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Phylogenetic Relationships within the Cyst-Forming Nematodes (Nematoda, Heteroderidae) Based on Analysis of Sequences from the ITS Regions of Ribosomal DNA
Abstract
The ITS1, ITS2, and 5.8S gene sequences of nuclear ribosomal DNA from 40 taxa of the family Heteroderidae (including the genera Afenestrata, Cactodera, Heterodera, Globodera, Punctodera, Meloidodera, Cryphodera, and Thecavermiculatus) were sequenced and analyzed. The ITS regions displayed high levels of sequence divergence within Heteroderinae and compared to outgroup taxa. Unlike recent findings in root knot nematodes, ITS sequence polymorphism does not appear to complicate phylogenetic analysis of cyst nematodes. Phylogenetic analyses with maximum-parsimony, minimum-evolution, and maximum-likelihood methods were performed with a range of computer alignments, including elision and culled alignments. All multiple alignments and phylogenetic methods yielded similar basic structure for phylogenetic relationships of Heteroderidae. The cyst-forming nematodes are represented by six main clades corresponding to morphological characters and host specialization, with certain clades assuming different positions depending on alignment procedure and/or method of phylogenetic inference. Hypotheses of monophyly of Punctoderinae and Heteroderinae are, respectively, strongly and moderately supported by the ITS data across most alignments. Close relationships were revealed between the Avenae and the Sacchari groups and between the Humuli group and the species H. salixophila within Heteroderinae. The Goettingiana group occupies a basal position within this subfamily. The validity of the genera Afenestrata and Bidera was tested and is discussed based on molecular data. We conclude that ITS sequence data are appropriate for studies of relationships within the different species groups and less so for recovery of more ancient speciations within Heteroderidae.
Supplementary resources (36)
... The ITS rRNA gene sequences are used to discriminate species including some representatives of the Schachtii group (Subbotin et al., 2010). Ferris et al. (1993) and Subbotin et al. (2001) compared the ITS rRNA gene sequences from several species of the Schachtii group and surprisingly few differences were found between them. Moreover, intensive PCR-ITS-RFLP and ITS rRNA sequence studies of H. schachtii, H. betae and H. trifolii populations revealed substantial heterogeneity of the rRNA clusters Wouts et al., 2001;Amiri et al., 2002). ...
... Alignments with the ITS rRNA, COI and hsp90 gene sequences were created using ClustalX 1.83 (Chenna et al., 2003) with default parameters. New sequences were aligned with corresponding published gene sequences (Subbotin et al., 2001;Madani et al., 2004Madani et al., , 2007Toumi et al., 2013;Vovlas et al., 2015;Cui et al., 2016;Guesmi-Mzoughi et al., 2018;Escobar-Avila et al., 2019;Kim et al., 2019;Powers et al., 2019;Sekimoto et al., 2019;Handoo et al., 2020;Jain et al., 2022;Peng et al., 2022 and others). Several alignments were created: i) ITS rRNA gene alignment containing only reference sequences of 13 species of the Schachtii group; ii) ITS rRNA gene alignment containing new and published sequences of the Schachtii group species published, except for H. cajani and H. dunensis; iii) COI gene alignment containing only reference haplotype sequences of valid and undescribed species of the Schachtii group; iv) several COI gene sequence alignments containing sequences of nine species: H. betae, H. ciceri, H. daverti, H. galeopsidis, H. glycines, H. medicaginis, H. mediter-ranea, H. schachtii and H. trifolii; and v) hsp90 gene alignment containing new and published sequences of the Schachtii group species. ...
... Heterodera cajani was first molecularly characterised by PCR-ITS-RFLP . Sequences of ITS rRNA (Subbotin et al., 2001), β-tubulin (Sabo & Ferris, 2004), the D2-D3 expansion segments of 28S rRNA (Subbotin et al., 2006) and COII (Riepsamen et al., 2011) genes were published for the Indian populations of this species. The sequences of the ITS rRNA and COI gene placed this species together with H. dunensis at the basal position of the Schachtii group. ...
Cyst-forming nematodes of the genus Heterodera are highly derived and economically important plant parasites. The Schachtii group of this genus is one of the largest ones with a total of 18 species parasitising dicotyledons. In this study, we provided comprehensive phylogenetic analyses of several hundred COI and ITS rRNA gene sequences of selected species from the Schachtii group, including H. betae, H. cajani, H. ciceri, H. galeopsidis, H. glycines, H. medicaginis, H. mediterranea, H. schachtii, H. sonchophila and H. trifolii, using Bayesian inference, maximum likelihood, and statistical parsimony. One hundred and twenty four new COI, 57 ITS rRNA and eight hsp90 gene sequences from 81 nematode populations collected in 19 countries were obtained in this study. Our study showed that the ITS rRNA gene has limited discrimination power compared to the COI gene. However, our analysis also revealed that partial COI gene sequences were identical for H. trifolii, H. betae and H. galeopsidis. Based on the results of phylogeographical analysis and age estimation of clades with a molecular clock approach, it was hypothesised that the majority of the Schachtii group species originated and diversified in the Mediterranean Basin biodiversity hotspot during the Pleistocene and then dispersed from this region across the world. The Sino-Japanese Floristic Region is likely one of the centres of diversification for the soybean cyst nematode, which showed distinct population structure. The possible role of hybridisation and polyploidisation in the evolution of species of the Schachtii group is discussed.
... The tylenchid family Heteroderidae Filipjev & Schuurmans Stekhoven, 1941, includes seven genera of "cyst nematodes," a monophyletic lineage of sedentary plant parasites united primarily by the form taken by adult females at the end of their life cycle, a hardened sac containing embryonated eggs (Luc et al., 1986;Baldwin, 1992;Subbotin et al., 2001Subbotin et al., , 2010aSubbotin et al., , 2010bBert et al., 2008). The largest genus, Heterodera Schmidt, 1871, comprises about 85 species, many of which are devastating pests of important crops, including cereals, legumes, vegetables, and a wide variety of other crops (Nicol et al., 2007;Subbotin et al., 2010aSubbotin et al., , 2010bToumi et al., 2013;Smiley et al., 2017). ...
... large subunit ribosomal RNA (28S rDNA), and the mitochondrial cytochrome oxidase subunit one (coxI) gene regions (e.g., Szalanski et al., 1997;Subbotin et al., 2000Subbotin et al., , 2010aSubbotin et al., , 2010bFerris et al., 2004;Mundo-Ocampo et al., 2008;Escobar-Avila et al., 2018;Powers et al., 2019). The number of sequences for species of Heterodera uploaded to the public database GenBank has accumulated rapidly since the late 1990s to early 2000s when these data first began to become available (e.g., Ferris et al., 1993;Szalanski et al., 1997;Subbotin et al., 2000Subbotin et al., , 2001. There have been some reports, however, of species pairs which cannot be differentiated using sequences of one or more of these genes (Subbotin et al., 2000(Subbotin et al., , 2001Waeyenberge et al., 2009;Vovlas et al., 2015;Sekimoto et al., 2017;Escobar-Avila et al., 2018). ...
... The number of sequences for species of Heterodera uploaded to the public database GenBank has accumulated rapidly since the late 1990s to early 2000s when these data first began to become available (e.g., Ferris et al., 1993;Szalanski et al., 1997;Subbotin et al., 2000Subbotin et al., , 2001. There have been some reports, however, of species pairs which cannot be differentiated using sequences of one or more of these genes (Subbotin et al., 2000(Subbotin et al., , 2001Waeyenberge et al., 2009;Vovlas et al., 2015;Sekimoto et al., 2017;Escobar-Avila et al., 2018). As we move into the genomic era, with all its implications for more rapid and better identification methodologies, it seems pertinent to assess the accuracy and utility of the pool of barcoding gene sequences that have accumulated over the last few decades as these data will undoubtedly be incorporated into the next generation of molecular diagnostic tools. ...
Difficulties inherent in the morphological identification of cyst nematodes of the genus Heterodera Schmidt, 1871, an important lineage of plant parasites, has led to broad adoption of molecular methods for diagnosing and differentiating species. The pool of publicly available sequence data has grown significantly over the past few decades, and over half of all known species of Heterodera have been characterized using one or more molecular markers commonly employed in DNA barcoding (18S, internal transcribed spacer [ITS], 28S, coxI ). But how reliable are these data and how useful are these four markers for differentiating species? We downloaded all 18S, ITS, 28S, and coxI gene sequences available on the National Center for Biotechnology Information (NCBI) database, GenBank, for all species of Heterodera for which data were available. Using a combination of sequence comparison and tree-based phylogenetic methods, we evaluated this dataset for erroneous or otherwise problematic sequences and examined the utility of each molecular marker for the delineation of species. Although we find the rate of obviously erroneous sequences to be low, all four molecular markers failed to differentiate between at least one species pair. Our results suggest that while a combination of multiple markers is best for species identification, the coxI marker shows the most utility for species differentiation and should be favored over 18S, ITS, and 28S, where resources are limited. Presently, less than half the valid species of Heterodera have a sequence of coxI available, and only a third have more than one sequence of this marker.
... H. ripae Subbotin, Sturhan, Rumpenhorst & Moens, 2003, H. turcomanica Kirjanova & Shagalina, 1965and H. vallicola Eroshenko, Subbotin & Kazachenko, 2001 Only two species from this group are considered as nematode agricultural pests: the hop cyst nematode, H. humuli on hop plants and the fig cyst nematode, H. fici on fig trees (Subbotin et al., 2010). Subbotin et al. (2001) showed that the willow cyst nematode, H. salixophila, shared a common ancestor with the Humuli group. These relationships were further supported by additional phylogenetic analysis (Handoo & Subbotin, 2018;Rezaee Danesh et al., 2020). ...
... Alignments with the ITS rRNA and COI gene sequences were created using ClustalX 1.83 (Chenna et al., 2003) with default parameters. New sequences were aligned with corresponding published gene sequences (Eroshenko et al., 2001;Subbotin et al., 2001, Tanha Maafi et al., 2003, Madani et al., 2004Toumi et al., 2013;Fatemy et al., 2017;Sun et al., 2017;Fanelli et al., 2019;Rezaee Danesh et al., 2020;Darling et al., 2021;Jiang et al., 2021 and others , 1997). Pairwise divergence between taxa was calculated as the absolute distance value and the percent of mean distance, with adjustment for missing data, using PAUP* 4b10 (Swofford, 2003). ...
... Phylogenetic relationships reconstructed using ITS rRNA and COI gene sequences for species of the Humuli group in this study are generally congruent to each other and similar to those published in previous works (Eroshenko et al., 2001;Subbotin et al., 2001;Tanha Maafi et al., 2003;Subbotin, 2010;Subbotin & Skantar, 2018;Rezaee Danesh et al., 2020). In this study we reconstructed phylogenetic relationships within the Humuli group using BI and ML analysis of COI gene sequences for the first time. ...
The Humuli group of the genus Heterodera contains species that parasitise dicotyledons and are characterised by a lemon-shaped cyst having a bifenestrate vulval cone (ambifenestrate for H. fici), long vulval slit and weak underbridge. Presently, the Humuli group includes seven species: H. amaranthusiae, H. fici, H. humuli, H. litoralis, H. ripae, H. turcomanica and H. vallicola. In this study we provided comprehensive phylogenetic analyses of COI and ITS rRNA gene sequences of species from the Humuli group using Bayesian inference, maximum likelihood, and maximum and statistical parsimony. All seven valid species from the Humuli group, one putatively new species belonging to this group and the willow cyst nematode, H. salixophila, sharing a common ancestor with the Humuli group, were analysed. Some 84 COI and 5 ITS rRNA new gene sequences from 37 nematode populations collected from 12 countries were obtained in this study. Our results confirmed that the COI gene is a powerful DNA barcoding marker for identification of populations and species from the Humuli group. Based on the results of phylogeographical analysis and age estimation of clades with a molecular clock approach, it was hypothesised that some species of the Humuli group primarily originated and diversified in Western and Middle Asian regions during the Pleistocene and Holocene periods and then dispersed from this region across the world. Two secondary diversification centres of the Humuli group were likely located in East and Southeast Asia, Russian Far East, and Oceania.
... A total of 14 nematode populations collected in nine countries were analysed in this study. Several valid and unidentified species characterised in the previous studies (Subbotin et al., 2001;Tanha Maafi et al., 2003, 2007Mundo-Ocampo et al., 2008) were also and morphometric studies, and phylogenetic and sequence analysis (Subbotin et al., 2010a). For scanning electron microscopy (SEM) of the vulval cone, cysts were prepared following the technique of Lax and Doucet (2002), after which they were mounted on microscope stubs and coated with goldpalladium (21 nm). ...
... Alignments with the ITS rRNA, the D2-D3 of 28S rRNA and COI gene sequences were generated using ClustalX 1.83 (Chenna et al., 2003) with default parameters. New sequences were aligned with corresponding published gene sequences (Subbotin et al., 2001;Tanha Maafi et al., 2003;Sekimoto et al., 2017;Li et al., 2020;Singh et al., 2020 and others). The sequence alignments were analysed with Bayesian inference (BI) using MrBayes 3.1.2 ...
... Phylogenetic relationships between Heterodera species are given in Figure 1 and they are congruent with those published by Subbotin et al. (2001) andTanha Maafi et al. (2003). Interspecific variation between H. bifenestra and Heterodera sp. 3 was 3.5-3.7% ...
The genus Heterodera presently contains 87 valid species, of which five species: H. bifenestra, H. cardiolata, H. cyperi, H. goldeni and H. sacchari are molecularly characterised in this study. Using molecular criteria, we also distinguished five putative new species: Heterodera sp. 1-sp. 5 from the Afenestrata, Cardiolata, Bifenestra and Sacchari groups. A total of 16 new ITS rRNA, four new D2-D3 expansion segments of 28S rRNA and 23 new partial COI gene sequences were obtained from 14 cyst nematode populations collected from nine countries: and Ukraine. Phylogenetic relationships within the genus Heterodera are presented using these three gene fragments. The study confirmed the conclusions that each cyst nematode species has a unique COI sequence or DNA barcode that enables its identification and separation from all other species.
... H. avenae is distinguished from several other species by morphology and morphometrics, except from H. australis (Subbotin et al. 2002), with which it shares most characters. Several biochemical and molecular methods distinguish the populations of CCN found in Australia: iso-electric focusing (IEF) (Gäbler et al. 2000;Rumpenhorst 1985;Sturhan and Rumpenhorst 1996), restriction fragment length polymorphism (RFLP), and sequences of the ITS rRNA gene, except from H. arenaria (Bekal et al. 1997;Rivoal et al. 2003;Subbotin et al. 1999Subbotin et al. , 2001Subbotin et al. , 2003. Two genotypes of the European CCN based on the ITS and COI gene sequences are now distinguished: types A and B. Type A is found widely across Europe and North America and type B is distributed in Asia and North Africa. ...
