Zoosyst. Evol. 96 (1) 2020, 175-193 | DOI 10.3897/zse.96.49022 yee BERLIN Description of Longidorus bordonensis sp. nov. from Portugal, with systematics and molecular phylogeny of the genus (Nematoda, Longidoridae) Carlos Gutiérrez-Gutiérrez', Margarida Teixeira Santos*, Maria Lurdes Inacio®, Jonathan D. Eisenback?, Manuel Mota! 1 NemaLab/ICAAM, Instituto de Ciéncias Agrarias e Ambientais Mediterrdnicas & Dept. de Biologia, Universidade de Evora, Nicleo da Mitra, Ap. 94, 7002-554 Evora, Portugal 2 Instituto Nacional de Investigagdo Agraria e Veterinaria (INIAV), Quinta do Marqués, 2780-159 Oeiras, Portugal 3 School of Plant and Environmental Science, Virginia Tech, Blacksburg, VA, USA http://zoobank. org/16388413-BF9F-4339-AF96-1179BB&CEDID Corresponding author: Carlos Gutiérrez-Gutiérrez (carlosg@uevora.pt) Academic editor: A. Schmidt-Rhaesa # Received 13 December 2019 # Accepted 27 March 2020 Published 5 May 2020 Abstract The genus Longidorus currently comprises 176 species of polyphagous plant ectoparasites, including eight species that vector nepo- viruses. Longidorus 1s one of the most difficult genera to accurately identify species because of the similar morphology and overlap- ping measurements and ratios among species. Sequences of ribosomal RNA (rRNA)-genes are a powerful level-species diagnostic tool for the genus Longidorus. From 2015 to 2019, a nematode survey was conducted in vineyards and agro-forest environments in Portugal. The populations of Longidorus spp. were characterized through an integrative approach based on morphological data and molecular phylogenetic analysis from rRNA genes (D2-D3 expansion segments of the 28S, ITS1, and partial 18S), including the topotype of L. vinearum. Longidorus bordonensis sp. nov., a didelphic species recovered from the rhizosphere of grasses, is de- scribed and illustrated. Longidorus vineacola, with cork oak and wild olive as hosts, is also characterized. This is the first time that L. wicuolea, from cork oak, is reported for Portugal. Bayesian inference (BI) phylogenetic trees for these three molecular markers established phylogenetic relationships among the new species with other Longidorus spp. Phylogenetic trees indicated that 1) L. bor- donensis sp. nov. is clustered together with other Longidorus spp. and forms a sister clade with L. pini and L. carpetanensis, sharing a short body and odontostyle length, and elongate to conical female tail, and ii) all the other species described and illustrated are phylogenetically associated, including the topotype isolate of L. vinearum. Key Words Bayesian inference, D2—D3 expansion segments of large ribosomal subunit 28S, genomic data, needle nematodes, internal tran- scribed spacer 1, partial small ribosomal subunit Introduction Dorylaimida Pearse, 1942 is one of the most diverse or- ders in terms of number of species within the phylum Nematoda (Jairajpuri and Ahmad 1992). Members of the genus Longidorus Micoletzky, 1922, commonly known as needle nematodes, belong to the subfamily Longidorinae within the family Longidoridae Thorne, 1935 (order Dorylaimida) and includes a large group of metazoan ectoparasites of herbaceous plants, bushes, and trees (Coomans 1996; Taylor and Brown 1997; Decrae- mer and Robbins 2007). Longidorus is one of the most species-rich genera, taking into account the species de- scribed by Lazarova et al. (2019) and Cai et al. (2019); it contains approximately 176 valid species. These nema- todes drill into the root by moving their needle-like stylet Copyright Carlos Gutiérrez-Gutiérrez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 176 Carlos Gutiérrez-Gutiérrez et al.: Description of Longidorus bordonensis sp. nov. from Portugal through root cells during feeding, which causes severe negative effects on the architecture of the root (Taylor and Brown 1997). In the field, the presence of this group is associated with poor plant growth, including reduced root systems which are characterized by severely stunted lateral and tap roots (Taylor and Brown 1997; Archido- na-Yuste et al. 2016). However, the major pest status of Longidorus spp. is earned because Longidorus is a vector of the subgroup B Nepoviruses (genus Nepovirus, sub- family Comovirinae, family Secoviridae) (Taylor and Brown 1997; Decraemer and Robbins 2007). In spite of being an extensive genus, only 4.6% of their members, i.e. eight species (LZ. apulus Lamberti & Bleve-Zacheo, 1977, L. arthensis Brown, Grunder, Hooper, Klinger & Kunz, 1994, L. attenuatus Hooper, 1961, L. diadecturus Eveleigh, 1982, L. elongatus (de Man, 1876) Micoletzky, 1922, L. fasciatus Roca & Lamberti, 1981, L. macroso- ma Hooper, 1961, and LZ. martini Merny, 1966), trans- mit 21% of the known Nepoviruses (Taylor and Brown 1997; Decraemer and Robbins 2007), which emphasize the need for establishing a selective strategy based on the correct identification of species. According to data collected from EU Directive 2000/29/EC and/or the Eu- ropean and Mediterranean Plant Protection Organization (EPPO) quarantine lists, some species are recommended for regulation as quarantine pests. Most Longidorus species have a restricted geograph- ical distribution. However, this genus is a cosmopolitan group, with South America being the less diverse region, followed in increasing order by Oceania, China, South Africa, North America, India, and Europe (Decraemer and Robbins 2007; Cai et al. 2019; Xu and Zhao 2019). Fifteen species of Longidorus have been reported in sev- eral cultivated and wild plants including angiosperms and gymnosperms in Portugal (Bravo and Lemos 1997; Gutiérrez-Gutiérrez et al. 2016): L. africanus Merny, 1966, L. alvegus Roca, Pereira & Lamberti, 1989, L. belloi Andres & Arias, 1988, L. carpetanensis Arias, Andres & Navas, 1986, L. crataegi Roca & Bravo, 1996, L. goodeyi Hopper, 1961, L. juvenilis Dalmasso, 1969, L. /usitani- cus Macara 1985, L. macrosoma, L. nevesi Macara, 1986, L. profundorum Hopper, 1996, L. reisi Roca & Bravo, 1993, L. unedoi Arias, Andres & Navas, 1986, L. vinea- cola Sturhan & Weischer, 1964, and L. vinearum Bravo & Roca, 1995. Most of these species appear to have an en- demic distribution limited to the Iberian