.6 (25.0-26.0) 25.6 1.0 (15.0-18.0) 18.1 1.3 (64.3-68.0) 59.5 2.9 (35.2-44.6) 43.4 1.3 (17.5-22.0) 18.8 3.3 (82.5-92.5) 84.1 1.4 (61.0-64.4) 61.7 1.1 (28.2-31.4) 28.5 ± 23.4 (705-780) 809 ± 0.7 (24.0-26.0) 28.4 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.6 (9-14) 12.5 1.6 (5-10) 4.7 0.6 (2.0-3.5) 3.0 0.4 (7.5-8.0) – 10.7 0.1 (6.0-6.5) 6.7 0.1 (3.5-4.0) 3.6 0.4 (5.4-6.4) 5.9 0.1 (0.9-1.3) 1.2 0.5 (5.5-7.0) 6.1 4.5 (112-127) 129 5.4 (104-121) 118 5.2 (130-147) 146 0.6 (12.5-14.0) 12.1 0.8 (25.0-27.5) 25.9 1.1 (15.0-19.0) 16.4 2.3 (60.0-68.0) 66.9 3.6 (35.5-45.7) 39.3 1.7 (16.0-21.0) 19.3 4.6 (71.3-85.0) 86.6 1.4 (59.6-63.8) 62.7 1.3 (26.2-30.2) 30.3 1.2 (23.0-27.5) 25 ± 51.3 (635-800) 756 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.9 (5-7) 7.7 1.9 (8-13) 11.8 1.3 (1.5-4.4) 2.7 0.1 (6.0-6.5) 6.3 0.3 (3.5-4.5) 3.8 0.6 (5.8-7.3) 5.8 0.1 (1.0-1.3) 1.1 0.1 (6.0-6.5) 6.3 8.6 (100-119) 121 0.2 (12.0-13.0) 12.9 0.5 (25.0-26.5) 26.7 1.2 (15.0-17.5) 16.4 1.5 (61.7-64.7) 65.2 4.3 (34.3-45.8) 39.8 1.2 (16.0-19.0) 17.8 8.3 (62.5-80.0) 79.1 2.0 (58.4-63.6) 61.6 1.2 (25.3-28.3) 27.4 1.7 (25.0-29.0) 25.8 ± ± 13.4 (116-144) 139 11.4 (81-112) 113 47.3 (642-778) 707 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± populations analysed in the present study. All measurements are in 1.3 (5-9) 6.0 1.5 (10-15) 10.5 0.7 (2.5-4.5) 2.9 0.1 (6.0-6.5) 6.4 0.1 (3.5-4.0) 3.8 0.3 (5.4-6.1) 6.5 0.1 (1.1-1.4) 1.1 0.3 (6.0-7.0) 6.2 8.7 (134-159) 132 6.0 (116-137) 107 5.5 (105-125) 96 0.2 (12.0-13.0) 12.5 0.6 (26.0-27.5) 26.1 1.0 (15.0-17.5) 16.0 2.2 (64.5-71.4) 62.9 2.3 (16.0-24.0) 17.3 3.1 (81.3-93.8) 68.4 1.0 (58.8-61.9) 60.8 1.5 (26.8-30.7) 26.4 0.7 (25.0-27.0) 27.1 ± 36.1 (692-805) 713 ± 4.4 (31.2-45.7) 41.5 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.9 (7-9) 6.6 1.8 (9-14) 12.5 0.7 (2.5-4.5) 3.2 0.4 (6.5-7.5) 6.4 0.3 (4.0-4.4) 3.8 0.6 (5.4-6.9) 5.7 0.1 (1.0-1.3) 1.2 0.6 (5.5-7.5) 6.4 8.7 (136-156) 146 7.9 (112-134) 131 3.9 (105-115) 118 0.8 (26.5-29.0) 26.9 0.7 (14.0-16.0) 16.3 4.6 (35.1-46.2) 37 4.7 (58.9-69.5) 67.3 1.9 (15.0-20.0) 20.3 5.5 (68.8-83.8) 88.3 1.5 (60.3-65.3) 60.7 1.6 (25.2-29.7) 28.7 0.9 (25.0-27.5) 25.9 ± ± 53.2 (675-817) 742 Helicotylenchus pseudorobustus 0.4 (12.5-14.0) 12.6 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Switzerland/ California, USA/ California, USA/ New Zealand/ Washington, USA/ California, USA/ ± ± CD796 (Type) CD785 CD881 CD704 CD914 CD1250 1.1 6.4 26.9 ∗ ∗ Morphometrics of Measured at vulval region.  DGO – – – 7.8 Pharynx 122 Pharyngeal glands 144 Conus length 13 Phasmid position 7.9 Tail annuli number 12.9 c Ventral peg length 3.4 Max. body diam. Stylet length 27.3 c 41.4 Lip diam. 6.8 Anal body diam. 15.7 MB 65.6 b6.0 Lip height 4.0 Lateral field width (range). a 27.2 V 62.6 Tail length 17.9 Median bulb 78.5 Table 2. CharacternL 71061089 734 Population/Code ∗ Excretory pore 111

