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Longidorid nematodes comprise more than 500 species, and Longidorus and Xiphinema are the most diversified, prevalent, and cosmopolitan genera within plant‐parasitic nematodes. This increases the risk of species misidentification. We conducted an integrative morphometric and genetic study on two longidorid species to elucidate the existence of new cases of cryptic speciation within the genera Longidorus and Xiphinema. Detailed morphological, morphometrical, multivariate, and genetic studies were carried out, and mitochondrial and nuclear haploweb analyses were used to differentiate species within the L. iliturgiensis and X. hispanum complexes. Species delimitation using multivariate and haplonet tools of L. iliturgiensis species complex clearly separated L. tabernensis sp. nov. from L. iliturgiensis and L. indalus, and X. subbaetense sp. nov. from X. hispanum and X. adenohystherum. D2‐D3, partial 18S, and partial coxI regions were used for inferring their phylogenetic relationships with other species in each genus. The present study provides new insights into the diversity of Longidorus and Xiphinema species detected in southern Spain, and new evidence of cryptic speciation in both genera. These results support our hypothesis that the biodiversity of Longidoridae in southern Europe is higher than previously supposed and is still not fully clarified. [ABSTRACT FROM AUTHOR]

The genus Paratrichodorus currently comprises 26 species of polyphagous ectoparasitic plant nematodes that are distributed worldwide and include a few tobravirus vector species. Paratrichodorus is one of the most difficult genera for species identification because it is phenotypically conserved, with morphometric characters that often overlap between species. From 2003 to 2016, nematode surveys were conducted in southern Spain, and the present study addresses 22 nematode populations of the Paratrichodorus hispanus group. Among them, two new species, Paratrichodorus almadenensis sp. nov. and Paratrichodorus ramblensis sp. nov. are described here morphologically and molecularly and compared with topotype specimens of Paratrichodorus hispanus. Furthermore, this analysis was complemented with the first molecular data for this species complex and for the genus Monotrichodorus. The phylogeny of family Trichodoridae showed the relationship of Monotrichodorus with Trichodorus, both of which are phylogenetically associated with the P. hispanus species group, including the two newly described species. Several phylogenetic hypotheses about the monophyly of the genera with molecular data composing the family Trichodoridae were tested using Shimodaira–Hasegawa and approximately unbiased tests. This work also includes the first observations of copulatory plugs in Paratrichodorus, which clearly differ from the different types of mating plugs described in Trichodorus species. [ABSTRACT FROM AUTHOR]

A new and a known longidorid nematode, Paralongidorus lusitanicus n. sp. and Paralongidorus plesioepimikis, are described and illustrated from populations extracted from soil associated with grapevine (Vitis vinifera L.) from Escaroupim and Pó (central-Western Portugal), respectively. The new needle nematode P. lusitanicus n. sp. is characterised by a very large body size (8072-12,022 μm), an expanded and rounded lip region, ca 30 μm wide, with a clear constriction followed by a depression posterior to the amphidial aperture, amphidial fovea very large (11.0-19.0 μm), stirrup-shaped, with conspicuous slit-like aperture as shown in scanning electron microscopy studies, a very long and flexible odontostyle (180.0-223.0 μm), guiding ring located at 28.0-41.5 μm from anterior end, vulva anterior to the mid-body (34-41%), a dorsally convex-conoid tail with rounded terminus (29-42 μm long), bearing two or three pairs of caudal pores and males common (ratio 1:1.6 females) with spicules ca 80 μm long. Morphological and morphometric traits for P. plesioepimikis fit well with the original description, and is reported for the first time in Portugal. Integrative diagnosis of both species was completed with molecular data obtained using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA and partial 18S-rDNA. The phylogenetic relationships of these species with other Paralongidorus spp. using these three molecular markers indicated that P. lusitanicus n. sp. clustered together with other Paralongidorus spp. forming a sister clade with P. plesioepimikis, both of them sharing a large body, long odontostyle, an anteriorly located vulva and an expanded and rounded lip region with a clear constriction followed by a depression posterior to the amphidial aperture. [ABSTRACT FROM AUTHOR]

