Phylogenetic Analysis of the Hoplolaiminae Inferred from Combined D2 and D3 Expansion Segments of 28S rDNA.
ABSTRACT: DNA sequences of the D2-D3 expansion segments of the 28S gene of ribosomal DNA from 23 taxa of the subfamily Hoplolaiminae were obtained and aligned to infer phylogenetic relationships. The D2 and D3 expansion regions are G-C rich (59.2%), with up to 20.7% genetic divergence between Scutellonema brachyurum and Hoplolaimus concaudajuvencus. Molecular phylogenetic analysis using maximum likelihood and maximum parsimony was conducted using the D2-D3 sequence data. Of 558 characters, 254 characters (45.5%) were variable and 198 characters (35.4%) were parsimony informative. All phylogenetic methods produced a similar topology with two distinct clades: One clade consists of all Hoplolaimus species while the other clade consists of the rest of the studied Hoplolaiminae genera. This result suggests that Hoplolaimus is monophyletic. Another clade consisted of Aorolaimus, Helicotylenchus, Rotylenchus, and Scutellonema species. Phylogenetic analysis using the outgroup species Globodera rostocheinsis suggests that Hoplolaiminae is paraphyletic. In this study, the D2-D3 region had levels of DNA sequence divergence sufficient for phylogenetic analysis and delimiting species of Hoplolaiminae.
Project description:The D2-D3 expansion segments of the 28S ribosomal RNA (rRNA) were sequenced and compared to predict secondary structures for Hoplolaiminae species based on free energy minimization and comparative sequence analysis. The free energy based prediction method provides putative stem regions within primary structure and these base pairings in stems were confirmed manually by compensatory base changes among closely and distantly related species. Sequence differences ranged from identical between Hoplolaimus columbus and H. seinhorsti to 20.8% between Scutellonema brachyurum and H. concaudajuvencus. The comparative sequence analysis and energy minimization method yielded 9 stems in the D2 and 6 stems in the D3 which showed complete or partial compensatory base changes. At least 75% of nucleotides in the D2 and 68% of nucleotides in the D3 were related with formation of base pairings to maintain secondary structure. GC contents in stems ranged from 61 to 73% for the D2 and from 64 to 71% for the D3 region. These ranges are higher than G-C contents in loops which ranged from 37 to 48% in the D2 and 33-45% in the D3. In stems, G-C/C-G base pairings were the most common in the D2 and the D3 and also non-canonical base pairs including A•A and U•U, C•U/U•C, and G•A/A•G occurred in stems. The predicted secondary model and new sequence alignment based on predicted secondary structures for the D2 and D3 expansion segments provide useful information to assign positional nucleotide homology and reconstruction of more reliable phylogenetic trees.
Project description:DNA sequence analyses of the nuclear ribosomal ITS1 region of the ribosomal DNA and D1-D3 expansion segments of the 28S gene were conducted to characterize the genetic variation of six amphimictic Hoplolaimus species, including H. magnistylus, H. concaudajuvencus, H. galeatus, Hoplolaimus sp. 1, Hoplolaimus sp. 2 and Hoplolaimus sp. 3, and two closely related parthenogenetic species, H. columbus and H. seinhorsti. PCR amplifications of the combined D1-D3 expansion segments and the ITS1 region each yielded one distinct amplicon. In the D1-D3 region, there was no nucleotide sequence variation between populations of H. columbus, H. magnistylus, Hoplolaimus sp. 2 and Hoplolaimus sp. 3, whereas the ITS1 sequences had nucleotide variation among species. We detected conserved ITS1 regions located at the 3' and 5' end of ITS1 and also in the middle of the ITS1 among Hoplolaimus species. These regions were compared with sequences of distantly related Heterodera and Globedera. PCR-RFLP and sequence analysis of ITS1 and 28S PCR products revealed that several haplotypes existed in the same genome of H. columbus, H. magnistylus, H. seinhorsti, H. concaudajuvencus and Hoplolaimus sp. 1. Maximum likelihood and maximum parsimony analysis using the combined ITS1 and D1-D3 expansion segment sequences always produced trees with similar topology; H. columbus and H. seinhorsti grouped in one clade and the other six species (H. galeatus, H. concaudajuvencus, H. magnistylus, Hoplolaimus sp. 1, Hoplolaimus sp. 2, Hoplolaimus sp. 3) grouped in another. Molecular analysis supports morphological schemes for this genus to be divided into two groups based on several phenotypic traits derived from morphological evolution.