42 Nematology Helicotylenchus pseudorobustus and genus phylogeny

Table 3. Morphometrics of Helicotylenchus microlobus populations analysed in the present study. All measurements are in μmandin the form: mean ± s.d. (range).

Character Population/Code Iowa, USA/CD989 California, USA/CD740 California, USA/CD743 China/718 n51256 L 783 ± 18.7 (752-797) 739 ± 44.5 (657-795) 704 ± 54.7 (642-780) 622 ± 55.5 (555-687) a 29.9 ± 1.1 (28.7-31.1) 26.6 ± 1.1 (24.8-28.5) 27.9 ± 1.4 (25.7-29.7) 25.9 ± 1.2 (24.7-27.5) b6.3± 0.1 (6.1-6.3) 5.2 ± 0.2 (4.7-5.5) 5.4 ± 0.3 (5.1-5.8) 4.7 ± 0.5 (4.4-5.3) c 48.5 ± 4.2 (43.9-54.7) 34.8 ± 2.0 (32.1-38.9) 32.7 ± 3.9 (28.3-38.9) 37.9 ± 4.8 (32.9-47) c 1.1 ± 0.2 (0.9-1.3) 1.3 ± 0.1 (1.2-1.5) 1.6 ± 0.2 (1.3-1.8) 1.2 ± 0.2 (0.9-1.4) V 60.1 ± 2.0 (57.9-62.2) 62.6 ± 1.4 (60.4-64.8) 61.9 ± 1.3 (60.4-63.6) 63.2 ± 1.7 (61.8-66.2) Lip height 4.4 ± 0.4 (4.0-5.0) 4.2 ± 0.4 (4.0-5.0) 3.6 ± 0.2 (3.0-4.0) 3.7 ± 0.1 (3.5-4.0) Lip diam. 6.7 ± 0.2 (6.5-7.0) 6.5 ± 0.2 (6.0-7.0) 6.3 ± 0.1 (6.0-6.5) 6.3 ± 0.2 (6.0-6.5) Stylet length 26.9 ± 0.6 (26.0-27.5) 28.9 ± 0.7 (27.5-30.0) 26.9 ± 0.6 (26.0-27.5) 25.3 ± 0.4 (25.0-25.5) Conus length 12.6 ± 0.2 (12.5-13.0) 13.2 ± 0.4 (12.5-14.0) 12.6 ± 0.3 (12.5-13.0) – Pharynx length 127 ± 3.1 (125-131) 140 ± 6.0 (125-145) 127 ± 3.1 (125-131) 128 ± 3.1 (125-131) Pharyngeal glands 148 ± 4.3 (144-154) 171 ± 9.0 (154-181) 147 ± 9.1 (139-163) 139 ± 2.7 (138-141) Excretory pore 110 ± 3.8 (105-115) 128 ± 6.3 (114-138) 117 ± 6.1 (110-125) 100 ± 6.3 (94-109) MB 62.5 ± 2.5 (59-65) 67.3 ± 2.1 (64.7-71.8) 66.9 ± 0.8 (66-68) 57.7 ± 1.2 (56.9-59) Max. body diam.∗ 26.3 ± 0.9 (25.0-27.5) 27.8 ± 1.2 (26.0-30.0) 25.3 ± 1.0 (24.0-26.0) 23.9 ± 1.7 (22.5-26.5) Lateral field width∗ 6.1 ± 0.3 (5.5-6.5) 6.3 ± 0.2 (6.0-7.0) 5.7 ± 0.4 (5.5-6.5) 5.9 ± 0.5 (5.0-6.0) Tail length 16.3 ± 1.7 (14.0-18.0) 21.3 ± 1.7 (19.0-23.0) 21.8 ± 3.0 (19.0-25.0) 16.6 ± 2.2 (12.5-19.0) Anal body diam. 14.3 ± 0.5 (14.0-15.0) 16.0 ± 1.0 (15.0-17.5) 13.8 ± 1.3 (12.5-15.0) 14.2 ± 0.6 (14.0-15.0) Ventral peg length 3.8 ± 1.2 (2.0-5.0) 6.3 ± 1.1 (4.5-7.5) 4.5 ± 0.6 (4.0-5.0) 4.6 ± 0.5 (4.0-5.0) Tail annuli number 9.6 ± 0.5 (9-10) 10.8 ± 1.3 (9-13) 12 ± 1.2 (10-13) 10.6 ± 2.0 (8-13) Phasmid position 7.2 ± 0.8 (6-8) 6.2 ± 1.3 (5-8) 2.4 ± 1.7 (1-5) 2.5 ± 2.1 (1-4)