A new dagger nematode, Xiphinema poasense n. sp., is described and illustrated from three populations extracted from soil associated with a combined plantation of Eucalyptus sp., Cupressus sp. and Pennisetum sp. and wild plants from a tropical premontane forest in Costa Rica. The new dagger nematode is characterised by a moderate body size 2612 (2416-3042) µm long, a rounded lip region 15.0 (13.5-16.5) µm broad, separated from the body contour by a shallow depression, amphidial fovea large, stirrupshaped, a very long odontostyle (175 (164-188) µm), stylet guiding ring located 167 (136-181) µm from anterior end, vulva situated anterior to mid-body (36-40%), anterior genital branch complete but strongly reduced, without uterine differentiation, female tail short, hemispherical to convex-conoid with a c' ratio = 0.7 (0.6-0.8) and bearing two pairs of caudal pores, and male absent. Integrative diagnosis was completed with molecular data using D2-D3 expansion segments of 28S rRNA, ITS1 region, partial 18S-rRNA and the partial mitochondrial gene cytochrome c oxidase subunit 1 (coxI). The phylogenetic relationships based on D2-D3 segments of this species with other Xiphinema spp. of the X. non-americanum group indicated that X. poasense n. sp. clustered with other species with a reduced anterior genital branch from the morphospecies Group 2, viz., X. costaricense and X. krugi. However, the phylogeny of coxI and partial 18S rRNA gene revealed that the new species did not cluster with Xiphinema species having the anterior genital branch absent or reduced (i.e., morphospecies Groups 1 and 2, respectively). [ABSTRACT FROM AUTHOR]

The genus Xiphinema constitutes a large group of ~275 species of polyphagous, plant-ectoparasitic nematodes that are distributed almost worldwide. Some species affect agricultural crops by feeding directly on root cells as well as by transmitting nepoviruses. Despite their agricultural importance, species discrimination in Xiphinema is difficult, leading to potential misidentification. Integrative taxonomy, based on the combination of molecular analyses and morphology, constitutes a new insight into Xiphinema species identification. In this study we describe two new species of Xiphinema from the Iberian Peninsula (X. macrodora, sp. nov. and X. oleae, sp. nov.) associated with cultivated and wild olive trees. Both species have specific rRNA sequences. Morphologically, Xiphinema macrodora, sp. nov. is characterised by a very long body (7.2-8.7 mm), a very long odontostyle and odontophore (190-206 and 105-120 µm, respectively), and a well developed pseudo-Z-organ, comprising 8 to 12 sclerotised bodies. Xiphinema oleae, sp. nov. is characterised by an odontostyle and an odontophore 136-149 and 65-80 µm long, respectively, and a well developed Z-organ with refractive inclusions (3-5), variable in shape. Additionally, X. macrodora, sp. nov. has the longest body size, and the longest odontostyle and odontophore of any Xiphinema, whereas X. oleae, sp. nov. is the first species with a well developed Z-organ from the Iberian Peninsula. [ABSTRACT FROM AUTHOR]

