Nematology 17 (2015) 27-52 brill.com/nemy Morphological and molecular characterisation of Helicotylenchus pseudorobustus (Steiner, 1914) Golden, 1956 and related species (Tylenchida: Hoplolaimidae) 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 nematodes 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, taxonomy. 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 NEMATODE 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 Vol. 17(1), 2015 Table 1. Helicotylenchus populations and species obtained for the present study and used for the phylogenetic analysis. Identification Previous Location Plant hosts Sample GenBank References or sources based on the identification codes accession number result of the ITS rRNA D2-D3 of present study 28S rRNA H. brevis H. brevis South Africa, Magaliesberg Solanum CD556 – HM014300 Subbotin et al. (2011) mauritianum H. broadbalkiensis Helicotylenchus USA, California, Death Valley Grasses CD363 KM506856 HM014276, S.A. Subbotin, spI-9 HM014277 Subbotin et al. (2011) H. broadbalkiensis – Spain, Jerez de la Frontera Ceratonia siliqua J94round KM506857, KM506832, P. Castillo KM506858 KM506833 H. depressus – New Zealand, Birdling Flat –