∗ Measured at vulval region. in eight populations. Nonetheless, anal body diam. in figuration, phasmids situated consistently at or close to a population from California (CD 740) was 16 μm. the inner ventral incisures, and tail projection not annu- Phasmids 1-8 annuli anterior to anus level. Tail longer lated or marked by lines). Sauer & Winoto (1975), For- than anal body diam., bearing 6-13 ventral annuli smaller tuner et al. (1984) and Firoza & Maqbool (1994) ac- than those on rest of body. Tail with pronounced ventral cepted the synonymisation proposed by Sher (1966) be- projection, usually rounded terminally, mucro absent. cause of the morphological variability observed in popu- Distal half of tail either without annulation or indistinctly lations of H. pseudorobustus from different origins, but annulated with marked curvature on dorsal contour. Krall (1978) and Siddiqi (2000) listed H. microlobus as a valid species. Taking into consideration the results of Male our integrative morphological and molecular analyses of Not found. the American and European populations previously iden- tified as H. pseudorobustus type B or other species, we REMARKS consider these populations as representatives of H. mi- crolobus rather than H. pseudorobustus or another taxon. Sher (1966) synonymised H. microlobus with H. pseu- Our data clearly indicates that these two species are valid dorobustus because he could not morphologically distin- and morphologically and molecularly different. Our de- guish the paratypes of both species. However, Siddiqi cision is supported by the results of multivariate analy- (1972) recognised both species as valid and proposed sev- sis of 41 populations of Helicotylenchus species, includ- eral characteristics to differentiate them (viz., lateral field ing H. microlobus topotypes published by Fortuner et al. not areolated on the tail, inner incisures of the lateral field (1984), which showed that H. microlobus topotypes clus- fused distally for about two annuli in a Y-shaped con- tered separately from H. pseudorobustus. Both the Amer-