The X iphinema americanum-group constitutes a complex of about 55 species of polyphagous plant-ectoparasitic nematodes with a worldwide distribution. This group of plant-parasitic nematodes is one of the most difficult dagger nematode species complexes for diagnosis because the morphology is very conservative and morphometric characters often overlap. We conducted nematode surveys in cultivated and wild olives in southern Spain from 2012 to 2014, from which we identified 16 nematode populations of the X . americanum-group, five of which were tentatively identified as belonging to three new species and are described herein as X iphinema plesiopachtaicum sp. nov., X iphinema vallense sp. nov., and X iphinema astaregiense sp. nov., and 11 populations belonging to nine known species: X iphinema brevisicum, X iphinema duriense, X iphinema incertum, X iphinema luci, X iphinema madeirense, X iphinema opisthohysterum, X iphinema pachtaicum, X iphinema parapachydermum, and X iphinema rivesi. A phenetic study based on multivariate factor analyses was developed to compare some of these related species by using morphometric features. In the factor analysis the first four factors accounted for 73.1% of the total variance of the selected characters, identifying body length, body length/maximum body width (a), body length/pharyngeal length (b), body length/tail length (c), and tail length/body width at anus (c′) ratios, distance from anterior end to vulva as percentage of body length ( V), stylet length, oral aperture-guiding ring distance, and lip region width as key morphometric characters to differentiate a restricted set of species within the X. pachtaicum-subgroup that includes X. plesiopachtaicum sp. nov. and X. vallense sp. nov. Multivariate analysis of variance using these specific characters allowed to differentiate species in the X. pachtaicum complex or groups of them using morphometric characters (body length, a, b, c, c′, V, stylet length, lip region width, oral aperture-guiding ring distance, female tail length, and hyaline region length). Phylogenetic analyses based on nuclear ribosomal DNA genes [ D2- D3 expansion segments of large ribosomal subunit 28S, and internal transcribed spacer 1 ( ITS1)] and the protein-coding mitochondrial gene, cytochrome c oxidase subunit 1 ( coxI) were congruent, showing two main clades separating most of the species of X. americanum-subgroup ' sensu stricto' from the X. pachtaicum-subgroup. Agreement between phylogenetic trees and some morphological characters ( viz. total stylet length, vulva position, and a ratio) were tested by reconstruction of their histories on rRNA-based trees using parsimony and Bayesian approaches. Thus, integrative taxonomy, based on a combination of multivariate morphological and molecular analyses constitutes a new insight into the identification of X. americanum-group species. © 2015 The Linnean Society of London [ABSTRACT FROM AUTHOR]

Sequence comparisons and molecular phylogenetic analyses were used to describe the nucleotide variability of the ITS containing regions of eighteen Pratylenchus species and several populations. Comparative analysis of nucleotide sequences of the rDNA internal transcribed spacers (ITS1 and ITS2) among Pratylenchus species used in the present study demonstrates that ITS sequences can widely vary in primary sequence and length. Alignment of eighty-seven Pratylenchus sequences and one outgroup taxon reveals the presence of ambiguous regions that have the greatest effect on phylogeny reconstruction. Phylogenetic analyses using Bayesian Inference, Neighbour Joining-LogDet, Maximum Likelihood and Maximum Parsimony, distinguished twelve highly or moderately supported major clades within Pratylenchus. Our results support the taxonomic usefulness of the ITS region to identify root-lesion nematode species of the genus Pratylenchus but the high nucleotide variability, sometimes, can preclude its use to resolve relationships among all members of the genus. In addition, the phylogenetic groupings are not congruent with those defined by characters derived by lip patterns and numbers of lip annuli. [ABSTRACT FROM AUTHOR]

During a recent nematode survey in natural environments of the Los Alcornocales Regional Park narrow valleys, viz., the renowned 'canutos' excavated in the mountains that maintain a humid microclimate, in southern Spain, an amphimictic population of Xiphinema globosum was identified. Morphological and morphometric studies on this population fit the original and previous descriptions and represent the first report from Spain and southern Europe. Molecular characterisation of X. globosum from Spain using D2-D3 expansion regions of 28S rRNA, 18S rRNA and ITS1-rRNA is provided and maximum likelihood and Bayesian inference analysis were used to reconstruct phylogenetic relationships within X. globosum and other Xiphinema species. A supertree solution of the different phylogenetic trees obtained in this study and in other published studies using rDNA genes are presented using the matrix representation parsimony method (MRP) and the most similar supertree method (MSSA). The results revealed a closer phylogenetic relationship of X. globosum with X. diversicaudatum, X. bakeri and with some sequences of unidentified Xiphinema spp. deposited in GenBank. [ABSTRACT FROM AUTHOR]