Peninsula (An- dres and Arias 1982; Roca et al. 1989; Bravo and Lemos 1997; Pefia-Santiago al. 2006; Gutiérrez -Gutiérrez et al. 2016; Archidona-Yuste et al. 2019), while some have a wider geographical distribution (Navas et al. 1990, 1993; Taylor and Brown 1997; Bravo and Lemos 1997; Gutiér- rez-Gutiérrez et al. 2016), such as L. juvenilis (Taylor and Brown 1997; Barsi and Lamberti 2004; Sirca and Urek 2009; Gutiérrez-Gutiérrez et al. 2016), L. goodeyi (Dal- masso 1969; Seinhorst and van Hoof 1981; Taylor and Brown 1997; Bravo and Lemos 1997; Gutiérrez -Gutiér- rez et al. 2016 ), L. africanus (Cohn 1969; Jacobs and Heyns 1987; Vadivelu and Muthukrishnan 1987; Zeidan zse.pensoft.net and Coomans 1991; Bravo and Roca 1995; Lamberti et al. 1996; Fadaei Tehrani and Kheiri 2005; Anwar and McKenry 2012; Subbotin et al. 2014; Guesmi-Mzoughi et al. 2017; Archidona-Yuste et al. 2019), L. macrosoma (Barsi 1989; Taylor and Brown 1997; Bravo and Lem- os 1997; Lamberti et al. 2001; Gutiérrez-Gutiérrez et al. 2016), and L. profundorum (Hooper 1965; Klingler et al. 1983; Andres and Bello 1984; Topham and Alphey 1985; Prikhodko 1988; Romanenko 1994; Bravo and Lem- os 1997; Taylor and Brown 1997; Lamberti et al. 2001; Liskova 2012; Gutiérrez-Gutiérrez et al. 2016). According to the morphological features and mor- phometric measurements of adult (mainly females) and that of juveniles [mainly first-stage juveniles (J1)], each Longidorus species 1s defined by 11 matrix codes (A—K) in the polytomous key published by Chen et al. (1997) and two additional supplements published by Loof and Chen (1999) and Peneva et al. (2013). However, the high intraspecific variability of some diagnostic features and the great diversity in phenotypic plasticity makes species identification based on metric and morphological data of external morphology and internal anatomy difficult and sometimes unreliable. Sequencing of RNA-based mark- ers 1s a powerful molecular diagnostic approach within this group (Archidona-Yuste et al. 2016, 2019; Palo- mares-Rius et al. 2017; Peraza-Padilla et al. 2017; Tzortz- akakis et al. 2017; Xu et al. 2018). Recently, several stud- ies (Archidona- Yuste et al. 2016, 2019; Roshan-Bakhsh et al. 2016; Esmaeili et al. 2017; Tzortzakakis et al. 2017; Xu et al. 2017, 2018; Barsalote et al. 2018; Cai et al. 2019; Lazarova et al. 2019) have shown the usefulness of a pair of molecular markers based on ribosomal RNA (rRNA) (D2—D3 domains of 28S gene and the ITS re- gions, particularly ITS1) for a fast and precise diagnosis of Longidorus species, even in extreme situations such as sibling and cryptic species. Subbotin et al. (2015) and Barsi and De Luca (2008) designed a PCR-RFLP of the D2—-D3 segments of the 28S rRNA and ITS. Both assays were based on five restriction enzymes for genotyping of species-specific variations: the first from L. orientalis Loof, 1982, and the other from L. pius Barsi & Lamberti, 2001 (Barsi and De Luca 2008; Subbotin et al. 2015). Ad- ditionally, studies have revealed that the mitochondrial marker gene, COJ, 1s useful for the delineation of closely related species within Longidoridae (Palomares-Rius et al. 2017; Archidona-Yuste et al. 2019; Cai et al. 2019). Thus far, more than half of the valid Longidorus species have molecular markers deposited in the GenBank data- base; however, only a small number belong to topotypes. Genomic data of topotypes are very useful for confirming identifications and clarifying the composition of species complexes within the Longidoridae (Gutiérrez-Gutiérrez et al. 2010, 2013; Kornobis et al. 2017; Archidona-Yuste et al. 2019; Fouladvand et al. 2019). Members of the genus Longidorus have not been studied in detail during the past 18 years in Portugal, and updated information on the present occurrence and distribution is lacking, as well as molecular data (Gutiér- Zoosyst. Evol. 96 (1) 2020, 175-193 rez-Gutiérrez et al. 2016). This prompted us to carry out surveys in vineyards and agro-forestry systems in Por- tugal, from 2015 to 2019. The objectives of the present work are: 1) to characterise 11 populations of Longidorus species through an integrative approach based on mor- phological, morphometric, and molecular data, including topotypes of L. vinearum and of L. bordonensis sp. nov.; 2) to establish phylogenetic relationships of the identified Longidorus species from the surveys with available se- quences of the known species. Methods Nematode population sampling, extraction, and morphological characterization Nematode surveys were conducted in spring and autumn from 2015 to 2019 in vineyards (Vitis sp.) and agro-forest- ry soils and included several host plants (Table 1). A total of 65 and 85 sampling sites of vineyards and agro-forest- ry areas, respectively, were arbitrarily chosen in Portugal. Field samples were taken in a zigzag pattern according to EPPO diagnostic protocols (QEPP/EPPO 2009). Each sample was collected using a drill from the upper 60 cm of the rhizosphere of 10—20 plants (sub-samples) from each field. Nematodes were extracted from 250 cm? of soil by a modification of Cobb’s decanting and sieving method (Flegg 1967). Additional soil was collected to guarantee enough specimens for morphological and/or molecular analyses. Nematodes were placed in a drop of water, killed in a hot fixative solution (4% formaldehyde + 1% glycerol + 85% distilled water), maintained for 48-72 h at room temperature (25 °C), and processed into pure glycerine by a modification of Seinhorst’s method (Seinhorst 1966). Specimens were examined using an Olympus BX50 light microscope with differential interference contrast (DIC) up to 1,000x magnification. Photographs were taken with an Olympus DP70 camera. Cell software (Olym- pus Software Imaging for Life Sciences) was used for image analysis and measurements. All measurements were expressed in micrometers (um). For line drawings of the new species, light micrographs were imported to CorelDraw v. X6 and the main features were outlined. All abbreviations are as defined in Jairajpuri and Ahmad (1992). According to metric (such as lip region length and width, body length, odontostyle length, maximum body width, guiding ring