Contributions to Zoology (2019) 1-41 CTOZ brill.com/ctoz Molecular phylogenetic analysis and comparative morphology reveals the diversity and distribution of needle nematodes of the genus Longidorus (Dorylaimida: Longidoridae) from Spain Antonio Archidona-Yuste Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menéndez Pidal s/n, 14004 Córdoba, Spain Carolina Cantalapiedra-Navarrete Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menéndez Pidal s/n, 14004 Córdoba, Spain Pablo Castillo Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menéndez Pidal s/n, 14004 Córdoba, Spain Juan E. Palomares-Rius Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menéndez Pidal s/n, 14004 Córdoba, Spain [email protected] Abstract The genus Longidorus constitutes a large group of approximately 170 species of plant-ectoparasitic nematodes that are polyphagous and distributed almost worldwide. Some of the species of this genus are vectors of plant viruses. Species discrimination in Longidorus is difficult because the morphology is very conservative, and morphometric characters often overlap, leading to potential misidentification. Integrative taxonomy, based on the combination of molecular analyses with morphology, is a useful and necessary approach in Longidorus species identification. In Spain from 2014 to 2017, we conducted nematode surveys among cultivated and wild plants, from which we identified 13 populations of Lon- gidorus, two of which appeared to represent new species and are described herein as L. iliturgiensis sp. nov. and L. pacensis sp. nov., and 11 populations belonging to eight known species: L. africanus, L. baeticus, L. carpetanensis, L. fasciatus, L. nevesi, L. cf. olegi, L. pini, and L. vallensis. Three species are new geographical records for Spain (L. nevesi, L. cf. olegi, and L. africanus). We report molecular data for L. nevesi, L. cf. olegi, © antonio archidona-yuste et al. 2019 | doi:10.1163/18759866-20191345 This is an open access article distributed under the terms of the prevailing cc-by license at the time of publication. Downloaded from Brill.com03/15/2019 10:07:45AM via Cons. Superior de Investig. Scientificas <UN> 2 archidona-yuste et al. L. carpetanensis and L. pini for the first time. Additionally, we describe the males of L. pini and the juveniles of L. cf. olegi. Keywords belowground diversity – Bayesian inference – CoxI – D2–D3 expansion domains of 28S rRNA gene – integrative taxonomy Introduction of seven out of 38 known nepoviruses (Taylor & Brown, 1997; Decraemer & Robbins, 2007) Nematodes include some of the most abun- of the Longidorus species vector nepoviruses, dant metazoans on earth with a global dis- including artichoke Italian latent virus (AILV), tribution and an estimated ~100,000 species carnation ringspot virus (CRSV), cherry rosette (Boucher & Lambshead, 1995; Blaxter et al., disease virus (CRDV), mulberry ringspot virus 1998; Coomans, 2000). In addition, among soil (MRSV), peach rosette mosaic virus (PRMV), fauna, nematodes are the most common and raspberry ringspot virus (RpRSV), and tomato diverse multicellular animals, being found in black ring virus (TBRV) (Taylor & Brown, 1997; many environments and representing one of Decraemer & Robbins, 2007). These transmis- the most ubiquitous animal phyla in the soil sions showed a marked specificity between (Ferris et al., 2001). Nematodes occupy all tro- plant viruses and their specific Longidorus phic levels within the soil food web, which vector species, except for L. elongatus trans- leads them to play a central role in numerous mitting tomato black ring virus (TomBRV) soil functions (Ferris et al., 2001). and raspberry ringspot virus (RRSV) (Taylor The needle nematodes of the genus Lon- & Brown, 1997), which confirms the need for gidorus Micoletzky, 1922 belong to the family accurate identification of Longidorus species. Longidoridae Thorne, 1935 comprising a wide Due to the highly conserved morphology, and diverse group of plant migratory ecto- with similar anatomical characteristics and parasitic nematode species (Coomans, 1996). high inter- and intraspecific morphometric Longidorus is considered cosmopolitan and variability, species delimitation in Longidorus contains approximately 170 species (Coomans, is a very complex and time-consuming task 1996; Archidona-Yuste et al., 2016a). Damage (Coomans et al., 2001; Archidona-Yuste et al., by Longidorus spp. to host plants is caused by 2016a). Accurate identification of needle nem- direct feeding on root cells as well as by trans- atodes is essential to establish an unequivocal mitting nepoviruses (genus Nepovirus, sub- diagnosis to discriminate virus vector spe- family Comovirinae) (Taylor & Brown, 1997). cies, select appropriate management strate- In addition, 8 species of this genus (6.5%) gies for preventing their spread and establish (L. apulus Lamberti & Bleve-Zacheo, 1977, efficient control measures. Recent studies L. arthensis Brown, Grunder, Hooper, Klinger using an integrative approach for identifi- & Kunz, 1994, L. attenuatus Hooper, 1961, cation highlighted the difficulty of correct L. diadecturus Eveleigh, 1982, L. elongatus (de identification at the species level within the Man, 1876) Micoletzky, 1922, L. fasciatus Roca genus Longidorus, as well as cryptic species & Lamberti, 1981, L. macrosoma Hooper, 1961, separation (Subbotin et al., 2015; Archidona- and L. martini Merny, 1966) are specific vectors Yuste et al., 2016a; DownloadedPeraza-Padilla from Brill.com03/15/2019 et al., 2017 10:07:45AM; via Cons. Superior de Investig. Scientificas <UN> DIVERSITY OF NEEDLE NEMATODES (LONGIDORUS) IN SPAIN 3 Xu et al., 2017, 2018). These studies provide Materials and methods molecular markers based on ribosomal RNA (rRNA) (D2–D3 expansion domains of 28S Nematode populations and morphological rRNA, ITS and 18S rRNA) and mitochondrial studies DNA (mtDNA) genes (cytochrome c oxidase Nematode surveys were conducted from the subunit I or CoxI) for precise and unequivo- spring of 2014 to 2017 in the soil around cul- cal diagnoses of some species of Longidorus, tivated and wild plants in Spain (supplemen- demonstrating that this genus is a complex tary fig. S1). Soil samples were collected with and taxonomically important group of plant- a shovel from the upper 50 cm of soil sur- parasitic nematodes. rounding four or five plants arbitrarily chosen Approximately forty species of Longidorus in each locality. Nematodes were extracted have been reported from the Iberian Penin- from 500 cm3 of soil by centrifugal flotation sula, including the recent descriptions of fif- (Coolen, 1979) and a modification of Cobb’s teen new species (Peña Santiago et al., 2003; decanting and sieving (Flegg, 1967) methods. Gutiérrez-Gutiérrez et al., 2013; Archidona- In some cases, additional soil samples were Yuste et al., 2016a). A survey of nematodes collected from the same locality for additional from cultivated and wild plants in Spain re- specimens for morphological and/or molecu- vealed the presence of thirteen unidentified lar identification. populations of needle nematodes belonging Specimens for light microscopy were killed to the genus Longidorus. Preliminary mor- by hot fixative using a solution of 4% formal- phological observations indicated that two of dehyde + 1% propionic acid and embedded in these populations appeared to be morphologi- pure glycerine using Seinhorst’s (1966) method. cally different from all other species described Specimens were examined using a Zeiss III in the genus, while the other populations compound microscope with Nomarski dif- were assigned to eight known species of Lon- ferential interference contrast at powers up gidorus. Detailed observations using light to 1,000x magnification. The morphometric microscopy and molecular characterisation study of each nematode population included indicated that these two populations should morphology-based diagnostic features in Lon- be assigned to two new species. In the present gidoridae (i.e., de Man body ratios (Jairajpuri study, we describe the two new species, Lon- & Ahmad, 1992)), lip region and amphid shape, gidorus iliturgiensis sp. nov. and Longidorus oral aperture-guiding-ring, odontostyle length pacensis sp. nov. and present phylogenetic and female tail shape) (Jairajpuri & Ahmad, data that confirm their species status. 1992). For line drawings of the new species, The objectives of this study were (1) to light micrographs were imported to CorelDraw morphologically and morphometrically char- ver. X7 and redrawn. In addition, a comparative acterise the two new species belonging to the morphological study on the type specimens of genus Longidorus and to compare them with one species was conducted with specimens previous records; (2) to characterise molecu- kindly provided by Dr. A. Navas from the Nem- larly the sampled Longidorus spp. popula- atode Collection of the Spanish National Mu- tions using the D2–D3 expansion domains seum of Natural Sciences-CSIC, Madrid, Spain of the 28S rRNA gene, ITS1, partial 18S rRNA (viz. L. pini Andrés & Arias, 1988). gene, and the partial mitochondrial CoxI gene Topotype specimens of L. carpetanensis sequences; and (3) to study the phylogenetic Arias, Andrés & Navas, 1986 were used for mor- relationships of the identified Longidorus spe- phological and molecular studies after verify- cies with available sequenced species.