Project description:Enchodorus yeatsi n. sp., a new species of the rare genus Enchodorus is described and illustrated based on its morphological and molecular characters. It was recovered from southern Iran. Females of the new species are characterized by having 1,511.3- to 1,792.5-µm long slender body, angular lip region having prominent papillae, 12- to 13-µm long odontostyle, double guiding ring, simple rod-like odontophore, didelphic-amphidelphic reproductive system, and 102- to 160-µm long elongate-conoid tail with rounded tip. Males of the new species are abundant and functional, characterized by 1,484- to 1,576-µm long body, 40- to 43-µm long spicules, 5 to 6 ventromedian supplements, and ventrally bent elongate conical tail. Compared to the type species, Enchodorus dolichurus, the new species has differences in its tail morphology and V value. These morphological differences and the separation of two species was further supported with basic differences in sequences of 28S rDNA D2/D3 and internal transcribed spacer 1 (ITS1) fragments. Compared to Enchodorus neodolichurus, it has basic differences in tail characters and spicule lengths. Molecular phylogenetic studies using partial sequences of 28S rDNA D2/D3 fragment of the new species and available sequences of Nordiidae members and several other dorylaim species/genera, revealed E. yeatsi n. sp. and E. dolichurus forming a clade with 0.81 Bayesian posterior probability (BPP). This clade forms a sister clade to the clade of Heterodorus sp. and Rhyssocolpus vinciguerrae, again with 0.81 BPP. In ITS1 tree, reconstructed using few available sequences, the new species and E. dolichurus formed a clade with 0.98 BPP.
Project description:During a survey in the Botanical garden of Ghent University, a new species Rotylenchus rhomboides n. sp. and a population of Rotylenchus buxophilus were found. Rotylenchus rhomboides n. sp. is characterized by the presence of a rhomboid-like widening of the mid-ridge of lateral field at the level of vulva, a feature previously unknown within the genus. The population of the new species, composed only by females, has a rounded labial region with 4 to 5 annuli, robust stylet (31-37??m long), short dorsal esophageal gland (9-19??m) overlap of the intestine, vulva located slightly posterior to mid-body, and hemispherical or rounded tail shape with large phasmids located 3 to 5 annuli anterior to the level of anus. The hierarchical cluster analysis based on morphological features indicated that the new species closely resembles R. corsicus, R. gracilidens, and R. rugatocuticulatus. The DNA analyses of the D2-D3 of 28S rDNA, ITS rDNA, and COI mtDNA sequences of Rotylenchus rhomboides n. sp. show a close relationship with R. buxophilus, R. goodeyi, R. laurentinus, R. pumilus, and R. incultus, all of which can also be differentiated from the new species by morphological features. The combination of morphological, morphometric, and molecular characteristics confirmed the new species and the first report of R. buxophilus on yam (Dioscorea tokoro) in Belgium.During a survey in the Botanical garden of Ghent University, a new species Rotylenchus rhomboides n. sp. and a population of Rotylenchus buxophilus were found. Rotylenchus rhomboides n. sp. is characterized by the presence of a rhomboid-like widening of the mid-ridge of lateral field at the level of vulva, a feature previously unknown within the genus. The population of the new species, composed only by females, has a rounded labial region with 4 to 5 annuli, robust stylet (31–37??m long), short dorsal esophageal gland (9–19??m) overlap of the intestine, vulva located slightly posterior to mid-body, and hemispherical or rounded tail shape with large phasmids located 3 to 5 annuli anterior to the level of anus. The hierarchical cluster analysis based on morphological features indicated that the new species closely resembles R. corsicus, R. gracilidens, and R. rugatocuticulatus. The DNA analyses of the D2-D3 of 28S rDNA, ITS rDNA, and COI mtDNA sequences of Rotylenchus rhomboides n. sp. show a close relationship with R. buxophilus, R. goodeyi, R. laurentinus, R. pumilus, and R. incultus, all of which can also be differentiated from the new species by morphological features. The combination of morphological, morphometric, and molecular characteristics confirmed the new species and the first report of R. buxophilus on yam (Dioscorea tokoro) in Belgium.