position, vulva position, pharyngeal length, and tail length and width) and non-metric (e.g., tail shape, size, and position of amphidial fovea, vulva size and shape, and lip region shape) morphological data of adult specimens and juveniles (J1—J4), each species was defined by the matrix code for the polytomous key (Chen et al. 1997; Loof and Chen 1999; Peneva et al. 2013). In addition, specimens of L. vinearum from its type locali- ty, Dois Portos, Torres Vedras, Portugal, were collected. After verifying that their morphology was consistent with LAS that of the original description, they were processed for their genotypic and phenotypic characterizations, and in- cluded as one of the 11 populations (Table 1). DNA extraction, PCR, and sequencing After nematodes were extracted from the soil, specimens were examined by light microscopy (LM) on temporary glass slide mounts and digital images were recorded. These photomicrographs were used to match each phe- notype with its associated genotype. Temporary slides were dismantled and individual nematodes were placed ina 2 ul drop of sterile water on the cover of a PCR tube, and the specimen was cut into six small pieces with a surface-sterilized scalpel. Subsequently, they were cen- trifuged in 18 ul of solution containing 10 ul ddH,O, 6 ul 10x PCR buffer, and 2 ul of proteinase K (20 mg/ml) (Nalgene), and frozen at —80 °C (15 min). Samples were mixed for 15 sec and PCR assays were conducted as de- scribed by Gutiérrez-Gutiérrez et al. (2018). The tubes were incubated at 57 °C (1 h), 65 °C (1 h), and 95 °C (15 min). Half of one ul of extracted DNA was transferred to an Eppendorf tube containing reaction mixtures of 25 ul NZYTaq 2x Green Master Mix (2.5 mM MgCl, 200 mM dNTPs, 0.2 U/ul DNA Polymerase) (NYZTech, Portu- gal), 0.4 ul of each primer (25 mM), and ddH,O was add- ed to make a final volume of 50 ul. The D2—D3 expansion segments of 28S rRNA gene, the ITS] region of rRNA, and a partial potion of 18S rRNA gene were amplified using several primer pairs (Suppl. material 3: Table S1). PCR cycle conditions for markers of ribosomal DNA included one cycle of 95 °C for 3 min; followed by 30 cy- cles of 94 °C for 30 s; an annealing temperature of 54 °C (D2A/D3B or 28LeX/ D3B), 53 °C (f(DNA1/18S), and 50 °C (988F/1912R, 1813F/2646R) for 30 s, 72 °C for 15-45 s; and one cycle of 72 °C for 7 min. PCR prod- ucts were purified after amplification using NZ YGelpure (NZYTech Genes & Enzymes, Portugal) following the manufacturer’s instructions and used as template for di- rect sequencing at Eurofins Genomic (Germany) using the primers listed (Suppl. material 3: Table S1). The se- quences were deposited in the GenBank database under accession numbers (Table 1) and used for constructing phylogenetic trees (Figs 3—5) were inferred using the methods descripted in the following section. Phylogenetic analyses D2—D3 expansion segments of 28S rRNA, partial 18S tRNA, and ITS1 rRNA sequences from L. bordonensis sp. nov. and all other known Longidorus spp. found in the survey (Table 1), together with additional accessions belonging to Longidorus spp. available in GenBank were used for phylogenetic reconstructions. Outgroup taxa for each dataset were chosen according to previously pub- lished data (Archidona-Yuste et al. 2016, 2019; Xu et al. zse.pensoft.net 178 Carlos Gutiérrez-Gutiérrez et al.: Description of Longidorus bordonensis sp. nov. from Portugal Table 1. Taxa sampled for Longidorus species, and sequences used in this study. Species Sample code Locality Host Genbank accessions D2D3 ITS1 18S Longidorus sp. 3 Silk Carregueira, Santarém cork oak tree MN082424 - - L. bordonensis sp. nov. 70-08-18 Bordonhos, S.Pedro Sul grass MNO082421 MN150062 MN1297570 MNO082422 “L. vinearum LISB-03-04 Dois Portos, Torres grapevine MNO082431 MN150065 - Vedras MN082432 MN150067 MN150068 L. vinearum “LISB-22 Picanceira, Mafra grapevine MN082434 - - L. vinearum LISB-13 Macheia, Ordasqueira, grapevine MN082433 - - Torres Vedras L. vinearum M3-OLV Valverde, Evora wild olive MN082430 MN150066 - L. vineacola 119/015 Q. da Amoreirinhas da cork oak tree MN082425 - - Cima , Montemor-o- Novo L. vineacola 120/015 Q. da Amoreirinhas da cork oak tree MNO082426 - - Cima, Montemor-o-Novo MN082427 L. vineacola TOF OLS Q. da Amoreirinhas da cork oak tree MN082428 MN150064 MN129758 Cima, Montemor-o-Novo L. vineacola M3-OLV Herdade da wild olive MNO082429 - - Mitra,Valverde, Evora L. wicuolea “Beja-16 Linhares, Beja cork oak tree MNO082423 MN150063 - * Topotype specimens “ Only one juvenile specimen was detected in this sample (—) Not obtained or not performed. 2018; Cai et al. 2019). The sequences were deposited in Genbank for each gene studied and they were aligned us- ing an online version of MAFFT v. 7 (Katoh and Stand- ley 2013) with default parameters. Sequence alignments were visualised with ClustalX2 (Thompson et al. 1997) and edited by Gblocks v. 0.91b (Castresana 2000) in Castresana Lab server (http://molevol.cmima.csic.es/cas- tresana/Gblocks_ server.html) using options for a relaxed selection of blocks (minimum number of sequences for a conserved position: 68 (D2—D3), 56 (18S) and 59 (ITS1); minimum number of sequences for a flanking position: 68 (D2—-D3), 56 (18S) and 59 (ITS1); maximum number of contiguous non-conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half). Homoge- neities of base frequencies and optional substitution mod- els for 28S rRNA, 18S rRNA, and ITS1 datasets were test- ed with Kakusan4 (Tanabe 2011). The homogeneity test indicated that the base composition of each dataset was significantly homogeneous. Bayesian inference (BI) trees of all molecular data were constructed with MrBayes v. 3.2.1 (Ronquist and Huelsenbeck 2003). For BI analysis, the substitution model was tested by the Bayesian Infor- mation Criterion and SYM model with a gamma-shaped distribution was selected for the three molecular regions studied. BI analysis was run for 5,000,000 generations, sampling every 100" tree and discarding ‘burn in’ first 25% of the sampled tree. Results Measurements and morphological images are presented here in the description of species. Molecular data also helped to discriminate