... From most species of the H. avenae complex, this species differs by longer mean body and mean tail lengths of J2s. The analysis studies did not reveal any differences in the ITS-rRNA gene sequences between H. arenaria and H. avenae type A (Clapp et al. 2000;Subbotin et al. 2001Subbotin et al. , 2003. However, H. arenaria differs from other species by IEF (Gäbler et al. 2000) and sequences of COI gene (Subbotin et al. unpublished). ...
... Auckland cyst nematode differs from H. avenae, H. australis, H. pratensis, H. sturhani, and H. riparia by a longer mean tail length in the J2s Wouts and Sturhan 1995). It can also be differentiated from other species of the H. avenae complex by IEF (Gäbler et al. 2000;Sturhan and Rumpenhorst 1996), PCR-ITS-RFLP, and sequence of the ITS-rRNA gene (Subbotin et al. 1999(Subbotin et al. , 2001. Restriction of PCR-ITS-rDNA products by several enzymes (CfoI, HinfI, PstI, and TaqI) distinguishes H. aucklandica from other species of the H. avenae complex . ...
This book outlines the economic importance of specific plant parasitic nematode problems on the major food and industrial crops and presents the state-of-the-art management strategies that have been developed to reduce specific nematode impacts and outlines their limitations. Case studies to illustrate nematode impact in the field are presented and future changes in nematode disease pressure that might develop as a result of climate change and new cropping systems are discussed.
... Chez les Heteroderinae, la phylogénie obtenue avec les séquences d'ITS « internal transcribed spacers » (Subbotin et al. 2001) Le premier signalement du nématode à kyste de la pomme de terre est de Kühn (1881), qui l'identifie comme le nématode à kyste de la betterave (Heterodera schachtii). Wollenweber (1923) est le premier à distinguer le nématode à kyste de la pomme de terre de celui de la betterave. ...
... First, no Globodera species is known to be native to any Gondwanan continent except South America. Second, molecular studies (Subbotin et al., 2001;Tanha Maafi et al., 2003;Picard, 2005) show that the genera Punctodera and Globodera form a monophyletic clade which splits into a Punctodera clade, an apparently Laurasian genus parasiting Poaceae (Ferris, 1979), and a Globodera clade which comprises a Eurasian subclade (with species such as G. achilleae and G. artemisiae, parasiting Asteraceae; Ferris, 1979) and a South American subclade (grouping species parasiting Solanaceae). This variety of hosts confirms that related cyst-nematode groups may parasite distantly related or unrelated plant taxa (Sturhan, 2002;Subbotin et al., 2004), suggesting that speciation among cystnematodes may also have occurred by opportunistic parasiting of new host groups. ...
... Other divergence age estimates can be obtained by using another chronological tie-point, namely by assuming that the divergence of Punctoderinae and Heteroderinae occurred before the breakup of Pangea (Ferris, 1979), thus before 185 Ma (Veevers, 2004). In this hypothesis, the largest sequence divergences among Heteroderidae (14.7 %; Subbotin et al., 2001) corresponds to this >185 Ma divergence. Applying this calibration date to our ITS1-5.8S-ITS2 ...
... T h e D 2 a n d D 3 e x p a n s i o n d o m a i n s o f t h e 2 8 S r R N A w e r e a m p l i f i e d u s i n g p r i m e r s D 2 A ( 5´-A C A A G TA C C G T G A G G G A A A G T T G -3´) a n d D 3 B ( 5´-T C G G A A G G A A C C A G C TA C TA -3´) ( D e Ley et al. 1999). The ITS region was amplified u s i n g f o r w a r d p r i m e r T W 8 1 ( 5´-G T T T C C G -TAGGTGAACCTGC-3´) and reverse primer AB28 (5´-ATATGCTTAAGTTCAGCGGGT-3´) (Subbotin et al. 2001). The COI gene was amplified using forward primer JB3 (5´-TTTTTTGGGCATCCTGAGGTTTAT-3´) and reverse primer JB5 (5´-AGCACCTAAACTTAA AACATAATGAAAATG-3´) (Bowles et al. 1992). ...
... The PCR cycling for COI primers was as follows: 95°C for 15 min, 39 cycles at 94°C for 30 s, 53°C for 30 s, and 68°C for 1 min, followed by a final extension at 72°C for 7 min. PCR volumes were adapted to 25 μL for each reaction, and primer concentrations were as described in De Ley et al. (1999), Subbotin et al. (2001) and Bowles et al. (1992). All PCR assays were . ...
A wide survey was conducted to study plant-parasitic nematodes (PPNs) associated with Prunus groves in Spain. This research aimed to determine the prevalence and distribution of PPNs in Prunus groves, as well as the influence of explanatory variables describing soil, climate and agricultural management in structuring the variation of PPNs community composition. A total of 218 sampling sites were surveyed and 84 PPN species belonging to 32 genera were identified based of an integrative taxonomic approach. PPNs species considered as potential limiting factors in Prunus production, such as Meloidogyne arenaria, M. incognita, M. javanica, Pratylenchus penetrans and P. vulnus, were identified in this survey. Seven soil physico-chemical (C, Mg, N, Na, OM, P, pH and clay, loamy sand and sandy loam texture classes), four climate (Bio04, Bio05, Bio13 and Bio14) and four agricultural management variables (grove-use history less than 10 years, irrigation, apricot seedling rootstock, and Montclar rootstock) were identified as the most influential variables driving spatial patterns of PPNs communities. In particular, younger plantations showed higher values for species richness and diversity indices than groves cultivated for more than 20 years with Prunus spp. Our study increases the knowledge of the distribution and prevalence of PPNs associated with Prunus rhizosphere, as well as on the influence of explanatory variables driving the spatial structure PPNs communities, which has important implications for the successful design of sustainable management strategies in the future in this agricultural system.
... The Kahramanmaraş and Bolu populations were classified into the second cluster of H. filipjevi, whereas Kilis and Mardin populations consisted of the third cluster of H. latipons. Subbotin et al. (2001) reported that different cyst nematode species were thought to be phylogenetically examined based on ITS sequences, and this region was thought to be useful in identifying species (Subbotin et al. 2001). ...
... The Kahramanmaraş and Bolu populations were classified into the second cluster of H. filipjevi, whereas Kilis and Mardin populations consisted of the third cluster of H. latipons. Subbotin et al. (2001) reported that different cyst nematode species were thought to be phylogenetically examined based on ITS sequences, and this region was thought to be useful in identifying species (Subbotin et al. 2001). ...
In this study, morphological and molecular characterizations of twenty-four Heterodera populations (cereal cyst nematodes, CCNs) collected from wheat production fields in Turkey were carried out. Light microscopy, species-specific markers, RFLP, and ITS sequencing were used to identify the nematode populations. The obtained CCN populations were identified as Heterodera avenae, H. filipjevi, and H. latipons according to the morphometric analysis, which was confirmed by the molecular techniques. The ITS region sequencing analysis confirmed the species identification, and phylogenetic analysis of this region grouped the populations with representative Heterodera populations from different origin countries deposited in GenBank. The simulation of four restriction enzymes, Alul, PstI, BsuRI (HaeIII), and Rsal, employed the ITS sequences of isolates to discriminate the Turkish Heterodera populations. ITS-RFLP patterns produced by endonuclease enzymes provided variations among Heterodera species. There was no intraspecific variation in populations of each Heterodera species in the ITS-RFLP analyses. The species-specific primers, AvenF-COI/AvenR-COI, HfF/HfR, and H-LatF/H-LatR, yielded 109 bp, 646 bp, and 204 bp products for H. avenae, H. filipjevi, and H. latipons populations, respectively. This is the first research to provide conclusive diagnostic tests for cyst nematode populations isolated from Turkey. These assays provide a sensitive, practical, and quick method for detecting Heterodera species and, therefore, have the potential to be utilized in the early identification of populations and monitoring of infestations without morphometric studies.
... The D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A (5 -ACAAGTACCGTGAGGGAAAGTTG-3 ) and D3B (5 -TCGGAAGGAACCAGCTACTA-3 ) primers [55]. The Internal Transcribed Spacer region (ITS) was amplified using forward primer TW81 (5 -GTTTCCGTAGGTGAACCTGC -3 ) and reverse primer AB28 (5 -ATATGCTTAAGTTCAGCGGGT -3 ) [56]. The COI gene was amplified using the primers JB3 (5 -TTTTTTGGGCATCCTGAGGTTTAT-3 ) and JB5 (5 -AGCACCTAAACTTAAAACA TAATGAAAATG-3 ) [57]. ...
... The COI gene was amplified using the primers JB3 (5 -TTTTTTGGGCATCCTGAGGTTTAT-3 ) and JB5 (5 -AGCACCTAAACTTAAAACA TAATGAAAATG-3 ) [57]. The PCR cycling conditions for all three molecular markers were as described in Clavero-Camacho et al. [5], De Ley et al. [55], Subbotin et al. [56] and Bowles et al. [57]. In all PCR reactions, we used 5× HOT FIREpol Blend Master Mix (Solis Biodyne, Tartu, Estonia). ...
Paratylenchus species are obligate ectoparasitic nematodes on cultivated and wild herbaceous and woody plants occupying numerous soil categories. Several species may cause damage to several crops (viz. P. dianthus, P. enigmaticus, P. microdorus, P. hamatus and P. epacris on carnation, lettuce, rose and walnut, respectively). This investigation proves and emphasizes the relevance of applying integrative taxonomy for the accurate detection of Paratylenchus species in mountainous wild environments in the Malaga province, Southern Spain. This research analyzed 45 soil samples of maritimus pine and one of green heather in southern Spain and identified fourteen Paratylenchus species, two of them are described herein as new species (P. paraaonli sp. nov., P. plesiostraeleni sp. nov.), six of them were first reports for Spain (P. canchicus, P. nainianus, P. neonanus, P. salubris, Paratylenchus sp. 2 SAS, and P. wuae), and six species (P. caravaquenus, P. microdorus, P. nanus, P. neoamblycephalus, P. sheri, and P. variabilis) have been already reported in Spain. Accordingly, these data increase the biodiversity of pin nematodes in Spain comprising a total of 47 species (33.1% out of 142 total species of this genus). Phylogenetic analyses based on ribosomal and mitochondrial markers (D2-D3, ITS, and partial COI) resulted in a consistent position for the newly described Paratylenchus species in this study (P. plesiostraeleni sp. nov., P. paraaonli sp. nov.). Paratylenchus plesiostraeleni sp. nov. grouped in a separated subclade as unequivocal species from the P. straeleni-complex species (including P. straeleni and P. parastraeleni), and P. paraaonli sp. nov. clustered with P. vitecus, but clearly separate from this species. This study indicates that Paratylenchus species diversity in natural environments may be higher than expected, and this study may help in accurate identifications.
... In modern nematode taxonomy, molecular systematics is a very important tool (Subbotin 2005, Ye et al. 2007). For a further appropriate understanding of agricultural pest biology, evolutionary relationship studies among nematode species are very imperative, though it is not essential for nematode taxonomy (Subbotin 2001). For evolutionary analysis of plant-parasitic nematodes, different genomic regions viz. ...
... The ability to identify soil nematodes permits species determinations to be conducted before planting. The specificity of the assay may allow monitoring species shifts in mixed Meloidogyne populations with single PCR amplification (Subbotin 2001). A survey of a lot of isolates ought to be conducted to verify the presence of all that necessary Meloidogyne spp. ...
Nematodes from plant-parasitic sources are ever-present and incidental to plant growth as well as crop production. The damage of tea gardens caused by nematode is often non-specific and easily confused with symptoms. The present study determined the parasitic and non-parasitic nematodes population in different tea gardens of the Sylhet region by their morphological and partial molecular characterization. Out of 13 tea gardens, it was observed that BTRI, Karimpur, Mathiura, and Tarapur tea garden has the highest number of parasitic and non-parasitic nematodes. After PCR amplification, DNA bands with desired amplicon size were detected by gel electrophoresis. Among thirteen soil samples, nematodes from Malnichara, Karimpur, BTRI, Mathiura , and Finlay had partially confirmed the presence of root-knot nematode (Meloidogyne spp.), root-lesion nematode (Pratylenchus brachyurus), burrowing nematode (Radopholus similis), reniform nematode (Rotylenchulus reniformis) and lance nematode (Hoplolaimus columbus) consequently based on approximately base pair of 1.7, 1.1 and 0.52 kb (different Meloidogyne spp.) 0.52, 0.52, 0.25 and 2.3 kb of specific genes. From evolutionary analysis, it might be said that Meloidogyne species are strongly related with each other making clusters except Meloidogyne natalie where this one is closely related with Hoplolaimus columbus in their evolutionary relationship as remaining others (Rotylenchulus reniformis, Radopholus similis, Pratylenchus brachyurus) are in different clusters in the same clade and this result could be confirmed after sequencing.
... The D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A (5 -ACAAGTACCGTGAGGGAAAGTTG-3 ) and D3B (5 -TCGGAAGGAACCAGCTACTA-3 ) primers [58]. The Internal Transcribed Spacer region (ITS) was amplified by using forward primer TW81 (5 -GTTTCCGTAGGTGAACCTGC -3 ) and reverse primer AB28 (5 -ATATGCTTAAGTTCAGCGGGT -3 ) [59]. The COI gene was amplified using the primers JB3 (5 -TTTTTTGGGCATCCTGAGGTTTAT-3 ) and JB5 (5 -AGCACCTAAACTTAAAACAT AATGAAAATG-3 ) [60]. ...
... The PCR cycling for COI primers was as follows: 95 • C for 15 min, 39 cycles at 94 • C for 30 s, 53 • C for 30 s, and 68 • C for 1 min, followed by a final extension at 72 • C for 7 min. PCR volumes were adapted to 25 µL for each reaction, and primer concentrations were as described in De Ley et al. [58], Subbotin et al., [59] and Bowles et al. [60]. We used 5× HOT FIREpol Blend Master Mix (Solis Biodyne, Tartu, Estonia) in all PCR reactions. ...
