An Integrated Approach for Synonymization of Rotylenchus rhomboides with R. goodeyi (Nematoda: Hoplolaimidae) Reveals High Intraspecific Mitogenomic Variation.

Rotylenchus is a widely distributed, economically important plant-parasitic nematode group whose species-level identification relies largely on limited morphological characters, including character-based tabular keys and molecular data of ribosomal and mitochondrial genes. In this study, a combined morphological and molecular analysis of three populations of Rotylenchus goodeyi from Belgium, Poland, and the Netherlands revealed important character variations of this species, leading to synonymization of R. rhomboides with R. goodeyi and a high nucleotide variation within cox1 gene sequences in these populations. Additional Illumina sequencing of DNA from individuals of the Dutch population revealed two variants of mitogenomes, each approximately 23 Kb in size, differing by approximately 9% and containing 11 protein-coding genes, 2 ribosomal RNA genes, and as many as 29 transfer RNA genes. In addition to the first representative whole-genome shotgun sequence datasets of the genus Rotylenchus, this study also provides the full-length mitogenome and the ribosomal DNA sequences of R. goodeyi.

Paratylenchus Ilicis n. Sp. (Nematoda: Paratylenchinae) Associated with Holly from the Netherlands and New Taxonomical and Phylogenetic Support for the Synonymization of Cacopaurus with Paratylenchus.

Paratylenchus ilicis n. sp. was found associated with holly in the Netherlands and was described based on morphology, morphometrics, rRNA and mitochondrial cox1 genes, phylogenetic relationships with other Paratylenchus species, host information and geographical distribution. This species can be morphologically diagnosed based on its light brown, slightly obese to obese females with tubercles on cuticle, lateral bands widening into an ovoid field around vulva level, stylet length of 70 µm to 100 µm, outstretched to reflexed ovary, rounded sperm-filled spermatheca, vagina opening into a thick-walled rounded space, absence of vulval flaps and vulva at 89% to 95% of body length, very short tail in all life stages, and a characteristic finger-like tail tip in juveniles (J2). The new species is morphologically closest to Cacopaurus pestis but differs based on the absence of a scutellum-like differentiation in the lateral field, the ovaries that can be outstretched or reflexed, and the finger-like tail tip in J2. Furthermore, both species were found to be molecularly distant from one another, found in different habitats, and are associated with different hosts. The high morphological similarity between Cacopaurus and Paratylenchus and our phylogenetic analyses, revealing that the former is embedded within different Paratylenchus clades and thus polyphyletic, provide new evidences for the synonymization of Cacopaurus with Paratylenchus.

PPNID: a reference database and molecular identification pipeline for plant-parasitic nematodes.

MOTIVATION: The phylum Nematoda comprises the most cosmopolitan and abundant metazoans on Earth and plant-parasitic nematodes represent one of the most significant nematode groups, causing severe losses in agriculture. Practically, the demands for accurate nematode identification are high for ecological, agricultural, taxonomic and phylogenetic researches. Despite their importance, the morphological diagnosis is often a difficult task due to phenotypic plasticity and the absence of clear diagnostic characters while molecular identification is very difficult due to the problematic database and complex genetic background. RESULTS: The present study attempts to make up for currently available databases by creating a manually-curated database including all up-to-date authentic barcoding sequences. To facilitate the laborious process associated with the interpretation and identification of a given query sequence, we developed an automatic software pipeline for rapid species identification. The incorporated alignment function facilitates the examination of mutation distribution and therefore also reveals nucleotide autapomorphies, which are important in species delimitation. The implementation of genetic distance, plot and maximum likelihood phylogeny analysis provides more powerful optimality criteria than similarity searching and facilitates species delimitation using evolutionary or phylogeny species concepts. The pipeline streamlines several functions to facilitate more precise data analyses, and the subsequent interpretation is easy and straightforward. AVAILABILITY AND IMPLEMENTATION: The pipeline was written in vb.net, developed on Microsoft Visual Studio 2017 and designed to work in any Windows environment. The PPNID is distributed under the GNU General Public License (GPL). The executable file along with tutorials is available at https://github.com/xueqing4083/PPNID. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Rotylenchus wimbii n. sp. (Nematoda: Hoplolaimidae) associated with finger millet in Kenya.