Vol. 17(1), 2015 43 S.A. Subbotin et al. 0.6 (6-7) 1.2 (5-9) 0.8 (9.0-12.0) 0.6 (3.0-5.0) 0.2 (4.3-5.0) 0.8 (6.5-8.0) 0.5 (5.0-6.0) 0.1 (1.0-1.4) 0.4 (3.5-5.5) 4.7 (110-126) 7.3 (134-158) 5.6 (104-126) 0.4 (12.0-13.0) 4.8 (40.3-54.9) 0.7 (25.0-28.0) 0.8 (11.0-14.0) 2.4 (25.1-32.9) 1.8 (12.0-18.0) 1.2 (59.0-63.0) 2.2 (50.9-59.6) 2.0 (22.0-29.0) ± ± 43.3 (609-758) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± m and in the form: μ 1.0 (6-8) 6.3 1.4 (8-12) 7.4 1.3 (8.5-14.0) 10.6 0.6 (2.0-4.0) 3.6 0.4 (4.3-5.8) 4.6 0.5 (6.0-7.0) 7.2 0.5 (5.0-6.0) 5.5 0.1 (1.1-1.4) 1.2 0.3 (5.0-6.0) 4.6 5.2 (111-131) 115 6.6 (139-164) 148 4.5 (109-124) 113 0.5 (12.0-14.0) 12.4 3.9 (37.2-49.5) 45.4 0.8 (25.0-28.0) 26.6 1.1 (12.5-16.0) 12.7 1.5 (25.2-31.0) 27.7 1.6 (15.0-21.0) 15.3 1.6 (59.0-63.0) 61.4 1.9 (51.7-58.1) 53.9 2.3 (22.0-31.0) 24.9 ± 63.5 (627-828) 687 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.4 (4-7) 6.8 1.3 (9-13) 9.9 7.8 (96-120) 120 0.6 (3.0-5.0) 3.1 0.5 (4.0-5.8) 4.9 0.5 (6.5-7.5) 6.5 0.5 (4.0-5.0) 5.5 0.1 (1.2-1.6) 1.2 0.8 (4.5-7.5) 5.2 9.1 (133-167) 152 4.1 (103-115) 118 0.6 (12.0-14.0) 12.9 0.9 (10.0-13.0) 11.4 2.5 (31.1-40.3) 42.5 0.5 (26.0-28.0) 26.8 1.4 (12.5-17.0) 14.2 1.7 (24.1-31.4) 28.4 1.4 (19.0-23.0) 17.7 1.2 (59.0-63.0) 60.6 4.6 (44.5-61.5) 54.8 1.7 (23.3-29.6) 26.4 ± ± 38.6 (663-798) 750 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.0 (6-8) 5.0 1.5 (7-13) 10.5 0.9 (2.0-5.0) 3.8 0.2 (4.4-5.2) 5.0 0.3 (6.0-6.5) 7.0 0.5 (4.0-5.0) 4.5 0.1 (1.1-1.5) 1.4 0.7 (3.0-5.5) 5.7 6.2 (105-125) 111 9.6 (126-159) 144 5.2 (101-118) 111 1.4 (6.5-11.5) 11.2 0.4 (11.5-13.0) 12.9 4.4 (33.6-48.0) 35.3 0.9 (25.0-27.5) 26.5 0.9 (12.0-15.0) 14.4 2.8 (24.0-33.1) 27.6 2.7 (14.0-23.0) 20.4 1.5 (60.0-64.0) 60.6 3.3 (51.1-62.3) 54.0 2.9 (21.0-30.5) 26.1 ± 42.9 (617-792) 718 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± populations from Spain and Italy analysed in the present study. All measurements are in 1.0 (6-8) 7.0 1.4 (6-11) 9.3 5.3 (95-114) 110 1.5 (8.0-14.0) 9.4 0.6 (3.0-5.0) 3.4 0.4 (4.1-5.7) 4.8 1.3 (5.0-8.0) 6.3 0.5 (4.0-5.0) 4.5 0.1 (0.9-1.4) 1.3 0.7 (3.5-6.0) 4.5 3.8 (101-114) 114 9.4 (124-157) 144 1.0 (10.0-14.0) 12.3 4.6 (35.8-55.4) 40.2 0.9 (25.0-28.5) 25.9 0.9 (12.5-16.0) 13.6 1.9 (24.2-30.0) 28.8 1.6 (12.0-18.0) 17.4 1.1 (59.0-63.0) 61.7 3.9 (47.0-63.4) 55.9 1.3 (24.0-27.0) 24.1 ± 35.0 (609-719) 690 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Spain/Locubin Spain/J94muc 1.1 5.1 Helicotylenchus microlobus Castillo de Locubín, Jerez de la Frontera, Andújar, Spain/ST20 Bari, Italy/Bari Palaggiano, Italy/Palag 25.2 ∗ ∗ Morphometrics of s.d. (range). ± Measured at vulval region.  Excretory pore 108 DGOPharynx 11.0 145 Pharyngeal glands 110 c Conus length 12.6 c 43.8 Phasmid position 7.3 b4.7 Tail annuli number 8.2 Stylet length 26.7 Ventral peg length 3.6 Lip diam. 6.5 a 26.9 Lip height 4.6 Anal body diam. 13.7 Max. body diam. Lateral field width mean Table 4. CharacternL 1414141414 677 Population/Code ∗ V 61.1 Tail length 15.6 MB 52.9

44 Nematology Helicotylenchus pseudorobustus and genus phylogeny

Table 5. Morphometrics of Helicotylenchus digonicus, H. paxilli and H. broadbalkiensis populations with location and code analysed in the present study. All measurements are in μm and in the form: mean ± s.d. (range).