species within the genus Longi- dorus in this study. For the description of the new spe- cies L. bordonensis sp. nov., DNA data were added to the zse.pensoft.net previously established morphological and morphometric analysis. Here, we provided metrical and non-metrical morphological data as well as molecular phylogenetic analyses of two known species previously reported from Portugal, L. vinearum and L. vineacola. Unfortunately, only one specimen was found in both L. wicuolea Ar- chidona-Yuste, Navas Cortés, Cantalapiedra-Navarrete, Palomares-Rius, & Castillo, 2016 and one unidentified Longidorus species (Longidorus sp. 3 isolate ST), which were used for sequencing of the D2—D3 expansion do- mains of 28S rRNA or/and ITS1. In this study, in addi- tion to the description of the new species, the molecular characterization of L. wicuolea comprises the first report of this species for Portugal. For both L. vineacola and L. vinearum, a brief description and comparison with the original description and other previous records are pro- vided; however, for the description of the second species, topotype material was included too. Paratypes of L. vin- earum were provided by Maria L. Inacio (INIAV, Oeiras, Lisbon, Portugal); however, they were not incorporated in this study because the specimens were 1n poor condition. Longidorus bordonensis sp. nov. http://zoobank.org/D753E7C6-512E-4A69-B9FE-326B9783D74C Figs 1(1—7), 211-10), Table 2, Suppl. material 4: Table S2) Holotype. Slide PLBOO1. Paratypes. 6 females and 6 males (slides PLB0O02-PLB 013) mounted on glass slides. Type repositories. The holotype (PLBO0O1) and 8 para- types (4 females and 4 males) (slides PLBO02—PLB005 and PLBOO8—PLBO11) are deposited in the Nematode Collection of the Nematology Lab, Institute for Mediter- 179 Zoosyst. Evol. 96 (1) 2020, 175-193 — — igen vee Qo ay oN OM OS Figure 1. Line drawings of Longidorus bordonensis sp. nov. paratypes from the rhizosphere of grass (unknown species) at Sao Pedro do Sul, Viseu district, northern Portugal (1 Spicule 4. Female lip region. 3. Female tail region. —7). 1. Female anterior end. 2 Detail of basal pharyngeal bulb. Scale bars: 23 um and lateral guiding piece of gubernaculum. 5. Vulva region. 6. Male tail region. 7 (1-3); 24 um (4, 6); 29 pm (5); 15 wm (7). zse.pensoft.net 180 Carlos Gutiérrez-Gutiérrez et al.: Description of Longidorus bordonensis sp. nov. from Portugal ranean Agricultural and Environment Sciences, ICAAM, University of Evora, Evora; 2 paratypes (1 female and 1 male) (slides PLB006 and PLBO12) in the Royal Belgian Institute of Natural Sciences, Brussels, Belgium; 2 para- types (1 female and | male) (slides PLBO07 and PLBO13) in the Istituto per la Protezione delle Piante (IPP) of Con- siglio Nazionale delle Ricerche (C.N.R.), Sezione di Bari, Bari, Italy. Type locality. Holotype and paratype specimens were ex- tracted from a soil sample collected from the rhizosphere of an unidentified grass species at Bordonhos, Sao Pedro do Sul, Viseu district, Beira Alta province, northern Por- tugal (40°45'53"N, 8°5'12"W) (Table 1) Etymology. The specific epithet of this species refers to the region of the type locality (Bordonhos) where the new species was found. Description of female. Short and slender body, slight- ly tapering at both ends, more pronounced in the tail. Curved in open J- or C-shaped relaxed by heat. Cuticle thin, appearing smooth under low magnifications, 1.8 + 0.3 (1.3-2.2) um thick at mid body, but thicker (9.1 + 0.7 (8.1—9.8) um) in hyaline region located at the end of tail region (Figs 1(3), 2(6, 7); Table 2). Lateral chord ca 11.1 um wide at mid-body or ca 34% of corresponding body diam. Lip region anteriorly flattened, expanded and rounded laterally, 10.1 + 0.4 (9.6—10.7) um wide and 4.1 + 0.5 (3.6-5.0) um high, set-off from body contour by a constriction (Figs 1(1, 2), 2(1, 2), Table 2). Amphidi- al fovea large asymmetrically bilobed pouch occupying from 2/3 to 3/4 of the distance from oral aperture to guid- ing ring (Figs 1(1, 2), 2(3), Table 2). Stylet guiding-ring single and posteriorly situated, 2.7—2.3 times lip region diameter from anterior end. Moderate and straight odon- tostyle, 1.5 + 0.1 (1.3—1.7) times as long as odontophore; weakly developed, with rather weak basal swellings (Figs 1(1), 2(4) Table 2). Nerve ring surrounding the tubular portion of the pharynx behind the odontophore base at 149.9 + 7.7 (138.1-157.7) um from anterior end. Ante- rior slender part of pharynx usually coiled in its poste- rior region. Basal bulb short and cylindrical, 79.9 + 6.2 (68.0—87.2) um long and 13.5 + 1.0 (12.1-14.7) um in diameter. Glandularium 71.9 + 3.0 (67.0—74.3) um long. Dorsal pharyngeal gland nucleus (DN) and ventro-sub- lateral nuclei (SVN) located at 33.1 + 3.2 (29.4-35.3)% and 53.1 + 0.7 (52.6-53.9)% of distance from anterior end of pharyngeal bulb, respectively. Cardia conoid to rounded, 8.6 + 2.7 (6.3-13.1) um long (Figs 1(7), 2(5), Table 2). Reproductive system with both genital branch- es equally developed, 8.4 + 0.5 (7.5-8.7) or 8.0 + 1.2 (6.7-9.5)% of body length (Table 2). Ovaries reflexed, variable in length, anterior ovary 71.4 + 14.6 (52.0-85.8 um long) and posterior ovary 82.3 + 13.6 (68.0-99.0 um long) (Table 2). Oviducts slightly longer than ovaries. Uteri cylindrical, quite variable in length, anterior uteri 283.9 + 94.0 (209.0-418.0 um long) and posterior uteri zse.pensoft.net 251.3 + 43.7 (194.0-295.0 um long); sphincter usually well developed, delimiting uterus and oviduct. Sperm commonly found in the uteri of female reproductive tract. Vulva transverse, located slightly anterior to the middle of the body, vagina perpendicular to body axis, 21.84 1.9 (19.0—24.6) um long (Figs 1(5), 2(9), Table 2). Prerectum visible, variable in length, 547.5 + 503.7 (162.0—1201.0) um long, and short rectum 20.0 + 2.9 (15.0— 23.0) um long or 1.2 (0.9-1.6) times anal body width. Tail long, bluntly conoid, slightly ventrally curved with rounded terminus, bearing three pairs of caudal pores (Figs 1(3), 2(6, 7); Table 2). Description of male. Males are as common as females. Appearance of body similar to female, except for repro- ductive organs. Male diorchic with