Molecular characterisation of four Schistonchus caprifici populations (two from Spain and two from Italy), using the partial 18S, the D2-D3 expansion segments of 28S of ribosomal DNA (rDNA) and the partial mitochondrial gene for cytochrome c oxidase I (mtCOI), is provided. Amplicons from partial mtCOI were analysed using 'Single Strand Conformation Polymorphism' (SSCP) analysis in order to rapidly screen for genetic (haplotypic) variability. SSCP analysis of mtCOI revealed a close relationship between the Spanish and Italian populations with low intra-population variability and only two haplotypes were detected. Mitochondrial COI and ribosomal genes were analysed using Maximum Likelihood and Bayesian inference to reconstruct phylogenetic relationships within S. caprifici and other members of the family Aphelenchoididae. The phylogenetic analysis based on mtCOI showed no geographic variability among Italian, Spanish and Turkish populations. The results of phylogenetic analyses based on the D2-D3 expansion segments of 28S rDNA region and the partial 18S rDNA genes revealed a closer phylogenetic relationship between S. aureus, S. laevigatus, S. virens and S. centerae, whilst S. caprifici and S. guangzhouensis clustered separately. The phylogeny of the genus Schistonchus was well related to some morphological characters, such as position of excretory pore, presence/absence of labial disc and number and position of caudal papillae. The monophyly of the genus Schistonchus was rejected by the Shimodaira-Hasegawa test based on tree topologies. [ABSTRACT FROM AUTHOR]

During recent nematode surveys in a muddy soil around undetermined graminaceous plants in El Rocío, Huelva Province, in southern Spain, and from the rhizosphere of date palm associated with graminaceous vegetation from Abadan, in Khuzestan Province, south-west Iran, populations of Longidorus orientalis were identified. Morphological and morphometrical studies on these populations fit the original description and represent the first report from Spain and Europe. Molecular characterisation of L. orientalis from Spain and Iran, using D2-D3 expansion regions of 28S rRNA, 18S rRNA and ITS-rRNA, is provided. Sequences of the D2-D3 expansion regions and partial 18S genes were analysed using maximum likelihood and Bayesian inference to reconstruct phylogenetic relationships within L. orientalis and other Longidorus species. The results revealed a closer phylogenetic relationship with L. goodeyi for the D2-D3 expansion region and with L. vineacola for the partial 18S region. [ABSTRACT FROM AUTHOR]