Character H. digonicus H. paxilli H. broadbalkiensis Kansas, USA/CD864 Florida, USA/CD694 Spain, Jerez de la Frontera/J94round n101212 L 689 ± 46.8 (632-780) 705 ± 35.9 (646-766) 656 ± 24.1 (629-701) a 27.1 ± 1.9 (23.5-29.7) 29.4 ± 3.0 (24.0-35.1) 25.9 ± 1.0 (23.7-27.0) b5.8± 0.3 (5.4-6.4) 4.7 ± 0.2 (4.5-5.0) 4.7 ± 0.1 (4.6-4.9) c 43.1 ± 4.7 (37.8-50.6) 44.1 ± 6.6 (31.3-55.5) 45.1 ± 4.8 (40.1-53.4) c 1.2 ± 0.2 (0.9-1.5) 1.2 ± 0.2 (0.9-1.5) 0.9 ± 0.1 (0.8-0.9) V 62.6 ± 0.7 (61.5-63.7) 62.9 ± 1.0 (61.0-65.0) 61.5 ± 0.7 (61.0-63.0) Lip height 3.7 ± 0.2 (3.3-3.8) 4.0 ± 0.4 (3.5-4.5) 4.3 ± 0.3 (4.0-5.0) Lip diam. 6.2 ± 0.1 (5.9-6.4) 6.1 ± 0.6 (5.5-7.0) 8.3 ± 0.3 (8.0-9.0) Stylet length 26.1 ± 0.8 (25-27.5) 26.4 ± 1.5 (23.5-30.0) 25.5 ± 1.0 (24.0-26.5) Conus length 12.5 ± 0.3 (12-13.1) 11.9 ± 0.7 (10.5-12.5) 12.3 ± 1.1 (11.0-14.0) DGO – 9.0 ± 2.0 (6.0-13.5) 10.5 ± 1.0 (9.0-12.0) Pharynx 122 ± 4.8 (115-129) 150 ± 6.0 (136-158) 139 ± 5.8 (130-148) Pharyngeal glands 139 ± 7.5 (130-151) 119 ± 2.2 (116-123) 107 ± 8.1 (95-117) Excretory pore 112 ± 5.8 (101-121) 116 ± 7.8 (101-128) 108 ± 4.6 (102-115) MB 67.7 ± 1.6 (65.0-69.4) 54.1 ± 1.8 (51.3-57.0) 54.7 ± 2.1 (51.6-57.5) Max. body diam.∗ 25.4 ± 1.1 (23.8-27.5) 24.2 ± 2.1 (20.5-29.0) 25.4 ± 0.9 (24.0-26.5) Lateral field width∗ 5.8 ± 0.3 (5.4-6.3) 5.2 ± 0.4 (4.5-6.0) 6.2 ± 0.3 (5.5-6.5) Tail length 16.3 ± 2.6 (12.5-20.0) 16.3 ± 2.6 (11.5-21.0) 14.7 ± 1.4 (12.5-16.0) Anal body diam. 14 ± 0.4 (13.8-15.0) 13.7 ± 0.8 (12.0-15.0) 16.2 ± 1.3 (13.5-17.5) Ventral peg length 1.0 ± 0.8 (0.1-2.5) 3.8 ± 0.6 (3.0-4.5) – Tail annuli number 11.1 ± 2.2 (8-15) 7.2 ± 2.3 (4-13) 16.0 ± 0.8 (15-17) Phasmid position 5 ± 1.4 (3-7) 8.8 ± 2.8 (6-12) 1.1 ± 0.8 (0-2)

∗ Measured at vulval region. ican and European populations of H. microlobus identi- Helicotylenchus paxilli Yuen, 1964 fied in our study had an overall number of ventral tail an- (Figs 3C; 5K-T; 7) nuli for the populations studied ranging from five to 13. This range was smaller than the average number of 14 re- MEASUREMENTS ported for the paratypes but within the range of those re- ported by Siddiqi (1972). It should be pointed out that the See Table 5. H. microlobus paratypes described by Perry et al. (1959) have the stylet knobs flattened anteriorly, whereas in our Female populations they were rounded. The populations of H. mi- Body in spiral and open C-shape in 93% and 7% of the crolobus that we examined differed morphologically from examined specimens, respectively, when heat-relaxed. Lip H. pseudorobustus in having the inner incisures of the region hemispherical with 4-6 annuli and separated from lateral field fused in a Y-shaped pattern vs U-, J- or M- body by a slight depression. Stylet robust, knobs slightly shaped in H. pseudorobustus and also had lower cumu- indented anteriorly. Hemizonid 3-4 annuli long, located lative means of the anal diam. which ranged from 12.7 1-4 annuli anterior to excretory pore. Pharyngeal gland to 14.4 vs 15.7 to 16.4 μminH. pseudorobustus.How- overlapping intestine ventrally. Anterior and posterior ever, the anal body diam. of California population (CD genital tracts similar in length at 157 ± 20 (131-193) and 740) did not differ from that of H. pseudorobustus.Inthis 157 ± 32 (121-228) μm. OV1 and OV2 also similar (25 ± study, we cannot consider the identification of the H. mi- (21-29) and 25 ± (19-31) μm). Spermatheca rounded crolobus populations definitive because of a lack of DNA (11 μm long, 12 μm diam.), empty, with a large cavity sequences from topotype specimens of this species. in centre and seen in 70% of examined specimens, off-set

Vol. 17(1), 2015 45 S.A. Subbotin et al.