testes paired and op- posed. Tail conoid, more convex-curved ventrally than that of the female, with rounded terminus at the end of tail (Figs 1(6), 2(8), Table 2). Spicules short, moderate- ly developed, and quite curved ventrally; lateral guid- ing pieces more or less straight, sometimes with slightly curved proximal ends (Figs 1(4, 6), 2(8, 10), Table 2). Large number of visible supplements, one pair of adanal and 9-11 mid-ventral supplements (Figs 1(3), 2(8)). Differential diagnosis. Longidorus bordonensis sp. nov. 1s characterized by a short body within the genus Longidorus (average = 4443 um and 4560 um in females and males, respectively), short odontostyle within the genus Longidorus (average = 70.0 um and 69.5 um in females and males, respectively), lip region anteriorly flattened, expanded (average = 10.0 um in both females and males) and rounded laterally, set-off from body contour by a constriction, asymmetrically bilobed amphidial pouches with lobes occupying from 2/3 to 3/4 part of the distance from oral aperture to guiding ring, tail long (average = 51.0 um and 55.0 um in females and males, respectively), bluntly conoid, slightly ventrally curved with rounded terminus, short to medium spicules (average = 37.0 um) with one pair of adanal and 9-11 mid- ventral supplements (Figs 1(1—7), 211-10), Table 2, Suppl. material 4: Table S2). According to the polytomous key of Chen et al. (1997) and two subsequent supplements (Loof and Chen 1999; Peneva et al. 2013), L. bordonensis sp. nov. has the following codes (codes in parentheses are exceptions): A2, Bl, C2, D4, E3, F2(3), G3, H(5)6, 12, J?, K?. On the basis of the diagnostic characters (body length, odontostyle length, lip region width, shape of anterior region, shape of amphidial pouch, oral aperture-guiding ring distance, tail length, spicule length, tail shape, a and Cc ratios, and frequency of males) used in the polytomous key by Chen et al. (1997), and supplements by Loof and Chen (1999) and Peneva et al. (2013), L. bordonensis sp. nov. is grouped with L. indalus Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares- Rius & Castillo, 2016, L. carpetanensis Arias, Andrés & Navas, 1986 , L. unedoi Arias, Andrés & Navas, 1986, L. juvenilis, L . pini Jacobs & Heyns, 1987, L. pisi Edward, Zoosyst. Evol. 96 (1) 2020, 175-193 ~- Figure 2. Light micrographs of Longidorus bordonensis sp. nov. paratypes from the rhizosphere of grass (unknown species) at Sao Pedro do Sul, Viseu district, northern Portugal (1-10). 1. Anterior region. 2. Odontostyle region. 3. Lip region showing amphidial fovea. 4. Odontophore region. 5. Detail of basal bulb. 6, 7. Female tail region. 8. Male tail region. 9. vulva region. 10. Detail of spic- ule region. Abbreviations: a anus, af amphidial fovea, ed cardia, gr guiding ring, ost odontostyle, odph odontophore, sp spicules, spl ventromedian supplements, V vulva, vg vagina. Scale bars: 23 um (1, 4); 15 um (2, 3, 5); 50 um (6, 7); 25 um (8, 10); 30 um (9). zse.pensoft.net 182 Carlos Gutiérrez-Gutiérrez et al.: Description of Longidorus bordonensis sp. nov. from Portugal Table 2. Morphometrics of females and males of Longidorus bordonensis sp. nov. from the rhizosphere of grass (unknown) at Bor- donhos, Sao Pedro do Sul, Viseu district, Beira Alta province, northern Portugal. Characters/ratios Holotype n 1 ks 4115.8 a 1267 b 12.8 ¢ 78.6 wn 2.3 2.3 + 0.4(1.9-3.1) VorT 45.6 G, (%) Fine, G,(%) 8.0 Odontostyle length 671 Odontophore length 45.6 Lip region width oo Oral aperture-guiding ring 2o.0) Tail length 5233 Spicules - Lateral accessory piece - J 8.0 Females 4443.0 + 593.1 (3671.4-5396.2) 135.8 + 13.1 (115.4-153.6) 13.8 + 1.8 (10.7-15.9) 88.0 + 13.7 (67.7-110.1) 46.7 + 2.0 (44.4-50.2) 8.4 + 0.5 (7.5-8.7) A 8.0 + 1.2 (6.7-9.5) = 69.8 + 2.4 (67.1-73.5) 47.0 + 3.7 (42.4-52.2) 10.1 + 0.4 (9.6-10.7) 24.7 + 1.4 (23.0-26.7) 51.0 + 6.8 (43.1-63.8) 9.2 + 0.7 (8.1-9.8) Males Y, 6 4560.0 + 357.4 (4188.6-5201.0) 155.3 + 11.5 (133.4-164.7) 13.9 + 1.1 (12.3-15.7) 83.4 + 9.7 (72.4-95.0) 2.3 O:2C1..9=275) 35.3 + 8.7 (24.5-42.0) 69.5 + 2.3 (67.1-73.4) 45.5 + 7.1 (37.0-54.6) 9.9 + 0.7 (8.8-10.8) 25.9 + 1.0 (24.8-27.6) 55.0 + 4.6 (46.9-59.5) _ 37.0 + 3.4 (32.6-41.8) = 8.4 + 0.5 (8.1-8.7) (n = 2) 9.1 + 0.6 (8.2-9.8) “Abbreviations are defined in Jairajpuri and Ahmad 1992. (—) Not obtained or not performed. Misra & Singh, 1964, and L. distintus Lamberti, Choleva & Agostinelli, 1983. Morphological and morphometric characters of the new species are compared with its closely related species (Suppl. material 4: Table S2). Longidorus bordonensis sp. nov. differs from paratypes of L. pini by small differences in the distance from the guiding ring to the anterior end (23.0—26.7 um vs 26-27 um), amphidial pouch shapes (asymmetrically bilobed with lobes occupying from 2/3 to 3/4 of the oa—gr distance vs symmetrically bilobed with lobes occupying from 1/3 to 2/3 of the oa—gr distance), tail shape (bluntly conoid, slightly ventrally curved with round terminus vs tail long, conical dorsally convex and ventrally concave, with the round terminus slightly subdigitate) and the frequency of males (common vs absent). Also, the new species differs from some previously cited species in measurements and ratios, including L, c’ and a ratios, odontostyle length, lip region diameter and shape, the distance from guiding ring to anterior, and tail length and shape (Suppl. material 4: Table S2). Longidorus vinearum Bravo & Roca, 1995 Suppl. material 1: Fig. S1(1-9), Suppl. material 5: Table S3 Remarks. Longidorus vinearum was originally described from around roots of grapevine (Vitis L.) in Dois Portos, Torres Vedras, Portugal (Bravo and Roca 1995). Subse- quently, Bravo and Roca (1998) reported it from the rhi- zosphere of olive trees (Olea europaea L.) in Matela, Vi- mioso, Portugal. Recently, Archidona- Yuste et al. (2016) found four populations of this species associated with wild olive trees in Andalusia (Spain) and characterized these populations molecularly. Three populations resembling this Longidorus species were detected parasitizing grape- vine roots at Dois Portos, Torres Vedras (type locality of L. vinearum), Ordasqueira, Torres Vedras, and Picanceira, zse.pensoft.net