Project description:Bursaphelenchus rainulfi isolated from dead pine trees in Zhejiang, China, is described and illustrated. It also provided some molecular characters of the Chinese population, including the PCR-RFLP and sequences of ITS region and D2-D3 expansion region of the large subunit (LSU) rRNA gene. Both the morphological characters and ITS-RFLP patterns match with the original description. The phylogenetic trees based on the 13 sequences of D2-D3 expansion region of the LSU rRNA gene and ITS region of Bursaphelenchus species were constructed, respectively, with the results showing the similar clades. The phylogenetic relationship based on the molecular data is similar to that with morphological characters. This is the first report of the species on pine wood in eastern China.
Project description:A population of Schistonchus caprifici was recovered in Iran during the present study. The molecular characters of this population were studied using partial sequences of large subunit ribosomal DNA (LSU rDNA D2-D3) for the first time, and its phylogenetic relationships with other isolates of the species were discussed. Compared to previous available data, no remarkable genetic distance was observed between the presently studied isolate and other isolates.A population of Schistonchus caprifici was recovered in Iran during the present study. The molecular characters of this population were studied using partial sequences of large subunit ribosomal DNA (LSU rDNA D2-D3) for the first time, and its phylogenetic relationships with other isolates of the species were discussed. Compared to previous available data, no remarkable genetic distance was observed between the presently studied isolate and other isolates.
Project description:Members of the genus Scutellonema can cause substantial crop losses to ornamental and cultivated plants directly by feeding ectoparasitically on plant roots (Bridge et al., 2005; Coyne et al., 2006). In May 2015, a soil sample from a house garden from Heraklion city in Crete, Greece, was sent for diagnosis of plant-parasitic nematodes. In this place, there had been cactus (Opuntia sp.) plants (probably imported), which were uprooted 3 to 4 years earlier. After that, the area was cropped with cucumber (Cucumis sativus L.) in spring-summer and leaf vegetables such as spinach (Spinacia oleracea L.) and chicory (Cichorium intybus L.) in autumn-winter. The soil was collected 1 mon after the end of chicory crop. A population density (ca. 30 individuals/100 cm(3) of soil) of spiral nematodes (Scutellonema sp.) was found by extracting soil with the wet sieving and decanting method (Cobb, 1918). Morphological and molecular analyses of females identified the species as Scutellonema brachyurus (Steiner, 1938) Andrássy, 1958. The morphology of females was characterized by a hemispherical lip region with four to six annuli, morphometric data for 12 females were L, 640 to 760 ?m; a, 24.6 to 30.6; b, 5.8 to 7.4; c, 69.1 to 99.3; c´ 0.5 to 0.6; stylet, 24.5 to 27.5 ?m with anterior part shorter than posterior; and spermatheca nonfunctional and male absent. The morphology agreed with the description of S. brachyurus (van den Berg et al., 2013). Alignment indicated that the D2-D3 and ITS sequences (KU059494 and KU059495, respectively) showed 99% and 100% to 99% similarity, respectively, to other sequences of S. brachyurus (type A) deposited in GenBank from the United States, Italy, and Korea (JX472037-JX472046, DQ328753, FJ485643; and JX472069, JX472070, JX472071, respectively), differing from one to six nucleotides. Phylogenetic analyses using Bayesian inference of these sequences placed the Scutellonema sp. in a highly supported (100%) clade that included all S. brachyurus (type A) sequences deposited in GenBank (van den Berg et al., 2013). All identification methods were consistent with S. brachyurus. To our knowledge, this is the first report of S. brachyurus for Greece. As the cucumbers and the leaf vegetables cultivated in the area were seed planted, we consider that the nematode originated most probably from the cactus plants which had been previously root ball planted. Scutellonema brachyurus may represent a threat for ornamental and cultivated plants production in Crete, Greece. The nematode has been already reported, mainly in greenhouses of six European countries (CABI and EPPO, 2006). In most of these cases, it is hypothesized that the nematode was introduced by imported plant material.
