28S D2-D3 Research Articles

Description of Pseudaphelenchus zhoushanensis n. sp. (Tylenchina: Aphelenchoididae) found in the wood of Pinus thunbergii at Zhoushan Islands, Zhejiang Province, China

Pseudaphelenchus zhoushanensisn. sp. was isolated from a deadPinus thunbergiiat Changgang Mountain, Zhoushan Islands, Zhejiang Province, China. It is characterised by the small to medium length body, cuticle slightly annulated, presence of three lateral lines, stylet 9.0-10.7 μm with small but conspicuous basal knobs, excretory pore located from same level as the metacorpus to slightly anterior to metacorpus, true bursa surrounding entire tail but inconspicuous, male tail conical with a single mucron, spicule with distinct condylus and rostrum strongly arcuate to a pointed end, female tail conical with annulation, strongly ventrally bent in distal part of tail, with terminus bluntly pointed or finely mucronate. Phylogenetic analyses using sequences of the18S and 28S D2-D3 regions of rDNA confirmed the status ofP. zhoushanensisn. sp. as a new species. Combining the molecular phylogenetic analyses, morphology and biology ofP. zhoushanensisn. sp. andTylaphelenchus jiaaeindicates thatT. jiaaeis a member ofPseudaphelenchusto which it is herein transferred asP. jiaaen. comb. (=T. jiaae).

Redescription of Bursaphelenchus parapinasteri (Tylenchina: Aphelenchoididae) isolated from Pinus thunbergii in China with a key to the hofmanni-group

Bursaphelenchus parapinasteriis redescribed morphologically and with new molecular characterisation. It was isolated from a deadPinus thunbergiion Changgang Mountain, Zhoushan Islands, Zhejiang Province, China. Detailed morphology of the spicule, female gonad, hemizonid position, arrangement of male caudal papillae, and female tail terminus shape are documented. The ITS-RFLP patterns and the ITS1/2, partial 28S D2-D3 and partial 18S gene sequences were characterised. The phylogenetic analyses revealed thatB. parapinasteribelongs to thehofmanni-groupsensuBraasch and is close toB. anamurius,B. hofmanni,B. mazandaranense,B. paracorneolus,B. pinasteriandB. ulmophilus. A diagnostic key to species of thehofmanni-group is presented.

Bursaphelenchus saudi n. sp. (Tylenchina: Aphelenchoididae) found in packaging wood from Saudi Arabia

Bursaphelenchus saudi n. sp., isolated in Ningbo, P.R. China, from Pinus packaging wood imported from Saudi Arabia, is described. It is characterised by four lateral lines, excretory pore located between metacorpus and nerve ring (anterior to median bulb before cultured), presence of a thick vulval flap, post-uterine branch ca two-thirds of the vulva-anus distance long, female tail hook-like (slightly ventrally bent before cultured), long and conical, tapering to a bluntly rounded terminus, spicules transversely striated with weakly developed rostrum and condylus, a single precloacal papilla and three pairs of ventro-sublateral papillae (one pair precloacal, two pairs postcloacal just anterior to bursal flap and adjacent to each other). It belongs to the africanus-group of the genus Bursaphelenchus and is most close to B. paraburgeri, B. burgermeisteri and B. obeche. It is distinguished from other Bursaphelenchus species by morphology, ITS-RFLP patterns and partial 18S, ITS and 28S D2-D3 rDNA gene sequencing results.

Description of Aphelenchoides huntensis sp. n. (Nematoda: Aphelenchoididae) isolated from Pinus sylvestris in western Iran

Aphelenchoides huntensissp. n. is described and illustrated from wood and bark samples of a dead scots pine in Kermanshah Province, western Iran. The new species has a body length of 507-673 μm (females) and 636-640 μm (males). The cuticle is weakly annulated with four lateral lines. Lip region offset. The stylet is 9.5 (9.0-10.0) μm long with small basal swellings. The excretory pore is locatedcaone body diam. posterior to the metacorpus valve. The spicules are large with the dorsal limb 37 or 38 μm long (n = 2), and have a prominent rostrum and rounded apex. The female tail is conical, the terminus having a complicated step-like projection, usually with many tiny nodular protuberances. The male tail bears six (2 + 2 + 2) caudal papillae and a well-developed mucron. The new species belongs to the Group 2 category ofAphelenchoidesspecies. Phylogenetic analysis based on the 18S and 28S D2-D3 regions of rDNA confirmed its status as a new species.