Spiral nematode species of the genus Rotylenchus have been reported on olive (Olea europaea L.) in several Mediterranean countries (Castillo et al., 2010; Ali et al., 2014). Nematological surveys for plant-parasitic nematodes on olive trees were carried out in Tunisia between 2013 and 2014, and two nematode species of Rotylenchus were collected from the rhizosphere of olive cv. Chemlali in several localities of Tunisia (Tables 1,2). Twenty-two soil samples of 3 to 4 kg were collected with a shovel from the upper 50 cm of soil from arbitrarily chosen olive trees. Nematodes were extracted from 500 cm³ of soil by centrifugal flotation method (Coolen, 1979). Specimens were heat killed by adding hot 4% formaldehyde solution and processed to pure glycerin using the De Grisse's (1969) method. Measurements were done using a drawing tube attached to a Zeiss III compound-microscope. Nematode DNA was extracted from single individuals and PCR assays were conducted as described by Castillo et al. (2003). Moderate-to-low soil populations of these spiral nematodes were detected (5.5-11.5, 1.5-5.0 individuals/500 cm³ of soil, respectively). This prompted us to undertake a detailed morphological and molecular comparative study with previous reported data. Morphological and molecular analyses of females identified these species as Rotylenchus eximius Siddiqi, 1964, and Rotylenchus incultus Sher, 1965. The morphology of R. eximius females (five specimens studied) was characterized by having a hemispherical lip region clearly off set, with four to five annuli, body without longitudinal striations, lateral fields areolated in the pharyngeal region only, stylet 32 to 36 mm long, and broadly rounded tail. The morphology of R. incultus females (51 females and 16 males; Table 2) was characterized by a hemispherical lip region with the basal annulus subdivided by irregular longitudinal striations, with three, rarely four annuli; stylet 21.5 to 27.5 mm long, female tail hemispherical with terminus regularly annulated; phasmids anterior to anus level (3-6 annuli above). The morphology of the isolated nematodes agreed with previous descriptions of R. eximius (Siddiqi, 1964; Castillo and Vovlas, 2005) and R. incultus (Sher, 1965; Castillo and Vovlas, 2005; Vovlas et al., 2008), respectively. A single individual was used for DNA extraction. Primers and PCR conditions used in this research were specified in Cantalapiedra-Navarrete et al. (2013), and a single amplicon of 800, 1,100, and 450 bp was obtained and sequenced for D2 to D3, ITS1, and cytochrome c oxidase subunit 1 (coxI), respectively. Sequence alignments for D2 to D3 (KX669231-KX669233), ITS1 (KX669238-KX669240), and cox I (KX669244-KX669245) from R. eximius, showed 99% to 97%, 98% to 94%, 93% similarity to other sequences of R. eximius deposited in GenBank (EU280794-DQ328741, EU373663-EU373664, JX015401-JX015402, respectively). Similarly, D2 to D3 (KX669234-KX669237), ITS1 (KX669241-KX669243), and coxI (KX669246-KX669249) sequence alignments from R. incultus, showed 99%, 99% to 95%, 99% to 90% similarity, respectively, to other sequences of R. incultus deposited in GenBank (EU280797, EU373672-EU373673, JX015403, respectively). The best fitted model of DNA evolution was obtained using µ Model Test v. 2.1.7 (Darriba et al. 2012) with the Akaike information criterion. BI analyses were performed under the general time reversible (GTR) with invariable sites and a gamma-shaped distribution of substitution rates (GTR + I + G) model for ITS1 and coxI. Phylogenetic analyses of ITS1 and coxI using Bayesian inference (BI) placed R. eximius and R. incultus from Tunisia in subclades that included all R. eximius and R. incultus sequences deposited in GenBank (Fig. 1), which agrees with previous results (Cantalapiedra-Navarrete et al., 2013). Morphology, morphometry, and molecular and phylogenetic data obtained from these samples were consistent with R. eximius and R. incultus identification. To our knowledge, this is the first report of R. incultus in Tunisia. Consequently, all these data suggest that spiral nematode species of the genus Rotylenchus are predominant in olive as previously reported in other Mediterranean areas (Ali et al., 2014). [ABSTRACT FROM AUTHOR]

Paralongidorus litoralis sp. n., a new bisexual species of the genus, is described and illustrated by light microscopy, scanning electron microscopy and molecular studies from specimens collected in a coastal sand dune soil around roots of lentisc (Pistacia lentiscus L.) from Zahara de los Atunes (Cadiz), southern Spain. Paralongidorus litoralis sp. n. is characterised by the large body size (7.5-10.0 mm), a rounded lip region, clearly offset from the body by a collar-like constriction, and bearing a very large stirrup-shaped, amphidial fovea, with conspicuous slit-like aperture, a very long and flexible odontostyle ca 190 μm long, guiding ring located at 35 μm from anterior end, and males with spicules ca 70 μm long. In addition, identification data of a Spanish population of P. paramaximus Heyns, 1965 recovered from sandy soil of a commercial citrus orchard at Alcala de Guadaira (Seville), southern Spain, agree very well with the original description of the species from South Africa. The 18S rRNA and D2 and D3 expansion regions of 28S rRNA gene sequences were obtained for P. litoralis sp. n. and P. paramaximus. Phylogenetic analyses of P. litoralis sp. n. and P. paramaximus rRNA gene sequences and of Longidoridae sequences published in GenBank were done using maximum likelihood and Bayesian inference. In trees generated from the 18S data set Paralongidorus clustered as an external clade from Longidorus, and in trees generated from D2-D3 of 28S dataset Paralongidorus was monophyletic and nested within Longidorus. Maximum likelihood test supported the hypothesis of validity of the Paralongidorus genus. [ABSTRACT FROM AUTHOR]