This study delves into the diagnosis of pin nematodes (Paratylenchus spp.) in Spain based on integrative taxonomical approaches using 24 isolates from diverse natural and cultivated environments. Eighteen species were identified using females, males (when available) and juveniles with detailed morphology-morphometry and molecular markers (D2-D3, ITS and COI). Molecular markers were obtained from the same individuals used for morphological and morphometric analyses. The cryptic diversity using an integrative taxonomical approach of the Paratylenchus straeleni-species complex was studied, consisting of an outstanding example of the cryptic diversity within Paratylenchus and including the description of a new species, Paratylenchus parastraeleni sp. nov. Additionally , 17 already known species were identified comprising P. amundseni, P. aciculus, P. baldac-cii, P. enigmaticus, P. goodeyi, P. holdemani, P. macrodorus, P. neoamblycephalus, P. pandatus, P. pedrami, P. recisus, P. sheri, P. tateae, P. variabilis, P. veruculatus, P. verus, and P. vitecus. Eight of these species need to be considered as first reports for Spain in this work (viz. P. amundseni, P. aciculus, P. neo-amblycephalus, P. pandatus, P. recisus, P. variabilis, P. verus and P. vitecus). Thirty-nine species of Par-atylenchus have been reported in Spain from cultivated and natural ecosystems. Although we are aware that nematological efforts on Paratylenchus species in Southern Spain have been higher than that carried out in central and northern part of the country, the present distribution of the genus in Spain, with about 90% of species (35 out of 39 species, and 24 of them confirmed by integrative taxonomy) only reported in Southern Spain, suggest that this part of the country can be considered as a potential hotspot of biodiversity.
... For plant-parasitic nematodes, molecular diagnostics not only improve speed and accuracy of nematode identification, but also have allowed a better understanding of the biology of nematodes as agricultural pests [10]. The genomic regions more often used to study phylogenetic relationships for plant-parasitic nematodes include DNA fragments from the 28S ribosomal DNA (rDNA), internal transcribed spacer (ITS), as well as mitochondrial DNA (mtDNA) [2,[10][11][12][13][14][15]. ...
... For plant-parasitic nematodes, molecular diagnostics not only improve speed and accuracy of nematode identification, but also have allowed a better understanding of the biology of nematodes as agricultural pests [10]. The genomic regions more often used to study phylogenetic relationships for plant-parasitic nematodes include DNA fragments from the 28S ribosomal DNA (rDNA), internal transcribed spacer (ITS), as well as mitochondrial DNA (mtDNA) [2,[10][11][12][13][14][15]. Ribosomal genes exhibit enough conserved inter-specific neutral genetic variation as to inform species delimitation without being prone to marker saturation [15][16][17][18]. ...
Potato cyst nematodes (PCN) from the genus Globodera spp. cause major losses in the potato (Solanum tuberosum) industry worldwide. Despite their importance, at present little is known about the status of this plant pathogen in cultivated potatoes in Colombia. In this study, a total of 589 samples collected from 75 geographic localities in nine potato producing regions of Colombia (Cundinamarca, Boyacá, Antioquia, Nariño, Santander, Norte de Santander, Tolima, Caldas and Cauca) were assayed for the presence of potato cyst nematodes. Fifty-seven percent of samples tested positive for PCN. Based on phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rRNA gene and D2-D3 expansion segments of the 28S rRNA gene, all populations but one were identified as Globodera pallida. Sequences of G. pallida from Colombia formed a monophyletic group closely related to Peruvian populations, with the lowest average number of nucleotide substitutions per site (Dxy = 0.002) and net nucleotide substitutions per site (Da = 0.001), when compared to G. pallida populations from Europe, South and North America. A single sample formed a well-supported subclade along with G. rostochiensis and G. tabacum from Japan, USA and Argentina. To our knowledge this is the first comprehensive survey of Globodera populations from Colombia that includes genetic data. Our findings on species diversity and phylogenetic relationships of Globodera populations from Colombia may help elucidate the status and distribution of Globodera species, and lead to the development of accurate management strategies for the potato cyst nematodes.
... Heterodera cajani was first molecularly characterised by PCR-ITS-RFLP (Subbotin et al., 2000). Sequences of ITS rRNA (Subbotin et al., 2001), β-tubulin (Sabo & Ferris, 2004), the D2-D3 expansion segments of 28S rRNA (Subbotin et al., 2006) and COII (Riepsamen et al., 2011) genes were published for the Indian populations of H. cajani. The real-time PCR assay with melting curve analysis using a species-specific ITS rRNA gene primer for this species was also developed by Katsuta et al. (2016) and tested with Myanmar populations of this species. ...
... Cysts of the pigeon pea cyst nematode, H. cajani, from India were provided by J. Rowe (Subbotin et al., 2000(Subbotin et al., , 2001. Several other cyst nematode species were also included in the tests (Table 1). ...
The pigeon pea cyst nematode, Heterodera cajani , is an important nematode pest of pigeon pea that is present in all major growing regions of this crop in India and reported from Pakistan, Egypt and Myanmar. In this study, a new real-time PCR assay for detection of H. cajani using a species-specific primer and a TaqMan probe was developed. The primers and a probe were designed to amplify the COI gene fragment. The specificity of the primer-probe set was tested in singleplex or multiplex reactions against target and non-target nematodes. In multiplex real-time PCR experiments with the specific and universal primer-probe sets, the signals were simultaneously observed for COI and D3 of 28S rRNA target genes. The results showed that the real-time PCR assay with species-specific primer and probe was sensitive enough to detect H. cajani DNA extracted from 0.003 egg or second-stage juvenile.
... The D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A (5′-ACAAGTAC-CGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) primers [58]. The Internal Transcribed Spacer region (ITS) was amplified by using forward primer TW81 (5′-GTTTCCGTAGGTGAACCTGC -3′) and reverse primer AB28 (5′-AT-ATGCTTAAGTTCAGCGGGT -3′) [59]. The COI gene was amplified using the primers JB3 ...
... The PCR cycling for COI primers was as follows: 95 °C for 15 min, 39 cycles at 94 °C for 30 s, 53 °C for 30 s, and 68 °C for 1 min, followed by a final extension at 72 °C for 7 min. PCR volumes were adapted to 25 μL for each reaction, and primer concentrations were as described in De Ley et al. [58], Subbotin et al., [59] and Bowles et al. [60]. We used 5x HOT FIREpol Blend Master Mix (Solis Biodyne, Tartu, Estonia) in all PCR reactions. ...
In previous studies, fifteen species of Paratylenchus, commonly known as pin nematodes, have been reported in Spain. These plant-parasitic nematodes are ectoparasites with a wide host range and global distribution. In this research, 27 populations from twelve Paratylenchus species from 18 municipalities in Spain were studied using morphological, morphometrical and molecular data. This integrative taxonomic approach allowed the identification of twelve species, four of them were considered new undescribed species and eight were already known described. The new species described here are P. caravaquenus sp. nov., P. indalus sp. nov., P. pedrami sp. nov. and P. zurgenerus sp. nov. As for the already known described species, five were considered as first reports for the country, specifically P. enigmaticus, P. hamatus, P. holdemani, P. israelensis, and P. veruculatus, while P. baldaccii, P. goodeyi and P. tenuicaudatus had already been recorded in Spain. This study provides detail morphological and molecular data, including the D2-D3 expansion segments of 28S rRNA, ITS rRNA, and partial mitochondrial COI regions for the identification of different Paratylenchus species found in Spain. These results confirm the extraordinary cryptic diversity in Spain and with examples of morphostatic speciation within the genus Paratylenchus.
... During the past 30 years, molecular data, including ITS-rDNA, D2-D3 region of 28S-rDNA, are more accurate tool for identification of cyst-forming nematode species. Sequence analysis of the ITS-rDNA and the D2-D3 region of 28S-rDNA of unknown species is sufficient to study the phylogenetic relationship and identify cyst-forming nematode species (Maafi et al., 2003;Subbotin et al., 2001Subbotin et al., , 2006. ...
... The 28 S D2-D3 region was amplified with the D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (De Ley et al., 2005;Ye et al., 2007). Detailed protocols for DNA extraction, PCR conditions used in this study were as described by Munawar et al. (2018), Maafi et al. (2003), and Subbotin et al. (2001). PCR products were separated on 1% agarose gels and visualized by staining with ethidium bromide. ...
A new cyst-forming nematode, Cactodera tianzhuensis n. sp. was isolated from the rhizosphere soil of Polygonum viviparum L. in Tianzhu county, China. Morphologically, the new species is characterized by lemon-shaped or rounded cysts that have protruding necks and vulval cones. The vulval cone of the new species appeared to be circumfenestrate without bullae and underbridge, vulval denticle present and anus distinct. Second-stage juveniles are vermiform, stylet well-developed with the rounded stylet knobs to slightly concave anteriorly. Lateral field with four incisures. Tail gradually tapering to a finely rounded terminus with a length of ca 54 (47-59) µm, outline of hyaline portion is V-shaped or U-shaped. Egg shells without visible markings or punctations. The phylogenetic analyses based on ITS-rDNA, D2-D3 of 28S-rDNA clearly revealed that the new species formed a separate clade from other Cactodera species, which further support the unique status of C. tianzhuensis n. sp. Therefore, it is described herein as a new species of the genus Cactodera.
... Molecular techniques based on the polymerase chain reactions (PCR) have been widely used to overcome these bottlenecks in nematode identification and have provided precise results (Toumi et al., 2013;Yan et al., 2013). Subbotin et al. (2001) indicated that the sequence variation in the internal transcribed spacer (ITS) region of ribosomal DNA provided useful information to identify many nematode taxa, including CCN. To our knowledge, the description of Algerian CCN populations has only been conducted using morphological tools (Mokabli et al., 2001(Mokabli et al., , 2002Haddadi et al., 2013); consequently, there is no information on genetic traits of CCN populations in Algeria. ...
... The ITS sequences of H. cruciferae populations were different in three nucleotides from those of the H. carotae populations in this study. Subbotin et al. (2001) and Madani et al. (2004) reported the high similarity between nucleotide sequences of H. cruciferae and H. carotae. Chizhov et al. (2009) indicating that the H. cruciferae population from Moscow grouped in a clade with a H. carotae population in GenBank. ...
Cereal cyst nematodes (CCN), Heterodera spp., are the most devastating plant-parasitic nematodes of cereals causing serious global economic losses. In this study, surveys to investigate plant-parasitic nematodes associated with wheat were performed in twenty fields in twelve provinces of Algeria in 2018. Cereal cyst nematodes were found in 41.6% of the investigated wheat fields. Forty-eight CCN populations from twenty locations were obtained and morphologically classified. To confirm the morphological classification, the internal transcribed spacer (ITS) of rDNA was amplified with F194/F195 primers, sequenced, and analyzed using BLASTn searches of the NCBI database. Populations were classified as Heterodera avenae, H. hordecalis, H. carotae, and H. cruciferae. Heterodera carotae and H. cruciferae are reported in Algeria for the first time from two and three surveyed locations, respectively. Heterodera carotae and H. cruciferae were grouped into a well-supported clade and close to populations from Italy and the Netherlands in the phylogenetic tree, respectively. Heterodera hordecalis and H. avenae were found in ten and five fields, respectively. Based on phylogenetic analysis, H. hordecalis showed high similarity to the Israeli population, whereas H. avenae populations from Algeria were found to have high similarity to the Spanish population. Due to the variation among the Algerian populations of H. hordecalis and H. avenae, it can be assumed they have been introduced into Algeria multiple times.
... The morphological characteristics are consistent with the Italian population of H. elachista through the comparison of the measurements. DNA was extracted from a single cyst randomly chosen (n = 5) using the protocol described by Subbotin et al. (2001). The ITS-rDNA and D2/D3 fragments of the 28S rRNA were respectively amplified with universal primers pairs TW81 and AB28 (Subbotin et al. 2001) and D2A and D3B (Castillo et al. 2003), and sequenced (Tsingke Biological Technology, Beijing, China). ...
... DNA was extracted from a single cyst randomly chosen (n = 5) using the protocol described by Subbotin et al. (2001). The ITS-rDNA and D2/D3 fragments of the 28S rRNA were respectively amplified with universal primers pairs TW81 and AB28 (Subbotin et al. 2001) and D2A and D3B (Castillo et al. 2003), and sequenced (Tsingke Biological Technology, Beijing, China). The consensus ITS sequence (GenBank Accession No. MN699476, 1037 bp) had 98.37 to 99.60% similarity with the H. elachista from corn in Italy (KC618469) and in China (MH712504), and from rice in China (HM560778) and in Iran (AF498391). ...
The cyst nematodes are important plant-parasitic nematodes and make detrimental economic impacts worldwide. Heterodera elachista attacks rice in Japan (Ohshima 1974), Iran (Tanha et al. 2004) and China (Ding et al. 2012) and also attacks corn in Italy (De Luca et al. 2013) and northeast China (Xiao et al. 2019). Considering this nematode can infect these two most important crops with high reproduction rates, special attention should be paid to avoid its spread to new areas by soil, water, and agricultural practices. A survey for cyst nematodes was performed in fields of corn seed for propagation in September 2018 in Zhangye City, Gansu Province, which is the biggest corn seed production base in China. One hundred seventy four samples were collected by the zig-zag method in about 25 ha corn fields. Each soil sample containing approximately 1 kg soil in total was from the mixture of 20 cores (20 cm in depth and 0.2 cm in diameter). Cysts were detected in 6 out of 174 samples using the sieving-decanting method (Byrd et al. 1983). The six samples from ~ 1.2 ha corn fields in which plants displayed symptoms of dwarf, yellow leaves and earlier wilting had an average of 14 ± 2.8 cysts per 100 g soil. The cysts (n = 30) were dark brown, spherical to lemon shape, protruding, tapered vulva with ambifenestrae separated by a vulva slit, obvious and medium-sized underbridge, few dark-brown vesicles and clear swollen protrusion around underbridge and cyst cuticle with a zigzag pattern. Morphological measurements of the cysts included body length (excluding neck) (range =359.4 to 498.6 μm, mean = 429.5 ± SD 39.3 μm), vulva slit length (34.3 to 42.8, 37.9 ± 2.5), fenestral length (31.2 to 43.8, 38.0 ± 4.4) and width (26.1 to 31.4, 29.3 ± 1.6), underbridge length (74.7 to 82.9, 78.3 ± 2.8). J2 (n = 20) had the following measurements: body length (386.6 to 454.9, 425.3 ± 21.8), stylet length (17.1 to 22.2, 19.2 ± 1.6), stylet knob width (3.4 to 4.3, 3.9 ± 0.3), tail length (50.5 to 60.5, 55.5 ± 3.3) and hyaline tail length (25.0 to 34.6, 29.1 ± 3.1). The morphological characteristics are consistent with the Italian population of H. elachista through the comparison of the measurements. DNA was extracted from a single cyst randomly chosen (n = 5) using the protocol described by Subbotin et al. (2001). The ITS-rDNA and D2/D3 fragments of the 28S rRNA were respectively amplified with universal primers pairs TW81 and AB28 (Subbotin et al. 2001) and D2A and D3B (Castillo et al. 2003), and sequenced (Tsingke Biological Technology, Beijing, China). The consensus ITS sequence (GenBank Accession No. MN699476, 1037 bp) had 98.37 to 99.60% similarity with the H. elachista from corn in Italy (KC618469) and in China (MH712504), and from rice in China (HM560778) and in Iran (AF498391). The D2/D3 sequence (GenBank Accession No. MN699477, 746 bp) exhibited 99.60 to 99.73% similarity with that of H. elachista from rice in China (JN202922, HM560842), and from corn in Italy (KC618462). This nematode population was identified as H. elachista according to the morphological and molecular features. Twenty-day-old corn seedling (cv. Tiannuo) was inoculated with 3000 J2 and eggs in a pot filled with sterile soil. Eight plants were inoculated and two uninoculated plants were used as controls. J2 were found in the stained roots at 10 d after inoculation and J4 were detected at 30 d. These inoculated plants exhibited yellowing and dwarfing similar to the symptoms observed initially in the field, and the control grew normally. After 60 d, the average of 28 cysts were detected from each inoculated plant. This is the first report of H. elachista in Gansu Province, northwest China.