Description of Sheraphelenchus parabrevigulonis n. sp. (Nematoda: Aphelenchoididae) in pine wood packaging from Italy and redescription of S. sucus in onion bulbs from South Korea, isolated at Ningbo, China

Two Sheraphelenchus populations were isolated from pine wood packaging from Italy and onion bulbs from South Korea at Ningbo Port, China, respectively. The former, on the basis of morphology and molecular sequencing, is described as S. parabrevigulonis n. sp., which is characterised by a stylet length of 11 μm, three lines in the lateral field, excretory pore at level with, or slightly posterior to, nerve ring, V = 93.1, and spicules with long condylus and membrane-like rostrum in a shallow triangular shape. The species from onion, also on the basis of morphology and sequence data, was identified as S. sucus, which is characterised by a stylet length of 13 μm (erroneously stated to be ca 6 μm in the type description), excretory pore at level of, or posterior to, median bulb, and V = 80.9. The phylogeny trees based on rDNA 18S, 28S D2-D3 and ITS1/2 sequences revealed that S. parabrevigulonis n. sp. and S. sucus clustered into a clear clade and grouped with Bursaphelenchus species. The study enriched the taxonomic information of Sheraphelenchus and confirmed its status as an independent genus.

Species delimitation using morphology, morphometrics, and molecules: definition of the Ophionscutellaris Thomson species group, with descriptions of six new species (Hymenoptera, Ichneumonidae).

The diverse genus Ophion is almost entirely undescribed in the Nearctic region. In this paper we define the Ophion scutellaris species group. This species group is well-supported by analysis of DNA (ITS2, COI, and 28S D2-D3) and morphology. It includes the Palearctic species Ophion scutellaris and the Nearctic species Ophion idoneus. An integrative analysis of DNA, geometric wing morphometrics, classical morphometrics and qualitative morphology indicates that this species group contains a minimum of seven species in North America, although the full diversity of the group has likely not been sampled. Ophion clave Schwarzfeld, sp. n., Ophion aureus Schwarzfeld, sp. n., Ophion brevipunctatus Schwarzfeld, sp. n., Ophion dombroskii Schwarzfeld, sp. n., Ophion keala Schwarzfeld, sp. n. and Ophion importunus Schwarzfeld, sp. n. are described, and a key to the known Nearctic species of the Ophion scutellaris group is provided.

Root-lesion and root-knot nematodes parasitizing potato

Root-lesion nematodes (RLN), Pratylenchus spp., are economically important plant-parasitic nematodes (PPN) of several crops, including potato. In Portugal, potato cyst nematodes (PCN), Globodera spp., are a frequent problem and root-knot nematodes (RKN), Meloidogyne spp., have been found coexisting with PCN. Although the diversity and pathogenicity of PCN on potato are well documented, little work has been done to evaluate the importance of other PPN. This study aimed at assessing the diversity of RLN and RKN in potato. Sampling encompassed fields located in four regions of Portugal: North, Centre, Lisboa and Algarve. Nematodes were extracted from 40 soil/root samples, using standard extraction techniques, identified at genus level and population densities quantified. RLN species were identified on basis of morphological characters, species specific primers, and D2D3 28S rDNA sequencing. RKN and PCN species were identified by esterase phenotype and PCR-RFLP, respectively. RLN were detected in all sampled regions in 83 % of soil and 78 % of root samples. Pratylenchus penetrans was the most abundant species and P. neglectus, P. crenatus and P. thornei were detected for the first time associated with potato in Portugal. Meloidogyne arenaria, M. hapla, M. incognita and M. javanica were found in 10 % of soil and 20 % of roots. Globodera pallida and G. rostochiensis were detected in 15 and 8 % of soil and roots. RKN and PCN were found in samples infected with RLN. The information obtained on the ubiquity and diversity of RLN and RKN reinforces the need for a careful monitoring of these nematodes in potato crops.

Occurrence of Meloidogyne fallax in North America, and Molecular Characterization of M. fallax and M. minor from U.S. Golf Course Greens.

Several species of root-knot nematodes (Meloidogyne spp.) are known to have significant presence on turfgrass in golf course greens, particularly in the western United States. Nematodes isolated from a golf course in King County, WA were identified as Meloidogyne minor based on analysis of the large ribosomal subunit (LSU 28S D2-D3 expansion segment), the internal transcribed spacers 1 and 2 (ITS rDNA), the intergenic spacer region 2 (IGS2), and the nuclear protein-coding gene Hsp90. Sequence-characterized amplified region (SCAR) primers that were originally designed to be specific for M. fallax were found to cross-react with M. minor. A population from California was determined to be M. fallax based on juvenile tail morphology and analysis of the ribosomal markers and Hsp90, comprising the first report of this species in North America. Using trees based on Hsp90 genomic alignments, the phylogenetic relationships of these populations and known root-knot nematode species were congruent with previous trees based on ribosomal genes. Resolution of M. fallax and M. chitwoodi using Hsp90 was equivalent to species separation obtained with 28S or 18S rDNA alignments. The strengths and weaknesses of ribosomal and Hsp90 markers, and the use of SCAR polymerase chain reaction as diagnostic tools are discussed.