(65%) or in-line (34%) in specimens examined which had Helicotylenchus digonicus Perry in Perry, a visible spermatheca. Phasmids 1.3 ± 0.3 (1.0-1.8) μm Darling & Thorne, 1959 in diam., located from two annuli posterior to 12 annuli (Figs 2M-O; 5F-J) anterior to anus level. Termination of inner two lines of lateral field on tail variable, usually joining in a Y- MEASUREMENTS (72%), V- (25%), U- (2%) or M- (1%) shaped pattern. See Table 5. The Y- and U-shaped patterns were the most common in paratypes of this species (Yuen, 1964). Tail longer than Female anal body diam., bearing 4-13 annuli (0.5-1.5 μm wide). Body in open C- to spiral-shape when relaxed. Lip Tail digitate with pronounced ventral projection usually region truncate with 4-5 annuli. Cephalic framework well rounded terminally with mucro in 66% of examined sclerotised, extending into body for 2-3 annuli. Stylet specimens. Ventral tail projection with surface smooth in knobs rounded or slightly indented anteriorly. Hemizonid 83% of examined specimens and with greatest curvature two or three annuli long, located one or two annuli on dorsal contour. One or two coarse annuli (1.9-2.2 μm anterior to excretory pore. Phasmids at anus level or wide), as compared to other body annuli (0.5 μm wide), two annuli anterior to anus. Inner and outer incisures observed on ventral tail projection in remaining 17% of of lateral field closed at termination on tail, joining in specimens. a U-shaped pattern. Tail longer than anal body diam., curved dorsally, bearing 8-15 ventral annuli. Tail terminus variable, without distinct annulation, sometimes with a Male short ventral projection, fasciculi absent. Not found. Male Not found.

REMARKS REMARKS

Morphological and morphometric characters of Heli- The DNA sequences of a spiral nematode population cotylenchus populations from Florida matched those pre- from Kansas, identified in the present study as H. digo- viously reported in the original description of H. pax- nicus, matched those for several samples previously iden- tified by Subbotin et al. (2011) as H. digonicus type B, illi by Yuen (1964) from the UK, and Wouts & Yeates H. pseudorobustus type C and type D, H. leiocephalus, H. (1994) from New Zealand. The congruence of the DNA platyurus, Helicotylenchus spI-5. In spite of the fact that sequences of these populations from Florida and another there are no morphological data supporting the molecu- from California with those of the H. paxilli topotypes lar congruity of these populations with the H. digonicus from the UK provided further evidence that these popu- population from Kansas, we consider all as representa- lations were representatives of H. paxilli. Morphology tives of H. digonicus. Characters of the Kansas population and morphometrics of H. paxilli topotypes and Florida of H. digonicus were generally similar to those published populations coincided with those in the original descrip- by Sher (1966), Anderson (1974) and Wouts & Yeates tion and also with those of type specimens of H. amplius (1994). Previously identified H. digonicus samples under Anderson & Eveleigh, 1982 from Canada (Anderson & letter codes C and A (Subbotin et al., 2011) are regarded Eveleigh, 1982) and a population from the Aleutian Is- here as unidentified species. Since no DNA sequences lands (Bernard, 1984), suggesting that H. amplius may of topotype specimens of H. digonicus are available, our be a junior synonym of H. paxilli. However, DNA se- characterisation of this species is not definitive. quences of topotype populations of H. amplius are needed to support this synonymy. A report of the occurrence of H. amplius from Florida (O’Bannon, 1988) needs confirma- Helicotylenchus broadbalkiensis Yuen, 1964 (Figs 2K-L; 3D; 5A-E) tion. There is also a report of the detection of H. paxilli in Florida (Lehman, 2002). This species, which was con- MEASUREMENTS sidered uncommon in the state, has very probably been confused with H. pseudorobustus for decades. See Table 5.