... The D2-D3 expansion domains of the 28S rRNA were amplified using the D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3') primers (De Ley et al. 1999). The ITS region was amplified using forward primer TW81 (5 0 -GTTTCCGTAGGTGAACCTGC-3 0 ) and reverse primer AB28 (5 0 -ATATGCTTAAGTTCAGCGGGT-3 0 ) (Subbotin et al. 2001). The partial 18S rRNA was amplified using the primers 988 (5 0 -CTCAAAGATTAAGCCATGC-3 0 ), 1912R (5 0 -TTTACGGTCA-GAACTAGGG-3 0 ), 1813F (5´-CTGCGTGAGAGGTGAAAT -3´), and 2646R (5´-GCTACCTTGTTACGACTTTT -3´) (Holterman et al. 2006). ...
During nematode surveys of natural vegetation in forests of La Cima de Copey de Dota, San José, San José province, Costa Rica, a Xenocriconemella species closely resembling X. macrodora and related species was found. Integrative taxonomical approaches demonstrated that it is a new species described herein as X. costaricense sp. nov. The new species is parthenogenetic (only females have been detected) and characterised by a short body (276–404 μm); lip region with two annuli, not offset, not separated from body contour; first lip annulus partially covering the second lip annulus. Stylet thin, very long (113–133 μm) and flexible, occupying 30.5–47.8% of body length. Excretory pore located from one or two annuli anterior to one or two annuli posterior to level of stylet knobs, at 42 (37–45) μm from anterior end. Female genital tract monodelphic, prodelphic, outstretched, and occupying 35–45% of body length, with vagina slightly ventrally curved (14–18 μm long). Anus located 6–11 annuli from the tail terminus. Tail conoid and bluntly rounded terminus, the last 2–3 annuli oriented dorsally. Results of molecular characterisation and phylogenetic analyses of D2-D3 expansion segments of 28S rRNA, ITS, and partial 18S rRNA, as well as cytochrome oxidase c subunit 1 gene sequences further characterised the new species and clearly separated it from X. macrodora and other related species ( X. iberica , X. paraiberica , and X. pradense ).
... The D2-D3 expansion domains of the 28S rRNA were amplified using the D2A (5′-ACA AGT ACC GTG AGG GAA AGTTG-3′) and D3B (5′-TCG GAA GGA ACC AGC TAC TA-3′) primers [25]. The ITS region was amplified by using forward primer TW81 (5′-GTT TCC GTA GGT GAA CCT GC-3′) and reverse primer AB28 (5′-ATA TGC TTA AGT TCA GCG GGT-3′) [26]. The partial 18S rRNA was amplified using the primers 988 (5′-CTC AAA GAT TAA GCC ATG C-3′), 1912R (5′-TTT ACG GTC AGA ACT AGG G-3′), 1813F (5´-CTG CGT GAG AGG TGA AAT -3´), and 2646R (5´-GCT ACC TTG TTA CGA CTT TT -3´) [27]. ...
The ring nematode genus Xenocriconemella De Grisse and Loof, 1965 comprises only one nominal species, Xenocri-
conemella macrodora (Taylor, 1936) De Grisse and Loof, 1965. The initial objective of the present study was to inves‑
tigate the morphological–morphometric and molecular diversity of 28 X. macrodora populations in the Iberian
Peninsula associated with tree forests (mainly Quercus spp.). However, a detailed integrative taxonomic analysis
(morphological–morphometric and molecular data) from each population and analysis of this data using principal
component analysis (PCA) for morphometric data (including these 28 populations and other 25 X. macrodora popula‑
tions around the world) and molecular and phylogenetic species delimitation methods revealed that X. macrodora
forms a species complex. This species complex is composed by species that are morphometricly and morphologically
similar, but clearly different at the molecular level. Three new species are described applying integrative taxonomy,
namely as Xenocriconemella iberica sp. nov., Xenocriconemella paraiberica sp. nov. and Xenocriconemella pradense sp.
nov. However, the molecular diversity of this species in USA and Italy confirmed that additional species are likely present in this species complex, and the diversity of this group may be higher than expected. The study of X. macrodora
topotypes can clarify the position of this species using molecular markers under an integrative approach.
... Mehaline et al. (2020) showed that while H. cruciferae and H. carotae are very close species to each other. On the other hand, the results show different of ITS1, ITS2 regions sequences capacity to making distinguish between different populations of the nematodes, are different(Subbotin et al., 2001; Mehaline et al., 2020). On the other hand, researchers likeEscobar et al. (2018) mention that ITS rRNA and COI partial sequences do not have potential to make differentiation between H. cruciferae and H. carotae and host range is very crucial for the species separation. ...
... Two universal nematode barcoding regions, the large subunit region (LSU rDNA, Nunn 1992) and the internal transcribed spacer region (ITS rDNA, Subbotin et al. 2001), were amplified using PCR with the primer pairs D2A (5'-ACAAGTACCGTGAGGGAAAGT-3') and D3B ...
... At present, molecular characteristics such as sequences of the ITS-rRNA gene and the D2-D3 region of the 28S-rRNA gene provide a new reference for accurate identification of Heteroderinae species. Morphological characteristics combined with sequence information and phylogenetic analysis have become the main method for the identification of species in Heteroderinae (Maafi et al., 2003;Skantar et al., 2021;Subbotin et al., 2001Subbotin et al., , 2006. ...
A new cyst-forming nematode, Cactodera guizhouensis n. sp., isolated from the roots and surrounding soil of potato in Guizhou, China, is the first species of genus Cactodera found from the potato rhizosphere. The new species was characterized by the L/W ratio of its cyst, being 1.4 ± 0.1 (1.3–1.8), with a fenestral diameter of 17.7–28.7 × 12.3–23.4 μm and being absent vulval denticlest; the stylet length of second stage juveniles is 26.5 ± 1.2 (24.1–29.0) μm, while the tail is 53.4 ± 3.9 (45.0–61.9) μm and the hyaline tail is 25.7 ± 2.4 (21.6–29.8) μm, the lateral field having four incisures and areolation; the eggshell having punctations. The internal transcribed spacer sequences of ribosomal RNA (ITS-rRNA), and sequences of the D2-D3 region of 28S ribosomal RNA (28S-rRNA) from the new species were amplified and sequenced. The phylogenetic tree based on the sequences showed that the new species clustered into a separate clade and could be distinguished from other species in genus Cactodera. A key to the species of Cactodera is also provided in this study.
... (5′-ATATGCTTAAGTTCAGCGGGT-3′) and TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) (Subbotin et al. 2001). PCR was performed in 20 µL PCR reaction mixture containing 40 ng of each template, 1.5 µL of each primer, 0.6 µL of dNTP, 0.6 µL of Taq polymerase, 2 µL of PCR buffer (Sileks M (Moscow, Russia)), and 13.8 µL of distilled water with ProFlex PCR system (Applied Biosystems). ...
Ikromov EE, Kuchboev AE, Ikromov EF, Sümer N, Yildirimhan HS, Amirov OO, Zhumabekova B. 2023. Morphological and molecular characteristics of the species Cosmocerca commutata and C. ornata (Nematoda: Cosmocercidae) in Uzbekistan. Biodiversitas 24: 4609-4616. In studies on the helminth fauna of amphibians conducted by numerous researchers, two species of the genus Cosmocerca, namely Cosmocerca ornata (Dujardin, 1845) Diesing, 1861 and C. commutata (Diesing, 1851), have emerged as the most common and extensively studied. This paper elucidates the morphology and molecular characteristics of two nematode species, Cosmocerca commutata and C. ornata, found in the intestines of Pelophylax sp. and Bufotes pewzowi in the Ferghana Valley of Uzbekistan. The captured amphibians were individually kept in plastic bags with water and vegetation until the examination. It is the first record of B. pewzowi as a host for both species C. commutata and C. ornata. Standard methods were used for the fixation and processing of nematodes. These nematodes мorphologically differ in body size, relative length of spicules and gubernaculum (C. commutata has twice the length of gubernaculum and spicules compared to C. ornata), shape and number of plectanes (C. commutata has 8 pairs of plectans and 4 pairs of caudal papillae, while C. ornata has 5 pairs of plectanes and 3 pairs of caudal papillae), and tail morphology and length. The morphological evidence supports that C. commutata and C. ornata commonly parasitize anurans in Uzbekistan. This study provides molecular data of both species and their sequences differed by 1.5% (6 bp) in the ITS1+5.8S+ITS2 region. Each species of them formed a well-supported clade in the phylogenetic tree. Our findings contribute to the comprehension of species and genetic diversity of the genus Cosmocerca in Uzbekistan.
... Tree was evaluated by Bootstrap test values based on 1000 replications (Felsenstein, 1985). If statistically significant values of such parameters were enumerated then the organisms proposed and validated as newer ones (Kaplan et al., 2000;Chilton et al., 2001;Subbotin et al., 2001;Elbadri et al., 2002;Ye et al., 2004;Shamsi et al., 2009;Upadhyay, 2012;Upadhyay, 2017aUpadhyay, , 2018. These studies were found to be helpful in understanding the phylogenetic relationships of helminths at familial, sub-familial, generic, and species levels. ...
... The use of molecular techniques to explore the internal transcribed spacer (rDNA-ITS) sequence characteristics of different populations has become a popular research tool for the molecular diagnosis of cyst nematodes in recent years. The ITS sequence is a versatile genetic marker that is located between repeating clusters of 18S and 28S ribosomal DNA genes and is separated by the 5.8S ribosomal DNA genes (Subbotin et al. 2001). Subbotin et al. (1999) analysed the ITS regions in the rDNA of cereal cyst nematode populations from several countries and regions of the world and found heterogeneity in the ITS region of CCNs. ...
Cereal cyst nematodes (CCNs) lead to major losses in the cereal crop industry worldwide and have been reported in many provinces of China. However, this plant nematode’s distribution and genetic differences are not fully understood. In the present study, 821 soil and host root samples were collected from 16 provinces in 2019–2022 to investigate the distribution of the CCNs. Heterodera avenae was detected in 56.39% of the total samples, primarily in Hubei, Henan, Hebei, Shandong, Shanxi, Gansu, Beijing, Tianjin, Inner Mongolia, Ningxia, Xinjiang, Qinghai, Anhui, Shaanxi, and Jiangsu. H. filipjevi was present in 21 samples, with a detection rate of 2.60%, and it was found mainly in Henan, Anhui, Jiangsu, Shandong, Shanxi, and Qinghai. A phylogenetic analysis of the internal transcribed spacer (ITS) region of the rRNA gene indicated that significant evolutionary and genetic differences existed between the Chinese populations and populations from other countries. Our results indicate that ITS1 can be used as a phylogenetic analysis and genetic target for H. avenae populations. The haplotypes of the ITS1 sequences of H. avenae populations from 14 countries were analyzed, and we speculate that H. avenae originated in a Middle East hotspot, then spread westwards to Europe and the United States and eastwards to China and Australia. Genetic differences between Asian and European populations suggest that the Himalayas and Kunlun Mountains formed a barrier that resulted in the formation of a separate evolutionary group in China. The phylogenetic and haplotype analysis results from different hosts showed significant differences among populations isolated from different hosts, and those isolated from weeds were distinct from those from other hosts, indicating that the rich genetic diversity of H. avenae populations is related to the large number of available hosts. Above all, geographic barriers, time of origin, and host adaptation might explain the current known distribution patterns of Chinese H. avenae populations.
... Subsequently, the tubes were incubated at 65°C for 1-2 h and the proteinase K was denatured at 95°C for 10 min. The ITS region was amplified with forward primer F194 (5 -CGT AACAAG GTA GCT GTA G-3 ) (Ferris et al., 1993) and reverse primer AB28 (5 -ATA TGC TTA AGT TCA GCG GGT-3 ) (Subbotin et al., 2001). PCR conditions were as described by Ye et al. (2007). ...
Aphelenchoides koreanus n. sp. isolated in Ningbo Port, P.R. China, from Pinus packaging wood from South Korea is described. It is characterised by the female length of 603 (511-687) μ m, stout body shape (a = 24.4 (21.9-26.7)). The lateral field has four incisures. The slender stylet is 12.3 (11.7-13.4) μ m long and has small basal swellings. The excretory pore is located posterior to the nerve ring. The hemizonid is immediately posterior to excretory pore, but sometimes invisible. The vagina is not sclerotised, and the vulva has strongly protruding lips and lacks a flap. The PUS is well developed and forms ca 30.6-41.9% of the vulva to anus distance. Tail sub-cylindrical, terminus bluntly rounded or slightly pointed, always bearing a very short mucron or a small projection, about 1 μ m long. Males absent. The new species belongs to the group 1 category of Aphelenchoides species sensu Shahina. Phylogenetic analysis based on ITS sequences confirmed its status as a new species.
... To confirm and validate the morphological and morphometric identification, H. filipjevi cysts were subjected to DNA extraction and PCR analysis to amplify the ITS1-5.8S-ITS2 of rRNA using the Direct PCR Master kit (Jena Bioscience) following the manufacturer's recommendations. PCR amplification was carried out with the primers TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') and AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') in a T100 thermal cycler (Bio-Rad) as described by Subbotin et al. (2001). The PCR was performed at initial denaturation of 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 1.5 min. ...