Rotylenchus wimbii n. sp. was found associated with finger millet in Kenya and is described based on light microscopy, scanning electron microscopy, and molecular information. Sequence analysis was performed on ITS, 18S, and D2-D3 of 28S of ribosomal DNA and COI of mitochondrial DNA. This new species is characterized by a moderate female body size of 0.6 to 0.8 mm, a continuous hemispherical lip region with four annuli, 3 to 4 irregular blocks on the basal lip annule, absence of longitudinal cuticular striations in anterior region, four lateral lines forming three equal bands which are areolated mainly at pharynx level, a robust stylet of 23 to 27 µm of which 45 to 53% is cone part, and with rounded to sometimes indented knobs, a secretory-excretory pore around level of pharyngo-intestinal junction, didelphic-amphidelphic reproductive system, vulva without distinct epiptygma, indistinct to empty spermatheca, tail usually truncated with 5 to 9 annuli, phasmids located at 7 to 17 annuli anterior to anus, and absence of males. Molecular phylogenies, in combination with species delimitation, supported the distinctiveness of Rotylenchus wimbii n. sp. and revealed some mislabeled Rotylenchus brevicaudatus sequences in GenBank.

Morphological and molecular characterization of Heterodera dunensis n. sp. (Nematoda: Heteroderidae) from Gran Canaria, Canary Islands.

Heterodera dunensis n. sp. from the coastal dunes of Gran Canaria, Canary Islands, is described. This new species belongs to the Schachtii group of Heterodera with ambifenestrate fenestration, presence of prominent bullae, and a strong underbridge of cysts. It is characterized by vermiform second-stage juveniles having a slightly offset, dome-shaped labial region with three annuli, four lateral lines, a relatively long stylet (27-31 µm), short tail (35-45 µm), and 46 to 51% of tail as hyaline portion. Males were not found in the type population. Phylogenetic trees inferred from D2-D3 of 28S, partial ITS, and 18S of ribosomal DNA and COI of mitochondrial DNA sequences indicate a position in the 'Schachtii clade'.

Plant-parasitic nematodes associated with sugarcane in Kilimanjaro, Tanzania.

Morphological and molecular analyses of plant-parasitic nematodes (PPN) from 12 sugarcane plantation sites of Tanganyika Planting Company (TPC) Limited in Kilimanjaro region of Tanzania revealed the presence of six PPN genera, i.e. Helicotylenchus, Hemicycliophora, Pratylenchus, Rotylenchulus, Scutellonema, and Tylenchorhynchus. The genera with the highest densities and present in virtually all samples were Pratylenchus and Rotylenchulus, and the most important species appeared to be R. parvus, P. zeae, T. crassicaudatus, and T. ventrosignatus. A total sequences of 11 partial ITS, 15 D2-D3 of 28S, and 6 partial 18S of rRNA gene, and 7 partial COI gene of mtDNA of these species were obtained in this study. Morphology and molecular data comparisons between the Tanzanian R. parvus and the South African R. parvus indicated that R. parvus is a cryptic species complex. Based on the results of morphological and molecular analyses of T. crassicaudatus and T. agri from China, Haiti, Indonesia, Iran, Niger and the USA, T. agri syn. n. is proposed as a junior synonym of T. crassicaudatus.Morphological and molecular analyses of plant-parasitic nematodes (PPN) from 12 sugarcane plantation sites of Tanganyika Planting Company (TPC) Limited in Kilimanjaro region of Tanzania revealed the presence of six PPN genera, i.e. Helicotylenchus, Hemicycliophora, Pratylenchus, Rotylenchulus, Scutellonema, and Tylenchorhynchus. The genera with the highest densities and present in virtually all samples were Pratylenchus and Rotylenchulus, and the most important species appeared to be R. parvus, P. zeae, T. crassicaudatus, and T. ventrosignatus. A total sequences of 11 partial ITS, 15 D2-D3 of 28S, and 6 partial 18S of rRNA gene, and 7 partial COI gene of mtDNA of these species were obtained in this study. Morphology and molecular data comparisons between the Tanzanian R. parvus and the South African R. parvus indicated that R. parvus is a cryptic species complex. Based on the results of morphological and molecular analyses of T. crassicaudatus and T. agri from China, Haiti, Indonesia, Iran, Niger and the USA, T. agri syn. n. is proposed as a junior synonym of T. crassicaudatus.