46 Nematology Helicotylenchus pseudorobustus and genus phylogeny

Female as belonging to H. pseudorobustus sensu stricto, includ- Body shape a closed spiral when heat-relaxed. Lip re- ing isolates from Germany, New Zealand and the USA. gion conoid-truncate, bearing 4-6 annuli. Cephalic frame- Intraspecific D2-D3 of 28S rRNA gene sequence variation work well sclerotised, extending 3-4 annuli into body. for H. pseudorobustus populations varied from 0.0-0.7% Stylet well developed, knobs with anterior surface slightly (0-4 bp), for H. microlobus – 0.0-1.2% (0-7 bp), for H. indented/concave. Hemizonid one or two annuli long, lo- paxilli – 0.0-0.5% (0-4 bp), for H. digonicus – 0.0-2.4% cated immediately anterior to excretory pore. Pharyngeal (0-14 bp), for H. broadbalkiensis – 0.5-1.6% (3-9 bp) gland overlapping intestine ventrally or ventrolaterally. and for H. dihystera – 0.0-2.3% (0-13 bp). Phylogenetic Pharyngo-intestinal junction often situated anterior to ex- relationships between Helicotylenchus species are given cretory pore, rarely posterior. Reproductive system with in Figure 9A, B. Eleven, mainly moderately and highly two branches equally developed, spermatheca absent. Tail supported, major clades can be distinguished within the conoid with curvature on dorsal side and tapering to a species studied. point ventrally, but flattening somewhat distally. Fasciculi The ITS of rRNA gene sequence alignment was Helico- absent. Phasmids from two annuli posterior to seven an- 1058 bp in length and contained 37 sequences of tylenchus nuli anterior to anus level. . Thirty-four sequences were newly obtained in the present study. Intraspecific ITS sequence variation for Male H. pseudorobustus sensu stricto populations varied from 0.0-0.6% (0-5 bp), for H. broadbalkiensis – 1.2-1.5% (12- Not found. 14 bp), for H. paxilli – 0.3-1.5% (3-15 bp), for H. mi- crolobus – 0.0-5.9% (0-57 bp) and for H. dihystera –0.3- REMARKS 2.6% (3-26 bp). Phylogenetic relationships between Heli- cotylenchus species are given in Figure 10. Seven highly After examining paratypes of H. broadbalkiensis and supported major clades can be distinguished within the H. digonicus, Sher (1966) synonymised H. broadbalkien- six studied species, including H. dihystera selected as an sis with H. digonicus, although he noticed a slight diffe- outgroup. Helicotylenchus microlobus samples were dis- rence in lip region shape, which was not considered suf- tributed into two subclades. ficient for species differentiation. The synonymy was ac- cepted by Krall (1978), Siddiqi (2000) and other authors. Helicotylenchus specimens obtained from samples from Discussion Spain and California (previously identified by Subbotin et al. (2011) as Helicotylenchus spI-9) fit well with the de- CONGRUENCE OF MORPHOLOGICAL AND scription of H. broadbalkiensis and differ from H. digo- MOLECULAR DIFFERENTIATION nicus by massive and slightly concave anteriorly project- ing stylet knobs, and tail shape variation. In this study In the design of this investigation we used an integrated we tentatively identify these samples as H. broadbalkien- approach with morphological and molecular datasets for sis. However, this identification should be confirmed by the delimitation of some species that are morphologically molecular comparison with topotype specimens. and genetically similar to H. pseudorobustus. Morpholo- gical and molecular studies of H. pseudorobustus popu- MOLECULAR CHARACTERISATION OF lations have previously been published by Fortuner et al. Helicotylenchus pseudorobustus AND ITS (1984) and Subbotin et al. (2011), respectively. PHYLOGENETIC RELATIONSHIPS WITH OTHER SPECIES Fortuner et al. (1984) conducted multivariate analyses on 28 populations of H. pseudorobustus, 11 populations The D2-D3 of 28S rRNA gene sequence alignment was of H. dihystera, and type populations of H. microlobus, 599 bp in length and contained 154 sequences of Heli- H. bradys Thorne & Malek, 1968, H. phalerus Ander- cotylenchus and two sequences of used as son, 1974, H. egyptiensis Tarjan, 1964 and H. africanus outgroup taxa. Sixty-one sequences were newly obtained (Micoletzky, 1916) Andrássy, 1958. Some morphological in the present study. The sequences from the topotype differences in the pattern of junction of the inner lines of H. pseudorobustus samples clustered with samples previ- lateral field on tail, the position of the phasmids and the ously named by Subbotin et al. (2011) as H. pseudorobus- dorsal gland opening were observed among the popula- tus type A, and thus confirmed the status of these samples tions of H. pseudorobustus. The analyses separated three

Vol. 17(1), 2015 47 S.A. Subbotin et al.

Fig. 9. A, B: Phylogenetic relationships of Helicotylenchus species and populations: Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the D2-D3 of 28S rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal to or more than 70% are given for appropriate clades. New sequences are indicated by bold font.