The study investigated the status of cereal cyst nematodes (CCN) in the main wheat-growing areas of Kyrgyzstan in 2020. Soil samples were taken from 69 different wheat fields located in Chuy and Issyk-Kul provinces. CCN were found in thirty-one out of the sixty-nine locations surveyed. The highest occurrence of CCN was in the Tyup location in Issyk-Kul province with 81 cysts (250 cm 3 soil)-1. The CCN populations were identified by both morphological and molecular analyses. According to the results, all populations were identified as Heterodera filipjevi. No variations in rDNA-ITS sequencing data were detected among the 31 cyst nematode populations, and the phylogenetic tree showed that Kyrgyz populations clustered with H. filipjevi populations from Belgium, Spain and Turkey, and separated populations from Germany, Iran, UK, Tajikistan, France and Russia. Therefore, the findings suggested the presence of only one species of CCN in the study areas of Kyrgyzstan, currently.
... The D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A (5 -ACAAGTACCGTGAGGGAAAGTTG-3 ) and D3B (5 -TCGGAAGGAACCAGCTACTA-3 ) primers [69]. The Internal Transcribed Spacer region (ITS) was amplified by using forward primer TW81 (5 -GTTTCCGTAGGTGAACCTGC-3 ) and reverse primer AB28 (5 -ATATGCTTAAGTTCAGCGGGT-3 ) [70]. The partial 18S rRNA was amplified using the primers 988 (5 -CTCAAAGATTAAGCCATGC-3 ) and 1912R (5 -TTTACGGTCAGAACTAGGG-3 ) [Holterman et al., 2006]. ...
Ring nematodes are obligate ectoparasites on crops and natural herbaceous and woody plants, and some species are of economic importance and cause damage to roots of several crops. Recent integrative taxonomical analyses recognized the existence of two cryptic species within the Criconema annuliferum morphotype in Spain. In this study, we corroborated that morphometric, morphological and a multi-locus analysis (including the ribosomal markers D2-D3 expansion segments of 28S rRNA, ITS rRNA, 18S RNA, and the mitochondrial DNA cytochrome oxidase I gene) identified a new lineage clearly separated from C. annuliferum, C. paraannuliferum and C. plesioannuliferum. The new lineage was described herein as Criconema pseudoannuliferum sp. nov., confirming that C. annuliferum species complex species complex comprises a hyper-cryptic species complex. This research analysed soil samples from the rhizosphere of maritime pine (Pinus pinaster Ait.) forests in Bermeja-Crestellina Mountain, located at the western part of Málaga province, southern Spain. The integrative taxonomical analyses revealed the occurrence of a new cryptic species identified using females, males and juveniles with detailed morphology, morphometry and molecular markers, described herein as Criconema pseudoannuliferum sp. nov. All molecular markers (D2-D3, ITS, 18S and COI) were obtained from the same individual that was also used for morphological and morphometric analyses. This research demonstrated the hidden diversity within the C. annuliferum species complex species complex can reach to four lineages under ribosomal and mitochondrial gene markers for one morphospecies group, which includes four species, viz. C. annuliferum, C. paraannuliferum, C. plesioannuliferum, and C. pseudoannuliferum sp. nov. Criconema pseudoannuliferum sp. nov. was detected in moderate soil density in two maritime pine forests (5 and 25 nematodes/500 cm3 of soil) suggesting that does not cause damage to maritime pine.
... Sequences from the samples were compared with GenBank accessions from other nematode species using the BLASTn homology search program (Madani et al. 2010;Subbotin et al. 2000Subbotin et al. , 2001. The published sequences of ITS, 28S, and COI from G. rostochiensis and other species were selected and downloaded. ...
On a global basis, potato cyst nematodes Globodera spp. Skarbilovich, 1959 (Behrens, 1975) are one of the most serious soil-borne pathogens in potato (Solanum tuberosum L.) production. In 2019-2020, 188 soil samples were taken from rhizosphere soil associated with the roots of stunted and chlorotic potato plants in the main potato-growing areas of Yunnan and Sichuan provinces of China. G. rostochiensis Wollenweber, 1923 (Skarbilovich, 1959) was recovered from 112 of the samples. Nematode identification was as confirmed by morphometric, light microscopy, electron microscopy and molecular methodologies. Population densities of G. rostochiensis ranged from 47.0 to 69.0 eggs/g soil. A BLASTn homology search program was used to compare the sequences of populations of G. rostrochienses from Yunnan and Sichuan provinces with populations of other Heteroderinae spp. and populations of G. rostochiensis from other nations. While potatoes have been grown in China for at least 400 years and the nation produces more potatoes than any other country, potato cyst nematodes were not reported in China until 2022.
... The D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGC-TACTA-3′) primers [69]. The Internal Transcribed Spacer region (ITS) was amplified by using forward primer TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and reverse primer AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′) [70]. The partial 18S rRNA was amplified using the primers 988 (5′-CTCAAAGATTAAGCCATGC-3′) and 1912R (5′-TTTAC-GGTCAGAACTAGGG-3′) [71]. ...
Ring nematodes are obligate ectoparasites on cultivated and wild herbaceous and woody plants, inhabiting many types of soil, but particularly sandy soils. This study explored the morpho-metrical and molecular diversity of ring nematodes resembling Criconema annuliferum in 222 soil samples from fruit crops in Spain, including almond, apricot, peach and plum, as well as populations from cultivated and wild olives, and common yew. Ring nematodes of the genus Criconema were detected in 12 samples from under Prunus spp. (5.5%
... The samples were incubated at 65 C for 1 h and then at 95 C for 15 min to deactivate the proteinase K. The following sets of primers were used for amplification of two DNA fragments in the present study: ITS (Internal Transcribed Spacer) containing regions using the forward primer TW81 (5 0 -GTTTCCGTAGGTGAACCTGC-3 0 ) and the reverse primer AB28 (5 0 -ATATGCTTAAGTTCAGCGGGT-3 0 ) (Subbotin et al., 2001); the D2-D3 expansion domains of 28S rRNA using the primer set D2A (5 0 -ACAAGTACCGTGAGGGAAAGTTG-3 0 ) and D3B (5 0 -TCGGAAGGAACCAGCTACTA-3 0 ) (Nunn, 1992); the 18S rDNA using the 18SnF (5 0 -TGGATAACTGTGGTAATTCTAGAGC-3 0 ) and 18SnR (5 0 -TTACGACTTTTGCCCGGTTC-3 0 ); the portion of the mitochondrial cytochrome oxidase c subunit 1 (mtCOI) gene was amplified with the primer set: COI-F1 (5 0 -CCTACTATGATTGGTGGTTTTGGTAATTG-3 0 ) and COI-R2 (5 0 -GTAGCAGCAGTAAAATAAGCACG-3 0 ) (Kanzaki & Futai, 2002). Amplification conditions were an initial denaturation at 94 C for 5 min, followed by 35 cycles of 94 C/50 s, 55 C/50 s 72 C/1 min with a final extension time of 7 min at 72 C. All products were examined by standard electrophoresis on a 1% agarose gel. ...
The recent invasion of the Japanese beetle Popillia japonica Newman (Coleoptera: Scarabaeidae) in northern Italy offered the opportunity to explore the entomopathogenic nematodes (EPNs) associated with the soil of hay meadows.
A total of 61 sites were considered for nematode sampling, and from 17 of them (27.9%) EPNs were isolated and further characterized with molecular and morphological techniques as well as with laboratory bioassays.
Two main species, Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) and Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae) were recorded with the same frequency together with a few other species (Rhabditida: Oscheius sp. and Phasmarhabditis sp.).
The newly isolated EPN populations were characterized for their activity (penetration rate) and infectivity to P. japonica grubs.
EPNs occurrence was related to the time of beetle invasion at each sampling site and there was evidence of a density‐dependent response of the EPNs community to P. japonica density.
The invasion of P. japonica apparently did not significantly affect the occurrence of native grubs, although a tendency to a decline was observed and should be further investigated.
... Three molecular markers were used: the internal transcribed spacer (ITS) region and the D2-D3 expansion segment of the 28S gene (28S) from ribosomal DNA, and the partial COI gene (COI) from the mitochondrial DNA genome. The primers TW81 (5'-GTTTCCGTAGGTGAACCTGC-3') and AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3') were used to amplify the ITS region (Subbotin et al., 2001). The primers D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B ...
... The existence of multiple conserved primers that amplify DNA from many species and phylogenetically informative sections make these regions particularly successful in detecting plantparasitic nematodes [55,145]. The GenBank genomes help identify most plant-parasitic nematode species [146][147][148]. However, DNA sequencing has been used to identify marine nematodes and determine population genetic structure [9,27,149]. ...
Nematodes are the most diverse but most minor studied microorganisms found in soil, water, animals, or plants. Either beneficial or pathogenic, they significantly affect human and animal health, plant production and ultimately affect the environmental equilibrium. Knowledge of their taxonomy and biology are the main issues to answer the different challenges associated with these microorganisms. The classical morphology-based nematode taxonomy and biodiversity studies have proved insufficient to identify closely related taxa and have challenged most biologists. Several molecular approaches have been used to supplement morphological methods and solve these problems with markable success degrees. The molecular techniques range from enzyme analysis, protein-based information to DNA sequence analysis. For several decades, efforts have been made to integrate molecular approaches with digital 3D image-capturing technology to improve the identification accuracy of such a taxonomically challenging group and communicate morphological data. This review presents various molecular techniques and provides examples of recent advances in these methods to identify free-living and plant-parasitic nematodes.
... In addition, the phenotypic adaptations of organisms may contribute to slight differences in physiology or structure that increase the difficulty of morphological species determination (Knight 1984). However, the advent of molecular techniques has made it possible to identify species of Trichuris (Subbotin et al. 2001). 18S ribosomal RNA (18S rRNA) has been sequenced in more than 1000 species of the phylum Nematoda, and the locus is the most widely used type of molecular data to research taxonomic relationships among members of the phylum (Blaxter et al. 1998;Callejon et al. 2013;Holterman et al. 2006). ...
Four adult female worms of Trichuris were isolated from an individual of the wild blue sheep (Pseudois nayaur) inhabiting the Helan Mountains, China, during an epidemiological survey of this wild ruminant. Although there were some differences among the worms in posterior end (rectum) morphology and egg shape, little information regarding species status could be inferred from their morphology. Phylogenetic trees were constructed based on sequences of the ITS1 segment of ribosomal RNA (rRNA), and the sequences of the four Trichuris specimens from wild blue sheep were divided into two distinct lineages (Clade A and Clade B). The two specimens in Clade A were named Genotype I, and had the closest relationship with Trichuris skrjabini; the two specimens in Clade B were named Genotype II and had the closest genetic relationship with a previously described Trichuris sp. In the two Trichuris genotypes identified in the present study, the 18S fragments (261 to 262 bp) of the newly obtained sequences were found to be highly conserved, with merely one insert mutation of a single nucleotide present. The genetic distance based on ITS1 between members of Clade A, composed of two T. skrjabini individuals and two Genotype I individuals, ranged from 0 to 0.0034. These distances are within the intraspecies variation of Trichuris (0–0.0272), suggesting that the Genotype I individuals infesting the wild blue sheep were T. skrjabini. In Clade B, the newly obtained sequences clustered with Trichuris sp. specimens isolated from ruminants (sheep and black goat) with strong support, and the genetic distance ranged from 0.0068 to 0.017, which is also within the intraspecies variation of Trichuris (0–0.0272). However, the genetic distances between the Clade A and Clade B were 0.0442 to 0.0578, which are higher than the intraspecies distances in Trichuris but lower than the interspecies distances (0.102–0.5078). These results implied that Clade A and Clade B most likely represent two subpopulations of T. skrjabini; however, the possibility that Clade A is T. skrjabini and Clade B is a Candidatus Trichuris could not be excluded.
... Dababat et al. (2020) also found an intraspecific polymorphism among H. filipjevi populations collected from northern Kazakhstan. The ITS sequences phylogenetically distinguished different cyst nematode species successfully in the previous study by Subbotin et al. (2001). However, Maafi et al. (2003) reported that the ITS sequence alignment of H. filipjevi populations from Iran and Russia clustered together with 100% nucleotide similarity. ...
Kazakhstan is one of the biggest wheat producers, however, its wheat production is far below the average international wheat production standard due to biotic and abiotic stressors. Plant-parasitic nematodes are devastating for cereal production systems worldwide. A comprehensive survey was conducted in 2019 to identify plant-parasitic nematodes associated with wheat in different locations of central, eastern, and south-eastern Kazakhstan. The results revealed 33 root-lesion and 27 cyst nematode populations from the 77 localities sampled. These two genera occurred in separate or in mixed populations. The root-lesion populations were identified as Pratylenchus neglectus and P. thornei while all cyst nematodes were identified as Heterodera filipjevi. The identification of nematodes was firstly performed based on morphological and morphometric features and confirmed by BLAST and phylogenetic analyses based on the internal transcribed spacer and the D2-D3 expansion located in the 28S gene of ribosomal DNA for CCN and RLN populations, respectively. Pratylenchus neglectus and P. thornei populations from Kazakhstan showed a high similarity with the American, European, and Asian populations. Heterodera filipjevi populations formed a well-supported cluster with the corresponding populations from different countries and showed a slightly intraspecific polymorphism. Kazakhstan populations of H. filipjevi may have multiple introductions in Kazakhstan due to the divergence among them. The results of this study are of great importance for breeding programs and will enable awareness to extension advisors to develop measures to control these nematodes in cereal cropping areas in Kazakhstan.
... A partial region of the 28S rRNA gene including the expansion domains D2 and D3 (D2-D3) was amplified by using the primers D2A (5 -ACAAGTACCGTGAGGGAAAGTTG-3 ) and D3B (5 -TCGGAAGGAACCAGCTACTA-3 ) [29]. The internal transcribed spacer region (ITS) was amplified using forward primer TW81 (5 -GTTTCCGTAGGTGAACCTGC-3 ) and reverse primer AB28 (5 -ATATGCTTAAGTTCAGCGGGT -3 ) [30]. The coxI gene was amplified using the primers JB3 (5 -TTTTTTGGGCATCCTGAGGTTTAT-3 ) and JB5 (5 -AGCACCTAAACTTAAAACATAATGAAAATG-3 ) [31]. ...
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.
... Cereal Cyst nematodes (CCNs) of genus Heterodera include approximately 70 species, having a multipart of 12 species known as from the group of Heterodera avenae (Rumpenhorst et al. 2003). Twelve species from Heterodera avenae species complex viz., H. australis, H. filipjevi, H. mani, H. pratensis, H. riparia, H. sturhani and H. ustinov, H. arenaria, H. latipons, H. avenae, H. mothi and H. hordecalis have been characterized by Subbotin et al. (2001). Maqbool (1981) noticed dominance of cereal cyst nematodes such as H. avenae, H. zeae and H. mothi in the soil samples of wheat fields. ...