Molecular characterization and species delimiting of plant-parasitic nematodes of the genus Pratylenchus from the penetrans group (Nematoda: Pratylenchidae).

Root-lesion nematodes of the genus Pratylenchus are an important pest parasitizing a wide range of vascular plants including several economically important crops. However, morphological diagnosis of the more than 100 species is problematic due to the low number of diagnostic features, high morphological plasticity and incomplete taxonomic descriptions. In order to employ barcoding based diagnostics, a link between morphology and species specific sequences has to be established. In this study, we reconstructed a multi-gene phylogeny of the Penetrans group using nuclear ribosomal and mitochondrial gene sequences. A combination of this phylogenetic framework with molecular species delineation analysis, population genetics, morphometric information and sequences from type location material allowed us to establish the species boundaries within the Penetrans group and as such clarify long-standing controversies about the taxonomic status of P. penetrans, P. fallax and P. convallariae. Our study also reveals a remarkable amount of cryptic biodiversity within the genus Pratylenchus confirming that identification on morphology alone can be inconclusive in this taxonomically confusing genus.

Diversity of Root-knot Nematodes Associated with Tubers of Yam (Dioscorea spp.) Established Using Isozyme Analysis and Mitochondrial DNA-based Identification.

The root-knot nematodes (RKN), Meloidogyne spp., represent an important threat to yam (Dioscorea spp.) production in West Africa. With the aim to establish the diversity of RKN species affecting yam tubers, for control and resistance screening purposes, surveys were conducted in the main yam producing areas of Nigeria. Galled tubers (N = 48) were collected from farmers' stores and markets in nine states in Nigeria and in one district in Ghana. RKN isolated from yam tubers were identified using enzyme phenotyping (esterase and malate dehydrogenase) and mitochondrial DNA (mtDNA) NADH dehydrogenase subunit 5 (Nad5) barcoding. Examination of 48 populations revealed that yam tubers were infested by Meloidogyne incognita (69%), followed by M. javanica (13%), M. enterolobii (2%), and M. arenaria (2%). Most of the tubers sampled (86%) were infected by a single species, and multiple species of RKN were detected in 14% of the samples. Results of both identification methods revealed the same species, confirming their accuracy for the identification of these tropical RKN species. In addition to M. incognita, M. javanica, and M. enterolobii, we report for the first time M. arenaria infecting yam tubers in Nigeria. This finding extends the list of yam pests and calls for caution when developing practices for yam pest management.

Small subunit ribosomal DNA-based phylogenetic analysis of foliar nematodes (Aphelenchoides spp.) and their quantitative detection in complex DNA backgrounds.

Foliar nematodes, plant-parasitic representatives of the genus Aphelenchoides, constitute a minority in a group dominated by fungivorous species. Distinction between (mostly harmless) fungal feeding Aphelenchoides species and high impact plant parasites such as A. besseyi, A. fragariae, A. ritzemabosi, and A. subtenuis is severely hampered by the scarcity of informative morphological characters, some of which are only observable in specific developmental stages. Poor description of a number of non-plant-parasitic Aphelenchoides species further complicates identification. Based on (nearly) full-length small subunit ribosomal DNA (SSU rDNA) sequences (≈1,700 bp), a phylogenetic tree was generated, and the four target species appeared as distinct, well-supported groups. Notably, this genus does not constitute a monophyletic group: A. besseyi and A. ritzemabosi cluster together and they are phylogenetically isolated from A. fragariae, A. subtenuis, and most other fungivorous species. A phylum-wide SSU rDNA framework was used to identify species-specific DNA motifs. For the molecular detection of four plant-parasitic Aphelenchoides species, polymerase chain reaction primers were developed with high, identical annealing temperatures (63°C). Within the molecular framework presented here, these primers can be used for the rapid screening of plant material and soil for the presence of one or multiple foliar nematode species.