Root-lesion nematode species rank third only to root-knot and cyst nematodes as having the greatest economic impact on crops worldwide. A survey of plant-parasitic nematodes associated with decaying raspberries (Rubus sp.) in northern Italy revealed that root-lesion nematodes were the most frequently occurring species among other phytonematodes. Several Pratylenchus species have been associated with Rubus sp. in Canada (Quebec, British Columbia) and USA (North Carolina, Maryland, New Jersey) including P. penetrans and P. crenatus. In the roots and rhizosphere of symptomatic raspberries, nematodes of two Pratylenchus spp. were detected. Detailed morphometrics of the two root-lesion nematode isolates were consistent with Pratylenchus crenatus and with an undescribed Pratylenchus species. The extracted nematodes were observed and measured as live and fixed materials and subsequently identified by integrative taxonomy (morphometrically and molecularly). The latter species is described herein as Pratylenchus vovlasi sp. nov., resulting morphometrically closest to P. mediterraneus and phylogenetically to P. pratensis. The molecular identification of Pratylenchus vovlasi sp. nov. was carried out by sequencing the ITS region, D2-D3 expansion domains of the 28S rRNA gene and a partial region of the nuclear hsp90 gene. ITS-RFLP and sequence analyses revealed that Pratylenchus vovlasi sp. nov. had species-specific restriction profiles with no corresponding sequences present in the database. The phylogenetic relationships with ITS and D2-D3 sequences placed the Pratylenchus vovlasi sp. nov. in a clade with P. pratensis and P. pseudopratensis. This research confirms the occurrence of cryptic biodiversity within the genus Pratylenchus as well as the need for an integrative approach to the identification of Pratylenchus species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Dagger nematodes of the genus Xiphinema include a remarkable group of invertebrates of the phylum Nematoda comprising ectoparasitic animals of many wild and cultivated plants. Damage is caused by direct feeding on root cells and by vectoring nepoviruses that cause diseases on several crops. Precise identification of Xiphinema species is critical for launching appropriate control measures. We deciphered the cryptic diversity of the Xiphinema hispanum-species complex applying integrative taxonomical approaches that allowed us to verify a paradigmatic example of the morphostatic speciation and the description of a new species, Xiphinema malaka sp. nov. Detailed morphological, morphometrical, multivariate and genetic studies were carried out, and mitochondrial and nuclear haploweb analyses were used for species delimitation of this group. The new species belongs to morphospecies Group 5 from the Xiphinema nonamericanum-group species. D2-D3, ITS1, partial 18S, and partial coxI regions were used for inferring the phylogenetic relationships of X. malaka sp. nov. with other species within the genus Xiphinema. Molecular analyses showed a clear species differentiation not paralleled in morphology and morphometry, reflecting a clear morphostatic speciation. These results support the hypothesis that the biodiversity of dagger nematodes in southern Europe is greater than previously assumed. [ABSTRACT FROM AUTHOR]

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 Longidorus, 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, L. carpetanensis and L. pini for the first time. Additionally, we describe the males of L. pini and the juveniles of L. cf. olegi. [ABSTRACT FROM AUTHOR]

Abstract: Knowledge of rRNA structure is increasingly important to assist phylogenetic analysis through reconstructing optimal alignment, utilizing molecule features as an additional source of data and refining appropriate models of evolution of the molecule. We describe a procedure of optimization for alignment and a new coding method for nucleotide sequence data using secondary structure models of the D2 and D3 expansion fragments of the LSU-rRNA gene reconstructed for fifteen nematode species of the agriculturally important and diverse family Hoplolaimidae, order Tylenchida. Using secondary structure information we converted the original sequence data into twenty-eight symbol codes and submitted the transformed data to maximum parsimony analysis. We also applied the original sequence data set for Bayesian inference. This used the doublet model with sixteen states of nucleotide doublets for the stem region and the standard model of DNA substitution with four nucleotide states for loops and bulges. By this approach, we demonstrate that using structural information for phylogenetic analyses led to trees with lower resolved relationships between clades and likely eliminated some artefactual support for misinterpreted relationships, such as paraphyly of Helicotylenchus or Rotylenchus. This study as well as future phylogenetic analyses is herein supported by the development of an on-line database, NEMrRNA, for rRNA molecules in a structural format for nematodes. We also have developed a new computer program, RNAstat, for calculation of nucleotide statistics designed and proposed for phylogenetic studies. [Copyright &y& Elsevier]