Background: Cereal Cyst Nematodes (CCN) are prevailing in all type of agricultural lands and responsible for enormous losses of cereal crops. The understandings on population densities and management of these nematode are required to improve crop health and productivity.Methods: A study was designed to assess the incidence of cereal cyst nematodes in wheat and rice monoculture cropping regions of eight districts of Punjab province, viz., Jhang, Khushab, layyah, Hafizabad, Sheikhupura, Narowal, Gujranwala and Sargodha in Pakistan. Population densities of cereal cyst nematodes were studied by extracting the cysts and second stage juveniles from soil samples and identified the nematode species. Result: The CCNs were found in 80% of the total 250 samples collected from wheat and rice monoculture fields of at least one crop. In the soil samples collected from wheat fields, an average of 7 to 38 cysts/100g of dry soil with eggs and J2 population of 142-771 were recorded. Whereas from the soil samples of rice fields, 17 to 25 cysts/100g soil were found with 345 to 508 eggs and juveniles. Among the wheat fields, the lowest incidence of 5.5% was recorded in kot–momin and highest incidence of 16.88% was recorded in Silanwali tehsils of Sargodha region. In rice fields, 24.69 to 27.00% incidence of CCNs was noticed in soil samples of various surveyed regions. Three different species of Heterodera genus were morphologically identified from the collections. The species includes Heterodera oryzae, Heterodera avenae and Heterodera graminophila. H. oryzae was more abundant in rice growing regions while H. avenae and H. graminophila were present dominantly in wheat growing regions. This study provides an inclusive information regarding cereal cyst nematode densities and species in wheat-rice growing regions of Punjab province of Pakistan.
... A partial region of the 28S rRNA gene including the expansion domains D2 and D3 (D2-D3) was amplified by using the primers D2A (5 -ACAAGTACCGTGAGGGAAAGTTG-3 ) and D3B (5 -TCGGAAGGAACCAGCTACTA-3 ) [29]. The internal transcribed spacer region (ITS) was amplified using forward primer TW81 (5 -GTTTCCGTAGGTGAACCTGC-3 ) and reverse primer AB28 (5 -ATATGCTTAAGTTCAGCGGGT -3 ) [30]. The coxI gene was amplified using the primers JB3 (5 -TTTTTTGGGCATCCTGAGGTTTAT-3 ) and JB5 (5 -AGCACCTAAACTTAAAACATAATGAAAATG-3 ) [31]. ...
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.
... ITS rRNA is a molecular barcode region for the diagnosis of Heterodera and Globodera spp. (Subbotin et al. 2001;Jones et al. 2011). ...
Global trading of plant materials, in combination with agricultural practices, may facilitate the spreading of cyst nematodes to so far non-infected areas. Recently Potato Cyst Nematode (PCN) was recognized to be present in Indonesia and both diversity and distribution require further study. Assessment of PCN populations was done by collecting soil samples, determination of morphological characteristics in combination with ITS rDNA and COI mtDNA sequencing. Thirty-seven soil samples were collected from potato fields in the Indonesia archipelago. The results showed the presence of Globodera rostochiensis in 22 out of 37 sampling fields, namely North Sumatra (6 fields), Central Java (12 fields), East Java (3 fields), and -for the first time- in Sulawesi (North Sulawesi) (1 field). The highest observed density was found in Banjarnegara (Central Java), i.e., 872 cysts 100 ml soil−1. Globodera pallida was not recovered. Both ITS and COI characterisation of Indonesian PCN (G. rostochiensis) revealed the virtual absence of sequence variation as compared to most PCN from the rest of the world; the COI sequences were identical to the most common and mostly distributed haplotype around the world. Microsatellite genotyping indicated a higher genetic diversity for populations from East Java than for populations from North Sumatra, suggesting that cysts at the origin of populations in North Sumatra were coming from populations in East Java. These data on species identification, population density, genetic diversity, and distribution of potato cyst nematode over the Indonesian archipelago constitute the very basis for the design of environmentally-sound and effective PCN control strategies.
Numerous plant parasitic nematodes (PPNs) have the potential to inflict considerable damage on agricultural crops. Through a comprehensive survey aimed at identifying PPNs affecting crops, cyst nematodes were isolated from the rhizosphere soil of buckwheat (Fagopyrum esculentum). Employing both molecular and morphological techniques, this cyst nematode was conclusively identified as Heterodera ripae. Notably, this represents the first documented occurrence of this particular cyst nematode species within the rhizosphere soil of F. esculentum.
In southern Manitoba, Canada, a survey was carried out in 2012 and 2013 to determine the presence of Heteroderidae cyst-forming plant-parasitic nematodes, with a focus on the soybean cyst nematode (SCN) (Heterodera glycines). A total of 48 fields having grown soybean were sampled. A modified Fenwick elutriation-flotation technique was used to extract cysts with a 75% cyst recovery efficiency. Cyst population density averaged 0.9 cysts kg −1 soil, with a total of 65 cysts recovered. Preliminary screening of cysts, based on general body shape and vulval cone top structure, showed the presence of cysts belong to circumfenestrate, and ambifenestrate groups of cyst-forming nematodes. Limited morphological data was accessible due to poor quality or insufficient cysts for analysis; however, generated DNA sequences for nuclear rDNA ITS and D2-D3 expansion region of the 28S rRNA were obtained for four samples and matched sequences in GenBank for the cyst nematodes Cactodera milleri, C. torreyanae, C. weissi, C. estonica and unknown Cactodera species. Only one of the ambifenestrate cysts with a cone top structure of Heterodera species yielded DNA for analysis and its identification was ambiguous for soybean cyst nematode (SCN). None of the cysts were positive through SCN diagnostic PCR. Cactodera is not a pest of soybean or other crops in Manitoba. These cyst nematodes are likely to be naturally associated with weeds and grasses in the sampled fields or may be introduced from neighbouring states of the USA. Further annual surveys are needed and recommended in the near future to encompass more soybean fields and corroborate the absence of the pest.
The potato rot nematode (Ditylenchus destructor) is a very economically important nematode in agronomic and horticultural plants worldwide. In this study, 43 populations of D. destructor were collected from different hosts across China, including 37 populations from Chinese herbal medicine plants. Obtained sequences of ITS-rDNA and D2-D3 of 28S-rDNA genes of D. destructor were compared and analyzed. 9 types of significant length variations in ITS sequences were observed among all populations. The differences in ITS1 length were mainly caused by the presence of repetitive elements with substantial base substitutions. Reconstructions of ITS1 secondary structures showed that the minisatellites formed a stem structure. 10 haplotypes were observed in all populations based on mutations and variations of helix H9. Among them, 3 known haplotypes (A-C) were found in 7 populations isolated from potato, sweet potato, and Codonopsis pilosula, and 7 unique haplotypes were found in other 36 populations collected from C. pilosula and Angelica sinensis compared with 7 haplotypes (A-G) of Subbotin' system. These unique haplotypes were different from haplotypes A-G, and we named them as haplotypes H-N. The present results showed that a total of 14 haplotypes (A-N) of ITS-rDNA have been found in D. destructor. Phylogenetic analyses of ITS-rDNA and D2-D3 showed that all populations of D. destructor were clustered into two major clades: one clade only containing Haplotype A from sweet potato and the other containing haplotypes B-N from other plants. For further verification, PCR-ITS-RFLP profiles were conducted on 7 new haplotypes. Collectively, our study suggests that D. destructor populations on Chinese medicinal materials are very different from those on other hosts and this work provides a paradigm for relevant researches.
Sugar beet is listed within the top ten most important crops in the world. The paleobotanic data suggest that the sea beet was grown in ancient times, while the beets with swollen roots were cultivated in the Middle Ages in Europe. Sugar beet cyst nematode, Heterodera schachtii, is an invasive organism causing high economic loss to sugar beets worldwide. The fundamental steps in the control of harmful organisms in plant protection and food safety are grounded on rapid detection of the causative agent and its proper identification. Prompt reaction before obvious symptoms occur can prevent devastating consequences. To confirm the identity of an invasive organism, the process demands a combination of identification techniques, such as morphology and molecular characterization. The phylogeography of available H. schachtii populations, based on matching historical data with phylogenetic analyses of the ITS rRNA region, pinpointed a possible place of origin of the European H. schachtii populations. Due to the long persistence of the parasite in soil, cysts harbor a large number of bacteria and fungi, the presence of which can lead to cyst death and population decline. Bacteria, fungi, and other antagonists, being an inevitable part of the soil ecosystem, are also part of those mechanisms in nature that limit the excessive number of invasive organisms and return the ecological system to its stable equilibrium.KeywordsAntagonistsBacteriaFungiIdentificationSugar beet cyst nematode
Ruehmaphelenchus taedae n. sp., isolated from Loblolly pine logs ( Pinus taedae L.) from the USA, is described and figured. It is characterised by a relatively slim body (a = 42 and 43 for males and females, respectively), three lines in the lateral field, male spicules relatively small (12-18 μ m) with high and dorsally bent condylus and weakly developed rostrum, bursal flap absent, short tail possessing a long terminal spike ending in a bluntly rounded tip and 8.7-13.3 μ m long, vulva positioned at ca 83% of body length, vulval flap absent, vulval lips slightly protruding, post-vulval uterine branch extending for less than half of vulva to anus distance, and female tail conoid, ca 3-4 anal body diam. long, with 13.7-18.5 μ m terminal projection. The new species can be separated from all other species of the genus by the male tail possessing a long terminal spike and the more anterior excretory pore. Detailed phylogenetic analysis based on 28S D2-D3 region sequences confirmed the status of this nematode as a new species.
A new species of cyst-forming nematode, Heterodera amaranthusiae n. sp., is described and illustrated from the weed, Amaranthus retroflexus , in a potato field in Yunnan Province, China. It is characterised by having canary to russet-brown and asymmetric lemon-shaped cyst, distinct neck, bifenestrate vulval cone, relatively short vulval slit of 29 (28-32) μ m, bullae absent and underbridge absent or weak if present. Second-stage juveniles are characterised by a well-developed stylet of 23 (22-25) μ m with robust shaft and basal knobs concave anteriorly, tail conoid, 51 (48-58) μ m long and hyaline region comprising 48 (41-53)% of its length. Morphologically and morphometrically it most resembles H. vallicola in the Humuli group. The ITS, 28S and COI gene sequences of H. amaranthusiae n. sp. clearly differentiate it from other Heterodera species. For diagnostic purposes, restriction enzyme analysis of the ITS region and three restriction enzymes, Alu I, Bsu RI ( Hae III) and Cfo I ( Hha I), were selected, clearly distinguishing H. amaranthusiae n. sp. from representative species in the Humuli group. Phylogenetic relationships with other species of the genus, inferred from two ribosomal regions and the cytochrome oxidase c subunit 1 region, based on Bayesian analysis, consistently showed that H. amaranthusiae n. sp. clustered with high support with other Humuli group species but with separate species status.
Ditylenchus gallaeformans is a plant parasitic nematode that induces galls on aboveground parts of Melastomataceae plants. It differs from most gall-inducing nematodes in that it is not an endoparasite, and has been considered as a possible biological control agent against invasive species of Miconia . Little is known about D. gallaeformans biology, genetic differences among populations and host preferences. This study examined the genetic differences among D. gallaeformans populations from different locations and host species and the phylogenetic relationships among them. Nematodes were collected from galls in plants from Costa Rica, Dominica, and Trinidad. The Cytochrome c oxidase 1 (cox1) region was sequenced from a total of 33 individual nematodes isolated from 33 different plant individuals, representing 21 species of Melastomataceae. Phylogenetic reconstructions, haplotype networks, and analysis of molecular variance showed that the species is monophyletic and has three major clades, which were mostly consistent with geographic location but not with host species. The first clade was composed by two subclades, one with individuals from Costa Rica and one with individuals from Dominica. The second and third clades comprised nematodes only from Trinidad. Overall, there is no evidence of host-species specialization in D. gallaeformans . Biocontrol efforts using the nematode against invasive Miconia could focus on geographical location matching but likely will not need to match host species.
Garcinia L. is a pantropically distributed genus with high species richness in South East Asia. It is a tropical evergreen plant with distinct morphological characteristics and has a high degree of endemism. Outstanding features of the Garcinia L. genus are monopodial growth, leafy texture, oil cavities containing yellow or light-colored resins present on all parts of the plant and polygamodioecious reproductive behavior. The current study was conducted to develop barcodes for different species of the genus Garcinia L., distributed widely in the Western Ghats of India. We assess the discrimination power of the plant DNA barcode (rbcL, matK, trnH-psbA, rpoB-trnCGAR and ITS), across major Garcinia L. species. Our results clearly demonstrate the value of plastid barcode data, previously unavailable for Garcinia L. species. Ten Garcinia L. species and three outgroup taxa selected from the Western Ghats of India for evaluation using four regions in the plastid genome (rbcL matK, trnH-psbA, rpoB-trnCGAR) and nuclear-transcribed spacer (nrITS) in order to discriminate them at the species level. A characteristic feature of all barcodes, maximum likelihood analysis, and Wilcoxon signed-rank test were used for species discrimination. The number of conserved sites were more using matK primer whereas more variables and informative sites found in rpoB-trnCGAR loci. For internal branches of species-specific clusters, maximum likelihood analysis showed a more resolved topology. Wilcoxon signed-rank test indicated a higher divergence for coding and non-coding regions. DNA barcoding was found to be a practical and rapid method for identifying more endemic species. These findings will potentially be helpful in delineating the various species of Garcinia L.
Two populations of Xiphinema persicum n. sp. belonging to the X. americanum-group, were recovered from Semnan province, and described and illustrated based upon morphological, morphometric and molecular data. The type population of the new species is characterized by 2233–2736 μm long females, offset lip region, anteriorly flat to slightly rounded and separated from the rest body by constriction, 82–87 μm long odontostyle, two equally developed genital branches with visible endosymbiont bacteria in ovaries under light microscope, vulva at 52.8–55.5%, 27–32 μm long dorsally convex and ventrally straight to slightly convex tail with a rounded tip, or having a wide mucro-like differentiation, rare male with six ventromedian supplements and four juvenile developmental stages. The new species was morphologically compared with six similar species, X. bricolense, X. californicum, X. incertum, X. pachtaicum, X. santos and X. simile. The latter species has closest morphology and phylogenetic affinities to the new species in both large subunit and internal transcribed spacer 1 (LSU and ITS1 rDNA) trees; but X. persicum n. sp. has four juvenile developmental stages (vs. three), longer odontostyle, and posteriorly located guiding ring compared to it. The phylogenetic relationships of the endosymbiont bacterium of the new species with other isolates was reconstructed using the partial sequences of 16S rDNA.