Integrative Taxonomy and Molecular Phylogeny of the Plant-Parasitic Nematode Genus Paratylenchus (Nematoda: Paratylenchinae): Linking Species with Molecular Barcodes.

Pin nematodes of the genus Paratylenchus are obligate ectoparasites of a wide variety of plants that are distributed worldwide. In this study, individual morphologically vouchered nematode specimens of fourteen Paratylenchus species, including P. aculentus, P. elachistus, P. goodeyi, P. holdemani, P. idalimus, P. microdorus, P. nanus, P. neoamblycephalus, P. straeleni and P. veruculatus, are unequivocally linked to the D2-D3 of 28S, ITS, 18S rRNA and COI gene sequences. Combined with scanning electron microscopy and a molecular analysis of an additional nine known and thirteen unknown species originating from diverse geographic regions, a total of 92 D2-D3 of 28S, 41 ITS, 57 18S rRNA and 111 COI new gene sequences are presented. Paratylenchus elachistus, P. holdemani and P. neoamblycephalus are recorded for the first time in Belgium and P. idalimus for the first time in Europe. Paratylenchus is an excellent example of an incredibly diverse yet morphologically minimalistic plant-parasitic genus, and this study provides an integrated analysis of all available data, including coalescence-based molecular species delimitation, resulting in an updated Paratylenchus phylogeny and the corrective reassignment of 18 D2-D3 of 28S, 3 ITS, 3 18S rRNA and 25 COI gene sequences that were previously unidentified or incorrectly classified.

Global Distribution of the Reniform Nematode Genus Rotylenchulus with the Synonymy of Rotylenchulus macrosoma with Rotylenchulus borealis.

Reniform nematodes of the genus Rotylenchulus are semi-endoparasites of numerous herbaceous and woody plant roots that occur largely in regions with temperate, subtropical, and tropical climates. In this study, we compared 12 populations of Rotylenchulusborealis and 16 populations of Rotylenchulusmacrosoma, including paratypes deposited in nematode collections, confirming that morphological characters between both nematode species do not support their separation. In addition, analysis of molecular markers using nuclear ribosomal DNA (28S, ITS1) and mitochondrial DNA (coxI) genes, as well as phylogenetic approaches, confirmed the synonymy of R. macrosoma with R. borealis. This study also demonstrated that R. borealis (= macrosoma) from Israel has two distinct rRNA gene types in the genome, specifically the two types of D2-D3 (A and B). We provide a global geographical distribution of the genus Rotylenchulus. The two major pathogenic species (Rotylenchulusreniformis and Rotylenchulusparvus) showed their close relationship with warmer areas with high annual mean temperature, maximum temperature of the warmest month, and minimum temperature of the coldest month. The present study confirms the extraordinary morphological and molecular diversity of R. borealis in Europe, Africa, and the Middle East and comprises a paradigmatic example of remarkable flexibility of ecological requirements within reniform nematodes.

Integrative taxonomy of root-knot nematodes reveals multiple independent origins of mitotic parthenogenesis.

During sampling of several Coffea arabica plantations in Tanzania severe root galling, caused by a root-knot nematode was observed. From pure cultures, morphology and morphometrics of juveniles and females matched perfectly with Meloidogyne africana, whereas morphology of the males matched identically with those of Meloidogyne decalineata. Based on their Cox1 sequence, however, the recovered juveniles, females and males were confirmed to belong to the same species, creating a taxonomic conundrum. Adding further to this puzzle, re-examination of M. oteifae type material showed insufficient morphological evidence to maintain its status as a separate species. Consequently, M. decalineata and M. oteifae are synonymized with M. africana, which is herewith redescribed based on results of light and scanning electron microscopy, ribosomal and mitochondrial DNA sequences, isozyme electrophoresis, along with bionomic and cytogenetic features. Multi-gene phylogenetic analysis placed M. africana outside of the three major clades, together with M. coffeicola, M. ichinohei and M. camelliae. This phylogenetic position was confirmed by several morphological features, including cellular structure of the spermatheca, egg mass position, perineal pattern and head shape. Moreover, M. africana was found to be a polyphagous species, demonstrating that "early-branching" Meloidogyne spp. are not as oligophagous as had previously been assumed. Cytogenetic information indicates M. africana (2n = 21) and M. ardenensis (2n = 51-54) to be a triploid mitotic parthenogenetic species, revealing at least four independent origins of mitotic parthenogenesis within the genus Meloidogyne. Furthermore, M. mali (n = 12) was found to reproduce by amphimixis, indicating that amphimictic species with a limited number of chromosomes are widespread in the genus, potentially reflecting the ancestral state of the genus. The wide variation in chromosome numbers and associated changes in reproduction modes indicate that cytogenetic evolution played a crucial role in the speciation of root-knot nematodes and plant-parasitic nematodes in general.