Mafra, and another population from around the roots of wild olive trees at Valverde, Evora, all in Portugal (Table 1, Suppl. material 5: Table S3). These populations prompted us to characterize them genotypically and phenotypical- ly, including the topotype specimens, in order to confirm their identification. Unfortunately, only one specimen was found at Picanceira, Mafra (Table 1) and used to complete the molecular analysis. These findings represent the third and fourth records of this species for Portugal and the Ibe- rian Peninsula, respectively. We confirm a wider geograph- ical distribution of this species in this geographical region. Longidorus vinearum populations are characterized by a lip region, which is broadly rounded frontally, and more so laterally, and almost totally continuous with the outline of the body; a vulva near mid-body; the amphidial fovea large and clearly asymmetrically bilobed; the odonto- style long and robust; short tail characterized by having a bluntly rounded to hemispherical shape, dorsal side quite more convex than ventral side with rounded terminus; males characterized by large-sized spicules (average = 112.0 um) and a large number of supplements, one pair of adanal and 18 or 19 mid-ventral supplements (Suppl. ma- terial 1: Fig. S1(1-9); Suppl. material 5: Table S3). Mor- phological and morphometrical traits of the topotype pop- ulation from Dois Portos, Torres Vedras (Suppl. material 5: Table S3) agree very well with the original description (Bravo and Roca 1995). Morphometric measurements of adult specimens of the topotype population are coincident with those provided in the original description (Bravo and Roca 1995) (Suppl. material 5: Table S3) except for minor differences in a and c’ ratios (69.2—79.8 vs 70.7—-101.3; 0.6-0.7 vs 0.5—0.8), lip region diameter (21.9-23.4 vs 18.0—27.5), length from the oral aperture to guiding ring (34.5—-43.2 vs 36.0-47.0), tail length (45.3-61.8 vs 38.0- 57.0), odontostyle length (113.8-126.4 vs 105.5—132.0), and odontophore length (65.3—82.5 vs 58.0—85.0) for the females (Suppl. material 5: Table $3), which may be due Zoosyst. Evol. 96 (1) 2020, 175-193 to intraspecific variability, as reported by Archidona- Yuste et al. (2016). Also, the topotype population shows simi- larity to four populations from Cordoba province, south- ern Spain (Archidona- Yuste et al. 2016); however, minor differences were detected in females such as L, a and c’ ratios, lip region diameter length, odontostyle and odonto- phore lengths, distance from oral aperture to guiding ring, and, in males, spicule length. In addition, the topotype population agrees closely with the morphological features and morphometric measurements of all Portuguese popu- lations examined (Suppl. material 5: Table S3), except for a higher V ratio (47.1—50.1 vs 45.8-51.4, 44.0), a longer odontostyle (113.8-126.4 vs 112.9-121.7, 116.8) um and a smaller J length (11.7—17.4 vs 13.1—21.3, 19.7) um (Suppl. material 5: Table S3). Nevertheless, these differ- ences further expand the intraspecific variation of the spe- cies and should be regarded as geographical intraspecific variation. According to the polytomous key by Chen et al. (1997) and its supplements (Loof and Chen 1999; Peneva et al. 2013), the topotypes and other studied Portuguese populations of this species have the following codes: A45, B45, C34, D2, E3, F45, G1, H1, 112, J?, K?. Unfortunate- ly, we did not detect the first juvenile-stage. However, this stage was characterized in the original description (Bra- vo and Roca 1995) and later by Archidona-Yuste et al. (2016), who also characterized this species molecularly. Longidorus vineacola Sturhan & Weischer, 1964 Suppl material 2: Fig. S2(1—11), Suppl.material 6: Table S4 Remarks. Three populations of L. vineacola from cork oak (Quercus suber L.) trees at Amoreirinhas da Cima, Montemor-o-Novo, Portugal and one population from wild olive (Olea europaea L. var. sylvestris) at Valverde, Evora, Portugal, are characterized morphometrically and morphologically: body medium-length to long (6.9— 9.6 mm in females and 6.4—-8.2 mm in males); odonto- style long (87.0—99.5 um in females and 91.5—100.9 um in males); lip region slightly set off from body contour by a depression; amphidial pouches asymmetrically bilobed; two equally developed female genital branches; females with broadly rounded tail usually as long as anal diam- eter; vulva posterior to mid-body; males with spicules well developed (69.9-79.9 um long), and supplements consisting of an adanal pair and 14 or 15 ventromedians (Suppl. material 2: Fig. S2(1—11); Suppl. material 6: Ta- ble S4). The morphological and metrical traits closely agree with the original description of the species (Sturhan and Weischer 1964) and subsequent records (Boag and Brown 1987; Brown and Taylor 1987; Andrés et al. 1991; Roca and Bravo 1996; Bravo and Lemos 1997; Brown et al. 1997; Gutiérrez-Gutiérrez et al. 2013, 2016; Archi- dona-Yuste et al. 2016), except for minor intraspecific variations in a and c ratios and length of odontostyle and spicules (Suppl. material 6: Table S4). This species was originally described parasitizing grapevine roots in Ger- many (Sturhan and Weischer 1964) and has since been 183 reported in a large number of records from various Eu- ro-Mediterranean countries from a wide range of herba- ceous and woody hosts (Bravo and Lemos 1997; Brown et al. 1997; Taylor and Brown 1997; Gutiérrez-Gutiérrez et al. 2013, 2016; Archidona- Yuste et al. 2016). The alpha- numeric codes for these populations of L. vineacola were applied in the diagnostic identification key for Longidorus spp. by Chen et al. (1997) and successive supplements by Loof and Chen (1999) and Peneva et al. (2013); they are (codes in parentheses are exceptions): A3(4), B3(4), C(2)3 ,D2, E3, F45, G23, H1, 12, J?, K?. Unfortunately, we detected no juvenile stages in our surveys; however, four juvenile stages were described by Sturhan and Weis- cher (1964) and by Roca and Bravo (1996). Additional- ly, Gutiérrez-Gutiérrez et al. (2013) using an integrative strategy characterized females, males, and first-stage Ju- veniles (J1) of several populations of