Distribution and genetic diversity of root‐knot nematodes (Meloidogyne spp.) in potatoes from South Africa

A molecular‐based assay was employed to analyse and accurately identify various root‐knot nematodes (Meloidogyne spp.) parasitizing potatoes (Solanum tuberosum) in South Africa. Using the intergenic region (IGS) and the 28S D2–D3 expansion segments within the ribosomal DNA (rDNA), together with the region between the cytochrome oxidase subunit II (COII) and the 16S rRNA gene of the mtDNA, 78 composite potato tubers collected from seven major potato growing provinces were analysed and all Meloidogyne species present were identified. During this study, M. incognita, M. arenaria, M. javanica, M. hapla, M. chitwoodi and M. enterolobii were identified. The three tropical species M. javanica, M. incognita and M. arenaria were identified as the most prevalent species, occurring in almost every region sampled. Meloidogyne hapla and M. enterolobii occurred in Mpumalanga and KwaZulu‐Natal, respectively, while M. chitwoodi was isolated from two growers located within the Free State. Results presented here form part of the first comprehensive surveillance study of root‐knot nematodes to be carried out on potatoes in South Africa using a molecular‐based approach. The three genes were able to distinguish various Meloidogyne populations from one another, providing a reliable and robust method for future use in diagnostics within the potato industry for these phytoparasites.

On the evolution of raptorial legs - an insect example (Hemiptera: Reduviidae: Phymatinae).

The presence of chelate and subchelate fore legs in Phymatinae (Hemiptera: Reduviidae), or ambush bugs, provides a unique opportunity to study the evolution of different types of raptorial legs in a closely related group of arthropods. Themonocorini have simple, possibly raptorial legs, Phymatini and Macrocephalini distinct subchelate fore legs, and the charismatic Carcinocorini are the only insects with a chelate fore leg apart from female dryinid Chysidoidea (Hymenoptera). Relationships between the four phymatine tribes are here analyzed in a cladistic framework thus permitting testable hypotheses on the evolution of raptorial legs. The presented analysis of phymatine tribal level relationships is based on a dataset comprising 11 species of Phymatinae and 54 non-phymatine Reduviidae and Heteroptera. The molecular data set consists of ∼3500 MAFFT aligned bases of 16S, 28S D2-D3, and 18S ribosomal genes. Parsimony and maximum likelihood analyses resulted in identical topologies for the ingroup with the relationships Themonocorini + (Phymatini + (Carcinocorini + Macrocephalini)) receiving high support values. Eleven morphological characters, eight of them derived from fore leg morphology, were optimized on the parsimony analysis. These optimizations indicate that the ancestral ambush bug had a simple raptorial leg; that size reduction of the tarsus, enlargement of the femur, curvature of the fore tibia, armature of tibia and femur with rows of tiny tubercles that allow for gripping of a prey insect, and the large process on the ventral surface of the femur arose in the common ancestor of Carcinocorini + Macrocephalini + Phymatini. The chelate leg in Carcinocorini is likely derived from a subchelate precursor similar to the one seen in recent Macrocephalini and may have evolved through elongation of the ventral, proximal portion of the fore femur and modification of the median process to form part of the digitus fixus. © The Willi Hennig Society 2010.

Molecular phylogeny and species recognition in the mealybug genusFerrisiaFullaway (Hemiptera: Pseudococcidae)