An early record of Meloidogyne fallax from Ireland.

Root-knot nematodes, Meloidogyne spp., cause huge economic losses worldwide. Currently, three Meloidogyne spp. are present on the quarantine A2 list of EPPO, Meloidogyne chitwoodi, Meloidogyne fallax and Meloidogyne enterolobii. As a quarantine organism, Meloidogyne fallax has been detected in England and Northern Ireland on sport turf in 2011, and in England on leek in 2013. However, its presence in Ireland has probably been overlooked since 1965, when Mr. John F. Moore and Dr. Mary T. Franklin had detected a new Meloidogyne species for that time. While the relevant data was recorded and a preliminary manuscript describing the species was prepared but never submitted for publication, and together with the original slides, pictures and drawings, it was restudied recently. We compared the population of Irish Meloidogyne sp. to other similar Meloidogyne spp. Careful observation and comparison shows that it belongs to Meloidogyne fallax. The characters found to be common for Irish Meloidogyne sp. and Meloidogyne fallax are female stylet length (14.6 µm) with oval to rounded basal knobs, oval shaped perineal pattern with moderately high dorsal arch, slender stylet in males (18.5 µm) with set off and rounded basal knobs, slightly set off male head with one post-labial annule and incomplete transverse incisures, and second-stage juveniles with large and rounded stylet basal knobs, and a gradually tapering tail (46.9 µm) with a broadly rounded tip and a clearly delimitated smooth hyaline part sometimes marked by constrictions (12.9 µm). The host test and gall formation also correspond to Meloidogyne fallax. The identification could not be additionally supported by molecular analysis, as we were unable to extract DNA from the old permanent slides. Nevertheless, our study reveals that the Meloidogyne species detected in Ireland in 1965 belongs to Meloidogyne fallax.

Morphological and molecular characterisation of Scutellonema species from yam (Dioscorea spp.) and a key to the species of the genus.

The yam nematode, Scutellonema bradys, is a major threat to yam (Dioscorea spp.) production across yam-growing regions. In West Africa, this species cohabits with many morphologically similar congeners and, consequently, its accurate diagnosis is essential for control and for monitoring its movement. In the present study, 46 Scutellonema populations collected from yam rhizosphere and yam tubers in different agro-ecological zones in Ghana and Nigeria were characterised by their morphological features and by sequencing of the D2-D3 region of the 28S rDNA gene and the mitochondrial COI genes. Molecular phylogeny, molecular species delimitation and morphology revealed S. bradys, S. cavenessi, S. clathricaudatum and three undescribed species from yam rhizosphere. Only S. bradys was identified from yam tuber tissue, however. For barcoding and identifying Scutellonema spp., the most suitable marker used was the COI gene. Additionally, 99 new Scutellonema sequences were generated using populations obtained also from banana, carrot, maize and tomato, including the first for S. paralabiatum and S. clathricaudatum, enabling the development of a dichotomous key for identification of Scutellonema spp. The implications of these results are discussed.

Disparate gain and loss of parasitic abilities among nematode lineages.