A new cyst-forming nematode, Heterodera microulae sp. n., was isolated from the roots and rhizosphere soil of Microula sikkimensis in China. Morphologically, the new species is characterized by lemon-shaped body with an extruded neck and obtuse vulval cone. The vulval cone of the new species appeared to be ambifenestrate without bullae and a weak underbridge. The second-stage juveniles have a longer body length with four lateral lines, strong stylets with rounded and flat stylet knobs, tail with a comparatively longer hyaline area, and a sharp terminus. The phylogenetic analyses based on ITS-rDNA, D2-D3 of 28S rDNA, and COI sequences revealed that the new species formed a separate clade from other Heterodera species in Goettingiana group, which further support the unique status of H. microulae sp. n. Therefore, it is described herein as a new species of genus Heterodera; additionally, the present study provided the first record of Goettingiana group in Gansu Province, China.
Potato cyst nematodes (PCN) from the genus Globodera spp. cause major losses in potato ( Solanum tuberosum ) industry worldwide. Despite their importance, at present little is known about the status of this plant pathogen in cultivated potatoes in Colombia. In this study, a total of 589 samples collected from 75 geographic localities from nine potato producing departments of Colombia were assayed for the presence of potato cyst nematodes. Fifty-seven percent of samples tested positive for PCN. All populations but one were identified as Globodera pallida , with conspicuous morphometric variation found among populations. Based on phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rRNA gene and D2-D3 expansion segments of the 28S rRNA gene, G. pallida from Colombia formed a monophyletic group closely related to Peruvian populations, with the lowest average number of nucleotide substitutions per site ( Dxy = 0.002) and net nucleotide substitutions per site ( Da = 0.001), when compared to G. pallida populations from South, North America and Europe. A single sample formed a well-supported subclade along with G. rostochiensis and G. tabacum from Japan, USA and Argentina. To our knowledge this is the first comprehensive survey of Globodera populations from Colombia that includes morphological and genetic data. Our findings on species diversity and phylogenetic relationships of Globodera populations from Colombia may help elucidate the status and distribution of Globodera species, and lead to the development of accurate management strategies for the potato cyst nematodes.
Heterodera riparia sp. n. from roots of Urtica dioica L. is described based on materials collected from plants growing at the sides of rivers, ponds and lakes in Russia, Germany and Belgium. The new species is similar to H. humuli, but differs by its smaller average cyst size (415-468 μm vs 452-524 μm in H. humuli) and shorter average fenestra length (46-52 μm vs 56-61 μm). The second stage juveniles of H. riparia sp. n. have a lower average body length (350-373 μm vs generally >375 μm), a shorter tail (40-47 μm vs 50-57 μm) and a shorter hyaline part of tail (18-23 μm vs generally >27 μm). One generation of the nematode developed during the vegetative season. Restriction enzyme analysis of ribosomal DNA sequences was used to distinguish H. riparia sp. n. from the related species H. humuli and H. fici. The pattern of restriction bands obtained with AluI clearly distinguished all species from each other and the enzymes CfoI and PstI also distinguished the new species from the other species. The distribution of the new species in Europe is reported.
A maximum likelihood method for inferring evolutionary trees from DNA sequence data was developed by Felsenstein (1981). In evaluating the extent to which the maximum likelihood tree is a significantly better representation of the true tree, it is important to estimate the variance of the difference between log likelihood of different tree topologies. Bootstrap resampling can be used for this purpose (Hasegawa et al. 1988; Hasegawa and Kishino 1989), but it imposes a great computation burden. To overcome this difficulty, we developed a new method for estimating the variance by expressing it explicitly.The method was applied to DNA sequence data from primates in order to evaluate the maximum likelihood branching order among Hominoidea. It was shown that, although the orangutan is convincingly placed as an outgroup of a human and African apes clade, the branching order among human, chimpanzee, and gorilla cannot be determined confidently from the DNA sequence data presently available when the evolutionary rate constancy is not assumed.
Nematodes are important: parasitic nematodes threaten the health of plants, animals and humans on a global scale; interstitial nematodes pervade sediment and soil ecosystems in overwhelming numbers; and Caenorhabditis elegans is a favourite experimental model system. A lack of clearly homologous characters and the absence of an informative fossil record have prevented us from deriving a consistent evolutionary framework for the phylum. Here we present a phylogenetic analysis, using 53 small subunit ribosomal DNA sequences from a wide range of nematodes. With this analysis, we can compare animal-parasitic, plant-parasitic and free-living taxa using a common measurement. Our results indicate that convergent morphological evolution may be extensive and that present higher-level classification of the Nematoda will need revision. We identify five major clades within the phylum, all of which include parasitic species. We suggest that animal parasitism arose independently at least four times, and plant parasitism three times. We clarify the relationship of C. elegans to major parasitic groups; this will allow more effective exploitation of our genetic and biological knowledge of this model species.
A non cyst-forming heteroderid nematode, Cryphodera brinkmani n. sp., is described and illustrated from the roots of Pinus thunbergii Parlatore from Japan; this new species is characterised by globose female with annulated cuticle and curved projecting neck; stylet 41.5 mu m long, with rounded knobs, set off from the shaft; excretory pore located 120 to 157 mu m from the anterior end, vulva terminal, with a Vulva-anus distance of 49 mu m and slightly protruding Vulval lips, vulva-anus area ranging from flat to concave, subcrystalline layer present, eggs retained in the body; males rare, with head region set off, two lip annules present, stylet 37.5 mu m long, rounded knobs set off, anteriorly slightly pointed, lateral field with three incisures, areolated; second-stage juvenile body 493 mu m long, head region set off, four cephalic annuli present, stylet 33.5 mu m long, with large anteriorly concave knobs, set off from the shaft, tail 70 mu m, tapering towards a pointed hyaline tail terminus of 37 mu m in length, areolated lateral field with three incisures, phasmids with a lens-like structure located 6 to 10 mu m posterior to the anus. A key for the Cryphodera species is proposed.
The relationships among a number of populations of Globodera pallida from Britian, the Netherlands, Germany, Switzerland, and South America were examined using PCR amplification of the ribosomal cistron between the 18S and 28S genes that include the two intergenic spacer regions (ITS1 and ITS2) and the 5.8S gene. Amplifications produced a similar-sized product of 1150 bp from all populations. Digestion of the amplified fragment with a number of restriction enzymes showed differences among the populations. The restriction enzyme RsaI distinguished the most populations. The RFLP patterns revealed by this enzyme were complex and could have arisen from heterogeneity between individuals within populations and from differences between the repeats of an individual. Sequence analysis from six of the populations, together with RFLP analysis of PCR products, shows that there is intraspecific variation in the rDNA of G. pallida.
Internal transcribed spacer 1 sequences were used to infer phylogenetic relationships among 8 of the 9 described species and one putative species of the entomopathogenic nematode genus Heterorhabditis. Sequences were aligned and optimized based on pairwise genetic distance and parsimony criteria and subjected to a variety of sequence alignment parameters. Phylogenetic trees were constructed with maximum parsimony, cladistic, distance, and maximum likelihood algorithms. Our results gave strong support for four pairs of sister species, while relationships between these pairs also were resolved but less well supported. The ITS1 region of the nuclear ribosomal repeat was a reliable source of homologous characters for resolving relationships between closely related taxa but provided more tenuous resolution among more divergent lineages. A high degree of sequence identity and lack of autapomorphic characters suggest that sister species pairs within three distinct lineages may be mutually conspecific. Application of these molecular data and current morphological knowledge to the delimitation of species is hindered by an incomplete understanding of their variability in natural populations.
DNA sequences and other molecular data compared among organisms may contain phylogenetic signal, or they may be randomized with respect to phylogenetic history. Some method is needed to distinguish phylogenetic signal from random noise to avoid analysis of data that have been randomized with respect to the historical relationships of the taxa being compared. We analyzed 8,000 random data matrices consisting of 10-500 binary or four-state characters and 5-25 taxa to study several options for detecting signal in systematic data bases. Analysis of random data often yields a single most-parsimonious tree, especially if the number of characters examined is large and the number of taxa examined is small (both often true in molecular studies). The most-parsimonious tree inferred from random data may also be considerably shorter than the second-best alternative. The distribution of tree lengths of all tree topologies (or a random sample thereof) provides a sensitive measure of phylogenetic signal: data matrices with phylogenetic signal produce tree-length distributions that are strongly skewed to the left, whereas those composed of random noise are closer to symmetrical. In simulations of phylogeny with varying rates of mutation (up to levels that produce random variation among taxa), the skewness of tree-length distributions is closely related to the success of parsimony in finding the true phylogeny. Tables of critical values of a skewness test statistic, g1, are provided for binary and four-state characters for 10-500 characters and 5-25 taxa. These tables can be used in a rapid and efficient test for significant structure in data matrices for phylogenetic analysis.
Molecular systematists generally rely on computer algorithms to establish the alignment of DNA sequences. However, when alignment regions are characterized by multiple insertions and deletions, these gap-filled stretches of DNA are often excised before phylogenetic reconstruction. This exclusion of systematic data is generally determined by subjective criteria. We explore a replicable methodology in which the comparison of several multiple sequence alignments can be used to eliminate regions of unstable sequence alignment. Using crocodilian and insect mitochondrial (mt) ribosomal (r) DNA as examples, we caution against the removal of sequence data prior to phylogenetic reconstruction.
No abstract available.
The family Heteroderidae of plant parasitic nematodes contains the cyst-forming species within the sub-family Heteroderinae, within which the numbers of genera (not all cyst-forming) and species described have increased greatly over the last five decades. When Franklin (1951) published her book on “The cyst-forming species of Heterodera”, cyst nematodes were already a major concern and were known to cause serious yield losses in important food crops such as potatoes, cereals, brassicas, tomatoes and sugar beet. The genus Heterodera Schmidt 1871 was, at that time, considered to be largely temperate, with about 12 species. Today, 67 valid species are recognized in the genus Heterodera alone (Table 1.1). After many studies and revisions in classification (Mulvey, 1972; Stone, 1977; Luc, Maggenti and Fortuner, 1988; Baldwin and Schouest, 1990), the specific characters of many other species that were once members of the genus Heterodera have been accommodated in new genera. These new genera of cyst-forming species are Globodera, Punctodera, Cactodera, Afenestrata and Dolichodera, which are listed with the species they contain in Table 1.2. The cyst-forming genera of the Heteroderinae seem, in general, to have hosts within particular plant families. For instance, the Poaceae support many species of Heterodera and Punctodera. On the other hand, there are examples of unusual host specificity within genera, such as Cactodera betulae, found normally on birch and occasionally alder whilst other species of Cactodera are found on members of the Cactaceae, Amaranthaceae and Chenopodiaceae.
The recent history, current status, and outlook for understanding the phylogeny and evolutionary relationships of the cyst nematodes as a group comprise the focus of this chapter. Arguments about taxonomic features and taxonomic nomenclature will be discussed only insofar as they are necessary to an understanding of broader issues of phylogeny and evolution. Other taxonomic details can be found in Chapter 4 on Taxonomy and Identification.
Knowledge of phylogeny of Heteroderidae is essential to achieving stable meaningful systematics. Stable systematics has been elusive. New genera are frequently described and genera are variously grouped into subfamilies and other taxa by a number of authors. Luc et al. (1978) pointed out “heterogenity and inconsistencies” within subfamilies and proposed abolishing existing subfamilies. They suggested that arrangement of genera into meaningful (i.e. phylogenetic) groups must await additional information. Currently, characters useful for interpreting phylogenetic relationships are scarce. Nevertheless, systematists have relied on these limited characters to propose several competing hypotheses of phylogeny of Heteroderidae (Wouts, 1973; Stone, 1977; Krall and Krall, 1978; Ferris, 1979, 1985). Further advancement requires testing, refining and retesting of these hypotheses.
Taxonomy is the theory and practice of classifying organisms. In its broadest sense this includes macrotaxonomy (Mayr and Ashlock, 1991) such as evolution, phylogeny, and systematics, which was covered for cyst nematodes in chapter 3. Our chapter is on microtaxonomy, including the classification and identification of cyst-forming nematodes.
The relationships among nematodes were studied by 18S rRNAgene sequencing. On the basis of phylogenetic trees and cladistic analysis of the secondary structure of helix 49, some orders of traditional Adenophorea should be ascribed to the Secernentea. The Chromadorida and Desmodorida should be grouped with nematodes of a complex consisting of the Monhysterida, Plectida and Secernentea. This taxon may be named Chromadoria, as was proposed earlier (Drozdovsky, 1981), since chromadorids are most closely related to the common ancestor of these groups. Hence, the class Adenophorea in a traditional sense is paraphyletic and should be revised.
Morphological and genome variation between 32 nematode isolates identified originally asPratylenchus coffeae, P. gutierrezi, P. loosi, and P. pseudocoffeae were characterized to estimate phylogenetic relationships among them. All isolates have numerous males, and two lip annuli. Viewed en face with scanning electron microscopy, the first lip annulus is divided into lateral and median sectors in several isolates from coffee (Central America and Indonesia), and in one From aster (Florida). The first lip annulus is smooth in all other isolates, including six from coffee (Brazil and Indonesia). Principal component analysis (PCA) of one morphological (smooth face vs divided face) and three weakly-allometric morphometric variables (V, a, length of stylet) revealed seven assemblages of isolates. The PCA-derived assemblages conform closely to phylogenetic relationships inferred from analysis of 28S rDNA sequences. Nevertheless, identity within the D2/D3 expansion segment was not absolute for all isolates within the morphological assemblages, indicating the possibility that several assemblages are species complexes. Based on face morphology, five isolates (smooth faces) from coffee near the type locality for P. coffeaein eastern Java, Indonesia are different species than preserved museum specimens (divided faces) collected in the same area. Moreover, the D2/D3 sequence of a Java isolate suggests that it may be conspecific with isolates of P. coffeae sensu lato (all with identical sequences and smooth faces) from citrus, banana, yam, aglaonema and cocoyam, but not with isolates from citrus in Oman, nor banana in Ghana. Morphology and D2/D3 sequence of a P. gutierrezi topotype isolate revealed the likelihood that it is not conspecific with two other isolates with divided faces from coffee in Central America, Morphology and genetic sequence data for isolates from coffee and citrus in Sao Paulo State in Brazil, indicate that they are one or more undescribed species. When compared to the morphology and D2/D3 sequence of P. loosi from tea in Sri Lanka, isolates recently described as P. loosi from Paspalum notatum and Panicum hemitomon in Florida are apparently two undescribed species.