Project description:Anguillonema amolensis n. sp. is described and illustrated based on its morphological, morphometric, and molecular characters. The new species is characterized by its 575 to 820 ?m long and wide body (body width at vulva = 30 to 59 ?m), irregularly ventrally curved after fixation, five to six lines in lateral fields, 6.0 to 7.5 ?m long stylet with small rounded knobs, pharynx lacking a median bulb, pharyngo-intestinal junction anterior to nerve ring and excretory pore, females with monodelphic-prodelphic reproductive system, 15 to 19 ?m long conical tail with broad rounded tip, and males absent. The new species is compared with two known species of the genus, Anguillonema poligraphi and A. crenati. Molecular phylogenetic studies of the new species using partial sequences of small subunit (SSU) rDNA revealed that it forms a clade with an unidentified nematode species and two species of the genus Howardula. In phylogenetic analyses using partial sequences of the 28S rDNA (D2-D3 segment), the new species formed a monophyletic group with species belonging to two genera Howardula and Parasitylenchus.
Project description:Heterodorus youbertghostai n. sp. is described and illustrated based on morphological, morphometric, and molecular data. The new species was found in two geographically distant points in northwestern Iran and is characterized by having angular lip region, separated from the rest body by a constriction, body length of 1,432.5 to 1,751.3 µm, odontostyle length of 24 to 28 µm, rod-like odontophore, 37.0 to 42.5 µm long, lacking flanges at base, double guiding ring at 14 to 16 µm distance from anterior end, pharyngeal bulb comprising 40% to 48% of pharynx, intestine usually containing green material, female reproductive system amphidelphic with less divided short uterus, specific structure of pars distalis vaginae, bluntly conical tail, dorsally convex and ventrally flat, with rounded tip and saccate bodies in ventral side. The new species comes more close to H. conicaudatus and H. irregularis by its morphology and morphometric characters. Compared to former, it has remarkable difference in vulva position and tail characters, and compared to the latter, it could be separated by shorter body, posteriorly located vulva, wider lip region, and longer tail. In phylogenetic analyses using partial sequences of 28S rDNA D2-D3, the new species formed a fully supported clade with several isolates of H. brevidentatus, prevalent in Iran. The other nordiid taxon, Enchodorus dolichurus, already reported from Iran, was also sequenced for the same genomic region and included in phylogenetic analyses.
Project description:Total DNA was isolated from individual nematodes of the species Longidorus helveticus, L. macrosoma, L. arthensis, L. profundorum, L. elongatus, and L. raskii collected in Switzerland. The ITS region and D1-D2 expansion segments of the 26S rDNA were amplified and cloned. The sequences obtained were aligned in order to investigate sequence diversity and to infer the phylogenetic relationships among the six Longidorus species. D1-D2 sequences were more conserved than the ITS sequences that varied widely in primary structure and length, and no consensus was observed. Phylogenetic analyses using the neighbor-joining, maximum parsimony and maximum likelihood methods were performed with three different sequence data sets: ITS1-ITS2, 5.8S-D1-D2, and combining ITS1-ITS2+5.8S-D1-D2 sequences. All multiple alignments yielded similar basic trees supporting the existence of the six species established using morphological characters. These sequence data also provided evidence that the different regions of the rDNA are characterized by different evolution rates and by different factors associated with the generation of extreme size variation.