southern Spain and assigned molecular markers for this species. Molecular results and phylogenetic relationships of Longidorus bordonensis sp. nov. and other Longidorus spp. Polymerase chain reaction (PCR) was used to amplify the D2—D3 expansion segments of 28S rRNA, ITS] rRNA, and partial 18S rRNA from L. bordonensis sp. nov. and four other Longidorus spp. For each of the species stud- ied, these three genes had an approximate size of 700— 800, 900-1000, and 1600 bp, respectively, based on vi- sualization of the band on the electrophoresis gel and the subsequent direct sequencing. D2-D3 sequences of L. bordonensis sp. nov. (MN082421—MN082422) matched well with the Longidorus spp. deposited in GenBank. Both D2—D3 sequences of L. bordonensis sp. nov. (MN082421 and MN082422) were almost identical, with a 99.31 % of se- quence similarity. D2—D3 sequences of the new species were 95, 95, and 87%, similar to L. pini (MH430028, Spain), L. carpetanensis (MH430019—MH430020, Spain), and Longidorus sp. 1 FG-2018 isolate (MG765547, Iran) , respectively, and differed in 27, 28-30, and 75 nucle- otides, respectively. ITS1 sequence of L. bordonensis sp. nov. (MN150062) appropriately matched with other Longidorus spp. deposited in GenBank. This ITS1 se- quence was 83-82, and 83% similar to L. carpetanenesis (MH429991—MH429993, Spain) and L. pini (MH430001, Spain), respectively. The variations among the ITS1 se- quences of these species were from 143 to 157 nucleotides. The partial 18S rRNA gene sequences of L. bordonensis sp. nov. (MN129757) showed a high homology (more than 99% similarity) with two sequences deposited in GenBank belonging to L. carpetanensis (MH430006, Spain) and L. pini (MH430011, Spain). The variations among the 18S sequences of these species were from 8 to 15 nucleotides. The D2—D3 expansion segments of 28S rRNA, ITS1 rRNA, and the partial 18S rRNA gene sequences ob- tained in this study for L. vinearum, L. vineacola, and zse.pensoft.net 184 Carlos Gutiérrez-Gutiérrez et al.: Description of Longidorus bordonensis sp. nov. from Portugal L. wicuolea matched well with sequences from the same species previously deposited in GenBank, increasing knowledge of the genotypic diversity in Longidorus (Ta- ble 1). For the species of Longidorus studied here, there were multiple failed attempts to sequence the ITS1 re- gion and a partial portion of 18S rRNA gene before our study was concluded (Table 1). The D2—D3 expansion segments of 28S rRNA gene sequences from L. vinearum (MN082430—MN082434) matched closely (99% similar- ity) to sequences of Spanish populations of this species in GenBank (KT308874, KT308876-KT308877); and the variations among these D2—D3 sequences ranged from 2 to 5 nucleotides. Intra-specific variation of D2- D3 detected among the populations of L. vinearum (three from grapevine and one from wild olive) (Table 1) was from 0 to 2 nucleotides (99% similarity and 0-1 indels). For L. vinearum, three ITS1 sequences (MN150065, MN150067, MN150068) from Dois Portos (LISB-03- 04, grapevine) and an ITS1 sequence (MN150066) from Evora (M3-OLYV, wild olive) were sequenced and showed a high similarity (99%), with some minor intra-specific variations among them (2-14 nucleotides and 0-1 in- dels). Our ITS1 sequences (MN150065—MN150068) had 98% similarity to others deposited in GenBank for L. vinearum (KT308892-KT308893, Spain); and the variations among them ranged from 17—23 nucleotides and 1-3 indels. The D2—D3 expansion segments of 28S rRNA gene sequences from L. vineacola (MN082425-— MN082429) also had 99% similarity to several sequenc- es of Spanish populations of this species in GenBank (JX445109-JX445111; KT308872, KT308873); and the variations among them ranged from 2 to 9 nucleotides. Intra-specific variation of the D2—D3 region among the four populations of L. vineacola (three from cork oak and one from wild olive) (Table 1) was low, varying from 0 to 6 nucleotides (99-100% similarity and 0 indels). For L. vineacola, the ITS1 region and the partial portion of 18S gene sequenced agree with results obtained from the D2— D3 fragments. The partial 18S rRNA gene sequence of L. vineacola (MN129758) was identical (100% similarity) to several L. vineacola sequences deposited in GenBank (AY283169, UK; JX445123, Spain), and 99% similar to L. onubensis Archidona-Yuste, Navas Cortés, Can- talapiedra-Navarrete, Palomares-Rius & Castillo, 2016 (KT308897, Spain), L. nevesi (MH430009, Spain), L. wicuolea (KT308900, Spain), L. fasciatus (MH430008, Spain; JX445122, Spain), and L. pacensis Archido- na-Yuste, Cantalapiedra-Navarrete, Castillo & Palo- mare-Rius, 2019 (MH430004—MH430005, Spain). The variations among the partial 18S rRNA gene sequences of these species varied from 8 to 16 nucleotides. Our ITS1 sequence of L. vineacola (MN150064) showed a varia- ble and low sequence homology with other sequences of L. vineacola in Genbank; the homology ranged from 97% (JX445094, Spain) to 94% (JX445096, Spain). The variations among the ITS1 sequences of these three se- quences ranged from 18 to 51 nucleotides. The D2—D3 expansion segments of 28S rRNA gene sequence from zse.pensoft.net L. wicuolea (MN082423) were 99% similar to three se- quences of Spanish populations of this same species in GenBank (KT308863—KT308865, Spain). The variations among these four D2—D3 sequences ranged from 1 to 5 nucleotides. For L. wicuolea, the ITS1 sequences agree with results from the D2—D3 region. Our ITS1 sequence of L. wicuolea (MN150063) was 100% identical to other two sequences of this species in Genbank (KT308887 and KT308889, Spain) and clearly different (90%—88% simi- larity) from L. silvestris Archidona-Yuste, Navas Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, 2016 (KT308884, Spain), and L. cf. olegi Kankina & Metlitskaya, 1983 (MH429999, MH430000, Spain); and the variations among these ITS1 sequences ranged from 18 to 51 nucleotides. The D2—D3 expansion segments of 28S rRNA gene sequence from Longidorus sp. 3 isolate ST (MN082424) matched well with several Longidorus spp. deposited in GenBank, including L. J/usitanicus (KT308869, Spain) as the closest with 98.55% similarity, followed by L. magnus Lamberti, Bleve-Zacheo & Ari- as, 1982 (JX445113 and KT308870, Spain), L. cratae- gi Roca & Bravo, 1996 (JX445114, Spain), L. goodeyi Hooper, 1961 (AY601581), and L. vinearum (KT308876, Spain) with 94-95% similarity; and the sequence varia- tions among the D2—D3 sequences of these species were from 11-42 nucleotides and 1-12 indels . Using Bayesian inference (BI), we compared the phy- logenetic position of L. bordonensis sp. nov. and other Longidorus spp. by using the D2—D3 expansion segments of 28S rRNA, the ITS1 region, and the partial 18S rRNA gene sequences (Figs 3—5). The BI tree (50% majority rule consensus tree) of the D2—D3 domains of 28S rRNA gene (Fig. 3) was based on a multiple-edited alignment (135 to- tal sequences) of 722 total characters and revealed a major clade containing the majority of these species, including L. bordonensis sp. nov. and the remaining Iberian popu- lations of Longidorus spp. (Fig. 3). The generated phy- logenetic tree, using sequences of these D2—D3 fragments (Fig. 3), showed a clearly congruent position of L. bordon- ensis sp. nov. (MN082421, MN082422). The clade, in- cluding L. bordonensis sp. nov., grouped morphologically related species characterized by a short body and odonto- style and elongate to conical female tail, such as in L. pini (MH430028, Spain) and L. carpetanensis (MH430019, MH430020, Spain). The D2—D3 tree showed a consistent position for L. vinearum (MN082430—MN082434), which was placed within a well-supported clade of available GenBank entries belonging to L. vinearum (KT308874, KT308876, Spain) and clearly separated from the new species and other morphologically related species, such as L. magnus (KT308870, KX445113, Spain), L. crataegi (JX445114, Spain), L. goodeyi (AY601581), L. onubensis (KT308857, KT308858, Spain), L. oakcrassus Cai, Archi- dona-Yuste, Cantalapiedra-Navarrete, Palomares-Rius & Pablo Castillo, 2019 (MK941187—MK941190), L. oak- gracilis Cai, Archidona-Yuste, Cantalapiedra-Navarrete, Palomares-Rius & Pablo Castillo, 2019 (MK941191- MK941193), L. wicuolea (KT308863—-KT308865, Zoosyst. Evol. 96 (1) 2020, 175-193 185 Miphinema index(HM921406) Figure 3. Phylogenetic relationships of Longidorus bordonensis sp. nov., L. wicuolea Archidona-Yuste, Navas-Cortés, Cantalapie- dra-Navarrete, Palomares-Rius & Castillo, L. /usitanicus Macara, 1986, L. vinearum Bravo & Roca and L. vineacola Sturhan & Weischer, 1964 within the genus Longidorus. Bayesian 50% majority rule consensus trees as inferred from D2—D3 expansion seg- ments of 28S rRNA sequences alignments under the SYM model. Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences in this study are coloured in purple. Scale bar: expected changes per site. Spain; MN022423, Portugal), L. andalusicus (JX445101, Spain), and L. vineacola (JX445111, Spain; MN082425— MN082429 Portugal). Likewise, this D2—D3 tree also showed congruence for the phylogenetic positions of L. vineacola sequences obtained here (MN082425— MN082429), as it was positioned within a well-supported clade together an available sequence in Genbank belonging to L. vineacola (JX445111, Spain). This clade, including all L. vineacola sequences, were separated from the new species and other phenotypically similar species, such as L. cf. olegi (MH430026—MH430027, Spain), L. silvestris (KT308859, Spain), L. /usitanicus (KT308869, Spain), L. oakcrassus (MK941187—MK941190), and L. wicuolea (KT308863—KT308865, Spain; MN022423, Portugal). In addition, Longidorus sp. 3 isolate ST (MN082424) was placed in a separated position within a well-supported sub- clade, clustering together to L. /usitanicus (KT308889, Spain) and L. crataegi (JX445114, Spain). Similarly, the BI tree (50% majority rule consen- sus tree) of a multiple-edited alignment, including 116 18S rRNA sequences and 1690 total characters (Fig. 4) and 116 ITS1 sequences and 570 total char- acters (Fig. 5), showed a topology similar to that of the D2—D3 fragments of the 28S gene. Both the par- tial 18S and ITS1 trees using BI (Figs 4, 5) showed a close phylogenetic relationship of L. bordonensis sp. nov. (18S, MN129757; ITS1, MN150062) with L. pini (18S, MH430011, Spain; ITS1, MH430001, Spain) zse.pensoft.net 186 Carlos Gutiérrez-Gutiérrez et al Xiphinema turdetanesis(KC567155) Xiphinema citricolum (AM086686) .. Description of Longidorus bordonensis sp. nov. from Portugal £. pini(MH430011) 0.81 fa L. carpetanensis (MH430006) L. bordonensissp. nov. (MN129757) bed L. iuglandis (3X445120) L. baeticus(3X445121) £. pacensis (MH430005) " L. pacensis (MH430004) L, fasciatus (MH430008) L. fasciatus (JX445122) ~L. oleae (3X445119) L. andalusicus (3K445118) L. vineacola (JX445123) + wy L. vineacola (MN129758) L. vineacola (AY283169) iL. magnus (KT308902) ” L. magnus (HM921345) L. crataegi (1X445124) L. artemisiae (KY¥119959) — L. artemisiae (KF242286) L. intermedius (KF242284) L. aetnaeus (KF242287) L. distinctus (KF242290) Longidorussp. 6 SAS-2014 (KF242289) Longidorussp. 1 SAS-2014 (KF242288) L. persicus (KU747176) iL. evonymus(EUS03144) L. evonymus(AY687995) iL. evonymus(KF242291) L. kheirii (EU503146) L. profundorum(EUS03143) Longidorussp. 9 Mile 3-30 (AY146472) L. breviannulatus(AY283161) L. crassus (AY283158) 0.99 L. biformis (AY283171) — L. americanum (AY283168) L. paralongicaudatus(AY283160) Longidorussp. DLP003 (AB477085) L. breviannulatus(GQ251040) Longidorussp. 3 SAS-2014 (KF242292) L. grandis (AY283165) : £. paravineacola (AY283159) L. paravineacola (AY283157) L. fragilis (AY283172) “ F] 0.94 0.98 moesicus (KI802899) L. sturhani(FI009677) Longidorussp. 4 SAS-2014 (KF242293) 0.89 0.97 L a L. rubi (YX445125) L. profundorum(3Q359005) L. polyae(MK172047) L. henanus(MF674015) i. henanus(MF674014) — Longidorussp. JH-2014 (KI741238) a Longidorussp. JA-2014 (KI755428) ‘_ Lengidorussp. JM-2014 (KI755427) L. asiaticus (KR351259) 2279) a Longidorussp. ITDL-2011 (HQ735099) L. ferrisi (AY283163) L. pisi (MK172049) L. caespiticola (KI567467) L. lignosus (KF242281) P. plesioepimikis (1Q673405) — P. lusitanicus (KY¥750569) 1 0.98 P. maximus (AI875152) P. litoralis (EU026159) P. (£u026157) 1 P. iranicus (JN032589) P. rex (K}427794) P. bikanerensis (11032586) 0.96 L. lgevicapitatus (KX136874) 1. laevicapitatu