Previous allozyme and DNA nucleotide sequence studies of the mealybug genus Ferrisia Fullaway (Hemiptera: Pseudococcidae), although limited, have suggested greater species diversity than is recognized by the current morphology-based taxonomy. Here we analyse nucleotide sequence data from one mitochondrial (cytochrome oxidase I) and two nuclear (EF-1α and 28S D2D3) genes and recover ten well-supported groups that allow us to reassess the taxonomic utility of morphological characters used for species recognition. We report on previously used morphological characters for which states are highly variable within genetic groups and identify new characters (of the wax-exuding cuticular ducts and pores) with taxonomically informative states. The widespread pest species F. virgata (Cockerell), commonly called the striped mealybug, should be diagnosed more narrowly. From samples identified as F. virgata, we recover six clades that we equate with species and that can be distinguished with the newly identified morphological characters. We determine that five of the ‘electrophoretic species' identified informally by the late Uzi Nur based on electrophoretic mobility of 20 enzymes correlate with four of our genetic groups. This matching of Nur's putative species with ours was possible only because some of Nur's slide-mounted voucher specimens were deposited in a museum and thus available for morphological study. Species confused with F. virgata are either new to science or were placed erroneously in synonymy with F. virgata by earlier authors: they will be described elsewhere. The most important characters of the adult female for distinguishing these species from F. virgata are the positions and characteristics of minute discoidal pore(s) associated both with the ventral oral-collar tubular ducts and with the sclerotized area surrounding each dorsal enlarged tubular duct, and the number of sizes of the ventral oral-collar tubular ducts. In addition, we determine that adult females of F. gilli Gullan from different populations on different host plants vary substantially in the number and size of clusters of small ventral oral-collar ducts on the body margins – features previously suspected to indicate distinct species.

A phylogenetic framework for root lesion nematodes of the genus Pratylenchus (Nematoda): Evidence from 18S and D2–D3 expansion segments of 28S ribosomal RNA genes and morphological characters

The root lesion nematodes of the genus Pratylenchus Filipjev, 1936 are migratory endoparasites of plant roots, considered among the most widespread and important nematode parasites in a variety of crops. We obtained gene sequences from the D2 and D3 expansion segments of 28S rRNA partial and 18S rRNA from 31 populations belonging to 11 valid and two unidentified species of root lesion nematodes and five outgroup taxa. These datasets were analyzed using maximum parsimony and Bayesian inference. The alignments were generated using the secondary structure models for these molecules and analyzed with Bayesian inference under the standard models and the complex model, considering helices under the doublet model and loops and bulges under the general time reversible model. The phylogenetic informativeness of morphological characters is tested by reconstruction of their histories on rRNA based trees using parallel parsimony and Bayesian approaches. Phylogenetic and sequence analyses of the 28S D2–D3 dataset with 145 accessions for 28 species and 18S dataset with 68 accessions for 15 species confirmed among large numbers of geographical diverse isolates that most classical morphospecies are monophyletic. Phylogenetic analyses revealed at least six distinct major clades of examined Pratylenchus species and these clades are generally congruent with those defined by characters derived from lip patterns, numbers of lip annules, and spermatheca shape. Morphological results suggest the need for sophisticated character discovery and analysis for morphology based phylogenetics in nematodes.

Direct optimization and multiple alignment of 28S D2–D3 rDNA sequences: problems with indels on the way to a molecular phylogeny of the cryptine ichneumon waSPS (Insecta: Hymenoptera)

AbstractA molecular phylogeny of the Cryptinae based on the D2–D3 region of the 28S rDNA gene, including 176 cryptines representing 139 genera and an additional 77 outgroup genera, is presented. Direct optimization and multiple alignment each with a range of gap/substitution parameters were employed. The informal higher grouping of the ichneumonids were supported, but the relationship of the Ichneumoninae and Cryptinae was not resolved. Cryptinae tribes were supported but many genera formed constant groups that did not follow earlier subtribal divisions. We investigated artifactual placements of, e.g., Agriotypinae and Brachycyrtinae, and showed that they do not possess molecular synapomophies with the Ichenumoninae where direct optimization places them. The sequences of anomalous placed taxa were markedly longer (or in some cases shorter) than those of most other taxa, and this is explained by homoplastic indels at “hot spots” in the absence of large numbers of informative substitutions. We show that multiple alignment generally resulted in taxa with large insertions or deletions being placed in more plausible positions. The relationship of the genusAlomyais also explored.© The Willi Hennig Society 2006.

Multiple displacement amplification (MDA) of total genomic DNA from Meloidogyne spp. and comparison to crude DNA extracts in PCR of ITS1, 28S D2-D3 rDNA and Hsp90

AbstractBecause the quantity of nematode specimens available for molecular analysis is often limited, the number of analyses possible is constrained by the availability of DNA. Multiple displacement amplification (MDA) was assessed for whole genome amplification of crude DNA from several Meloidogyne species. MDA produced microgram quantities of template that resulted in successful amplification of the ribosomal internal transcribed spacer (ITS1) and 28S D2-D3 expansion regions, producing PCR results that were comparable to template generated by the single nematode smash method. MDA greatly improved degenerate primer PCR of single-copy Hsp90, a gene which is more sensitive than multi-copy ribosomal genes to limited DNA template. MDA should expand the number of molecular analyses possible for single nematodes. MDA will also be useful for archiving DNA from valuable specimens and provide a way for laboratories to share identical genetic material for nematode diagnosis.