48 Nematology Helicotylenchus pseudorobustus and genus phylogeny

Fig. 9. (Continued.) groups of samples among the samples studied: i)a‘pseu- Instead, these authors accepted it as a geographical variant dorobustus’ group containing the topotype sample, mostly of H. pseudorobustus and noted that morphological dif- European populations, and populations from Africa and ferences were too slight to warrant the creation of a sub- New Zealand, was characterised by stylet length of 26.0- species. The results of the present molecular analysis of 27.0 μm, ratio V = 61, L = 700-750 μm, DGO at 9 μm, partial 28S rRNA and ITS rRNA gene sequences clearly phasmids 7-8 annuli anterior to anus, inner lines of lat- distinguished H. pseudorobustus from H. microlobus and eral field joining mostly in a U-shape (but Y-shape was H. dihystera, and all three from each other, and thus might also present), areolations on body/tail in some specimens be congruent with the results of the multivariate analyses and tail always ending in a ventral projection of vari- presented by Fortuner et al. (1984; their Fig. 3). able length; ii)a‘microlobus’ group including the para- In this study, using molecular datasets, we tried to use types of H. microlobus, H. bradys, H. phalerus and the a tree-based method and sequence analysis for species North American populations of H. pseudorobustus, and delimiting of Helicotylenchus. This approach is not al- differing from the first group mostly in the phasmids be- ways straightforward and might give ambiguous results, ing closer to the anus (four annuli), DGO further from because the delimiting results depend on thresholds for the stylet (11 μm), and the junction of the inner lines intra- and inter-specific sequence divergences. It has been always Y-shaped; and iii)aH. dihystera group, clearly shown by several studies that for some well-defined separated from the first two by shorter stylet of 24.0- species the rRNA gene sequences might not form a dis- 26.5 μm, more posterior vulva (V = 62.5-65.0), often tinct clade in the best tree but instead be distributed among shorter body of 600-750 μm, DGO sometimes more pos- several clades with intra-specific sequence divergences terior at 10-15 μm, junction of the inner lines of the lat- reaching several percentage points. In this study, involv- eral field always Y-shaped and the tail with or without a ing a geographically wide range of samples, we proposed ventral projection. Fortuner et al. (1984) did not propose tentative species delimitation which should be tested fur- the microlobus group as a valid taxon as some samples ther using other independent datasets. from Europe, California and Venezuela were intermediate As a concluding remark, we would like to add that this between the microlobus and pseudorobustus groups, and study has confirmed the wide distribution in different con- also because of the general variability of the characters. tinents of species that were considered junior synonyms

Vol. 17(1), 2015 49 S.A. Subbotin et al.

Fig. 10. Phylogenetic relationships of Helicotylenchus pseudorobustus and closely related species: Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the ITS rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal to or more than 70% are given for appropriate clades. New sequences are indicated by bold font. of H. pseudorobustus and H. digonicus, such as H. mi- AGR-136 from ‘Consejería de Economía, Innvovación y crolobus and H. broadbalkiensis, respectively. The con- Ciencia’ from Junta de Andalucía and Union Europea, firmation of the occurrence of H. paxilli in Florida has Fondo Europeo de Desarrollo regional, ‘Una manera de economic relevance since this species is a parasite of turf hacer Europa’, and the Russian Foundation of Basic Re- grasses and has been implicated in turf grass decline un- search, project number 14-04-00953. The authors thank der the incorrect name of H. pseudorobustus (Crow et al., Drs R. Fortuner (France) and J.A. Navas-Cortés (IAS- 2013). CSIC, Spain) and for comments and suggestions during manuscript draft preparation, C. Cantalapiedra-Navarrete, Acknowledgements J. Martín Barbarroja and G. León Ropero (IAS-CSIC) for their excellent technical assistance, Charles Spriggs This research was partially supported by grant (FDACS) for sample collection, and Drs G. Tylka, C. AGL2012-37521 from ‘Ministerio de Economía y Com- Blomquist, J. Stack, W. Ye, M. Pridannikov and R.V. Khu- petitividad’ of Spain, grant P12-AGR 1486 and grant sainov for providing nematode materials. Authors also

50 Nematology Helicotylenchus pseudorobustus and genus phylogeny acknowledge Dr A. MacDonald (the Lawes Agricultural applications in molecular barcoding. Philosophical Transac- Trust and Rothamsted Research) for access to samples tions of the Royal Society of London, B: Biological Sciences from Broadbalk. The Rothamsted Long-term Experiments 360, 1945-1958. National Capability (LTE-NCG) is supported by the UK Firoza, K. & Maqbool, M.A. (1994). A diagnostic compendium Biotechnology and Biological Sciences Research Council of the genus Helicotylenchus Steiner, 1945 (Nematoda: Hop- and the Lawes Agricultural Trust. lolaimidae). Pakistan Journal of Nematology 12, 11-50. Fortuner, R. (1984). Morphometrical variability in Helicotylen- chus Steiner, 1945. 6: Value of the characters used for specific References identification. RevuedeNématologie7, 245-264. Fortuner, R. (1985). Helicotylenchus pseudorobustus. CIH De- scriptions of plant-parasitic nematodes, Set 8, No. 109. Farn- Anderson, R.V. 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