Plant parasitism has arisen time and again in multiple phyla, including bacteria, fungi, insects and nematodes. In most of these organismal groups, the overwhelming diversity hampers a robust reconstruction of the origins and diversification patterns of this trophic lifestyle. Being a moderately diversified phylum with ≈ 4,100 plant parasites (15% of total biodiversity) subdivided over four independent lineages, nematodes constitute a major organismal group for which the genesis of plant parasitism could be mapped. Since substantial crop losses worldwide have been attributed to less than 1% of these plant parasites, research efforts are severely biased towards this minority. With the first molecular characterisation of numerous basal and supposedly harmless plant parasites as well as their non-parasitic relatives, we were able to generate a comprehensive molecular framework that allows for the reconstruction of trophic diversification for a complete phylum. In each lineage plant parasites reside in a single taxonomic grouping (family or order), and by taking the coverage of the next lower taxonomic level as a measure for representation, 50, 67, 100 and 85% of the known diversity was included. We revealed distinct gain and loss patterns with regard to plant parasitism per se as well as host exploitation strategies between these lineages. Our map of parasitic nematode biodiversity also revealed an unanticipated time reversal in which the two most ancient lineages showed the lowest level of ecological diversification and vice versa.

Description of Basiria birjandiensis n. sp (Nematoda: Tylenchidae) from South Khorasan province with a checklist of the family Tylenchidae Örley 1880 from Iran.

In order to identify plant-parasitic nematodes (family Tylenchidae Örley 1880) associated with Ziziphus zizyphus in Iran, 360 soil and root samples were collected from South Khorasan province during 2012-2014. Herein, a new species of Basiria and several known members of the family Tylenchidae are reported. B. birjandiensis n. sp. is characterized by short body length (584-748 µm [660.6±72.3]), lip region with flat apex, stylet 11-12 µm (11.3±0.5), excretory pore position varying from isthmus level to the middle of the basal bulb (78-91 µm from the anterior end of the body), post-vulval uterine sac 8-14 µm (10.7±1.9) long, filiform tail (151-181 µm, c= 3.7-4.2, c´= 14.3-17.2) and body annuli 0.5-1 µm (0.6±0.1) wide. A checklist of Tylenchidae species from Iran is also presented.

Mitochondrial coding genome analysis of tropical root-knot nematodes (Meloidogyne) supports haplotype based diagnostics and reveals evidence of recent reticulate evolution.

The polyphagous parthenogenetic root-knot nematodes of the genus Meloidogyne are considered to be the most significant nematode pest in sub-tropical and tropical agriculture. Despite the crucial need for correct diagnosis, identification of these pathogens remains problematic. The traditionally used diagnostic strategies, including morphometrics, host-range tests, biochemical and molecular techniques, now appear to be unreliable due to the recently-suggested hybrid origin of root-knot nematodes. In order to determine a suitable barcode region for these pathogens nine quickly-evolving mitochondrial coding genes were screened. Resulting haplotype networks revealed closely related lineages indicating a recent speciation, an anthropogenic-aided distribution through agricultural practices, and evidence for reticulate evolution within M. arenaria. Nonetheless, nucleotide polymorphisms harbor enough variation to distinguish these closely-related lineages. Furthermore, completeness of lineage sorting was verified by screening 80 populations from widespread geographical origins and variable hosts. Importantly, our results indicate that mitochondrial haplotypes are strongly linked and consistent with traditional esterase isozyme patterns, suggesting that different parthenogenetic lineages can be reliably identified using mitochondrial haplotypes. The study indicates that the barcode region Nad5 can reliably identify the major lineages of tropical root-knot nematodes.

SEM Observations on the Marine Nematode Dracognomus simplex (Gerlach, 1954) Allen and Noffsinger, 1978 (Draconematidae: Prochaetosomatinae).

The free-living marine nematode Dracognomus simplex (Gerlach, 1954) Allen &Noffsinger, 1978 was studied by scanning electron microscopy (SEM). The morphology of males and females is described and illustrated in detail. In addition to the typical and modified adhesion tubes, a new type of posterior adhesion tube was discovered. A neotype is proposed for Dracognomus simplex, and D. simplex sensu Decraemer &Gourbault, 1986 is renamed as Dracognomus americanum n. sp. Additionally, a key toward the Dracognomus species is proposed.

Phylogenetic Analyses of Meloidogyne Small Subunit rDNA.

Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species.