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
Heterodera urticae Cooper, 1955, widespread in Northern Ireland on nettles (Urtica dioica L.), is described and accepted as a valid species. It resembles related species of the goettingiana group in that the mature cysts possess a vulval cone which is ambifenestrated and without conspicuous bullae. The cysts are small, averaging 492 X 435 μ; the egg-sac is small and never contains extruded eggs; and the larvae are long, averaging 541 μ, with a mean stylet length of 27 μ and a clear tail/stylet length ratio averaging 1.1. The gubernaculum has a beaded margin — such ornamentation has not been previously reported for any Heterodera species.
Cladistic analysis of free-living soil nematodes of the Leptonchoidea (Nematoda: Dorylaimida) resulted in groupings different from those obtained by traditional methods. We can interpret distributions of species groups obtained by phyletic analysis in relation to plate tectonic events. Similar techniques are applicable to plant parasitic nematodes. Grouping on the basis of synapomorphies produced a cladogram of genera of the family Heteroderidae (Nematoda: Tylenchida) in which Meloidodera and Cryphodera appear to be the most ancestral genera and the cyst forming genera the most derived. A cladogram of groups of species in Heterodera sensu lato showed a major division, with the round cyst nematodes and the Cacli group in one grouping and the rest of the Heterodera species in the second. I interpret present-day distributions by a strict vicariance view and suggest potential falsifiers; and also discuss ancient dispersal routes as alternative ways of thinking about nematode distribution.
Molecular examination of the ribosomal internal transcribed spacer (ITS) region in potato cyst nematodes (PCN) is described. The ITS was amplified and sequenced from a number of PCN collections. A low level of sequence variation was found between Globodera rostochiensis, G. pallida, and a Peruvian PCN collection, but no variation within Australasian collections of species was noted. Polymerase chain reaction (PCR) primers based upon the G. rostochiensis–G. pallida sequence differences were designed and successfully used to identify mixed PCN species in a single PCR reaction.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
The family Heteroderidae and the subfamilies Heteroderinae and Meloidoderinae are redefined. The subfamily Meloidogyninae is raised to family Meloidogynidae. The genus Meloidoderita Poghossian, 1966 is transferred to the family Meloidogynidae. Ataloderinae n. subfam. is proposed and diagnosed in the family Heteroderidae. A key to the three subfamilies is presented and a possible phylogeny of the family Heteroderidae is discussed.
Canonical discriminant analysis of four morphometric characters of juveniles and restriction enzymes analysis of ribosomal DNA sequences were used to distinguish Heterodera arenaria, H. aucklandica, H. avenae, H. filipjevi, H. hordecalis, H. iri, H. latipons, H. litoralis, H. schachtii and an undescribed species from grasslands. The results of unweighted pair group cluster analysis showed that H. avenae populations formed three groups and H. filipjevi two groups at the 80% level of similarity. Intraspecific polymorphism was revealed by rDNA-RFLP studies and two types of ITS regions within H. avenae populations can be distinguished. The pattern of restriction bands obtained with BsuRI, PstI and TaqI clearly distinguished populations of H. filipjevi from other species of the H. avenae group. Further enzymes and their combinations distinguished the other species. There are no enzymes which differentiate European populations of H. avenae from H. arenaria. Morphometrics, restriction endonuclease cleavage maps of ITS regions and a dendrogram of putative phylogenetic relations of several cyst-forming nematode species are given.
The subfamily Meloidoderinae and its genera Meloidodera Chitwood, Hannon & Esser, 1956 and Cryphodera Colbran, 1966 are redefined. M. floridensis Chitwood, Hannon & Esser, 1956, M. charis Hopper, 1960 and C. eucalypti Colbran, 1966 are recognized as the only valid species. Their descriptions are emended and a new Meloidodera species and three new species belonging to a new genus, which is defined, are described and illustrated. A key to the genera and species is presented and their interrelationships are discussed.
Scanning electron microscope observations of heads of second stage juveniles of five of the genera of the Heteroderidae are described and the systematic significance of the different forms discussed. Within the genus Heterodera differences in head morphology generally accord with the recognised sub-divisions of the genus.
Detailed descriptions are given of the amphimictic nematode strains PS1158, PS2052 and PS2160, which are unusual in that they only differ in predominant body handedness. Although these strains are morphologically identical in all other respects, published reproductive data and new DNA sequence data of the D2/D3 region of the large subunit rRNA gene show that they do represent two separate species. On the basis of comparison with type material, the left-handed strains PS1158 and PS2160 are identified as Acrobeloides bodenheimeri, and the right-handled strain PS2052 as A. camberenensis, which is re-instated as a valid species. A. bodenheimeri and its relatives exhibit various types of diagnostic and taxonomic problems at species level, and it is shown that D2/D3 sequence data provide an important new diagnostic tool for addressing these problems. Phylogenetic analysis shows that two right-handed parthenogenetic strains identified as A. maximus represent a third species which is more closely related to A. camberenensis than to A. bodenheimeri.
Heterodera aucklandica sp. n. is described from the New Zealand native grass Microlaena stipoides (Labill.) R.Br. The cyst nematode species belongs to the H. avenae group, a heterogeneous group of species characterised by a vulval slit length in the range 4.5–20 μm. Within the H. avenae group, H. aucklandica resembles most closely Heterodera avenae Wollenweber, 1924 from which it can be distinguished by the longer tail and longer and thinner hyaline part of the tail, and by the flat anterior face of the stylet knobs of the second stage juvenile. The taxonomic status of the species of the H. avenae group is discussed and their key taxonomic features are presented in a table.
Ampli ed ITS region products of rDNA from 25 valid species and one unidenti ed species from the genus Heterodera and from Meloidodera alni were digested by 26 restriction enzymes. A combination of seven enzymes clearly separated the agriculturally most important species from each other and from their sibling species. Species specii c digestion pro les of ITS regions and a table with approximate sizes of digested fragments for several identi cation enzymes are given. Heterogeneity of ITS regions was revealed for some cyst forming nematode species. Résumé – Identi cation de nématodes à kystes du genre Heterodera (Nematoda: Heteroderidae) basée sur les RFLP du DNA ribosomal – Des fragments ampli és de la région de l'ITS du rDNA de 25 espèces valides et d'une espèce non identii ée du genre Heterodera et de Meloidodera alni ont été soumis à une digestion par 26 enzymes de restriction. La combinaison de sept enzymes a permis une séparation nette des espèces les plus importantes en agriculture, tant les unes par rapport aux autres que par rapport aux espèces jumelles. Sont donnés les proo ls spécii ques de digestion des régions de l'ITS et un tableau regroupant les tailles approximatives des fragments digérés pour plusieurs enzymes d'identii cation. L'hétérogénéité des régions de l'ITS a été révélée chez quelques espèces de nématodes à kyste.
The D3 expansion region of the 28S gene and the ITS1-5.8S-ITS2 region of rDNA sequences from Globodera rostochiensis, G. pallida, G. tabacum tabacum, G. tabacum virginiae and G. tabacum solanacearum have been aligned and compared, There are no nucleotide differences in the D3 region sequences between G. rostochiensis and G, pallida. Sequence analysis and RFLPs of ITS-PCR products showed that several haplotypes are present in the genomes of G. rostochiensis and G. pallida populations. Restriction patterns of PCR products for eight enzymes for differentiation of these two species are given. Phylogenetic analysis of 41 ITS region sequences obtained from populations and species of the subfamily Punctoderinae revealed four distinct main clades within Globodera parasitising solanaceous plants: G. rostochiensis, G. tabacum, G. pallida and an undescribed Globodera sp. from South America. The utility of RFLP profiles and sequences of the rDNA are discussed for diagnostics and phylogeny of Globodera.
Taxonomic schemes for the Heteroderinae Filip'ev & Schuurmans Stekhoven, 1941, sensu Luc et al., (1988) have been unstable due to the large number of genera and the paucity of known reliable characters. Reliable characters are essential when using phylogenetic inference in developing a natural classification. Morphological and developmental studies using light, scanning and transmission electron microscopy have revealed the new characters of host response, en face patterns, phasmid structure and female cuticular layers. These techniques also gave us insight into the homoplasy and polarity of many characters, revealed previously undetected character states and clarified misinterpreted character states. A matrix with the 19 most reliable characters is proposed for 20 operational taxonomic units (OTUs) and we employ this matrix for comparing computer generated phylogenetic analyses of the PHYLIP and PAUP packages. PAUP was deemed the more reliable parsimony algorithm for phylogenetic analysis of the Heteroderinae (Fink, 1986; Platnick, 1987). Monophyly of Atalodera + Sherodera + Thecavermiculatus (tribe Ataloderini), and Cactodera + Heterodera + Afenestrata, as well as Punctodera + Globodera + Dolichodera is supported by both programs. Most importantly, analyses strongly support monophyly of all cyst-forming genera (tribe Heteroderini) contrary to previous hypotheses of repeated evolution of the cyst (Wouts, 1985). In addition, monophyly of the Heteroderini with the Ataloderini is demonstrated. PAUP indicates monophyly of Sarisodera + Rhizonema + Bellodera + Hylonema and Ekphymatodera (tribe Sarisoderini new rank). Monophyly of the Sarisoderini was at first only weakly supported, but, subsequently, the reduced width of the submedial lips of second stage juveniles and males was recognized as a synapomorphy which strengthened subsequent PAUP trees and monophyly of the tribe. The present study rejects as paraphyletic or polyphyletic several previously proposed combinations, including Thecavermiculatus sequoiae (versus Rhizonema sequoiae), Sarisodera africana (versus Afenestrata africana), Dolichodera andinus (versus Thecavermiculatus andinus). The question whether T. andinus is a distinct genus, was not resolved due to insufficient data. PAUP supports our previous observations that Cactodera betulae is intermediate in a transformation series between other Cactodera and Heterodera: it also indicates these species as bring monophyletic with Heterodera + Afenestrata, but not with other Cactodera. Although these phylogenetic analyses strongly support some relationships, they indicate unresolved alternative hypotheses for others. Meloidodera (tribe Meloidoderini) and Cryphodera (tribe Cryphoderini) must be investigated for consideration of a possible synapomorphy not included in the present data matrix. Future studies are proposed to more clearly define the monophyly of the Heteroderini, as well as the Sarisoderini. Tests are also proposed to clarify questions of the monophyly of Verutus (tribe Verutini new rank) with the Heteroderinae versus other Tylenchida.
The family Heteroderidae is revised. On the basis of shared, derived characters sister groups are established and arranged in a phylogenetic tree. A hypothetical, primitive ancestor for the family is defined. The genus Verutus has a large equatorial vulval slit and is considered to be the most primitive form. The genus Meloidodera developed by a reduction in vulval size. Genera which developed later exhibit a subterminally located vulval slit and progressively lost the annulation of the female cuticle. In this process four evolutionary lines emerge: (i) a posterior shift of the vulva and the formation of more or less distinct vulval lips gave rise to the genera Zelandodera and Cryphodera; (ii) changes in the lip configuration of the second-stage juvenile gave rise to the genera Hylonema, Afrodera n.g., Heterodera and Bidera; (iii) changes in the composition of the female cuticle resulted in the genera Thecavermiculatus, Atalodera, Sherodera, Sarisodera and Bellodera n.g. and; (iv) a reduction in vulval slit size led to the development of the genera Dolichodera, Globodera, Cactodera and Punctodera. The genera Ephippiodera and Rhizonema are synonymized with Bidera and Sarisodera respectively. Verutus and Meloidodera are recognized as subfamilies Verutinae and Meloidoderinae and the genera in the four evolutionary lines are recognized as subfamilies Cryphoderinae, Heteroderinae, Ataloderinae and Punctoderinae respectively.
Two new genera, Afrodera and Bellodera, are erected for species originally described in Sarisodera and Cryphodera. Both new genera are characterized by a depressed vulval slit and the anus located on the dorsal side of the vulval cone. Differences in lip configuration of the infective juvenile and a postulated difference in female cuticle justifies their placement in different subfamilies. The lip configuration of the infective juveniles in the subfamilies Verutinae, Meloidoderinae, Cryphoderinae, Ataloderinae and Punctoderinae remains basically unchanged. The possible development of this character in the subfamily Heteroderinae is discussed and illustrated. The family Heteroderidae, its six subfamilies and 17 genera are defined or redefined, and for each of the genera the nominal species and their synonyms are listed. New synonyms introduced are: Heterodera rumicis and H. scleranthi of H. trifolii, H. ustinova of Bidera avenae and H. mali of Globodera chaubattia. Cactodera acnidae (Schuster & Brezina, 1979) n. comb. and Dolichodera andinus (Golden, Franco, Jatala & Astogaza, 1983) n. comb. are transferred from Heterodera and Thecavermiculatus respectively. Keys are provided for all taxa for which no suitable keys are available in the literature. Species inquirendae are listed. ac]19840606
DCSE provides a user-friendly package for the creation and editing of sequence alignments. The program runs on different platforms, including microcomputers and workstations. Apart from available hardware, the program is not limited in the size of the alignment it can handle. It deviates more from classical text editors than other available sequence editors because it uses a different approach towards editing. It shifts characters or entire blocks of aligned characters, rather than inserting or deleting gaps in the sequences. Alignment of a new sequence to an existing alignment is partly automated. Although DCSE can be used on protein sequence alignments, it is especially targeted at the examination of RNA. The secondary structure for every sequence can be incorporated easily in the alignment. DCSE also has extensive built-in support for finding and checking secondary structure elements. A sophisticated system of markers allows notation of special positions in an alignment. This system can be used to store information such as the position of hidden breaks, introns and tertiary structure interactions.
Diagnoses of the cyst-forming genera of Heteroderidae (viz., Heterodera, Sarisodera, Globodera, Punctodera, Cactodera, and Dolichodera) and distribution and morphometrics of the 34 known cyst species in the Western Hemisphere are presented along with an illustrated key for the identification of these genera and species. The key is based mainly on cysts and larvae, and important morphological and diagnostic features are extensively shown by LM and SEM illustrations. The genus Bidera is placed as a new synonym under the genus Heterodera.
Systematic contributions to Heteroderidae include description of Cactodera eremica n. sp., an emended diagnosis of Sarisodera Wouts and Sher, 1971, and proposal of a new genus and new combination, Afenestrata africana (synonym Sarisodera africana Luc et al., 1973). Cactodera eremica, from the roots of shadscale in Utah, most closely resembles Cactodera thornei (Golden and Raski, 1977) but differs by the presence of a finely striated cuticle, a fine surface pattern on eggs, a shorter female stylet, distance of the DGO from the stylet, vulval slit, and smaller diameter of circumfenestra, as well as a shorter tail in second-stage juveniles. The response of the host to C. eremica is similar to other Heterodera sensu lato including a large syncytium with wall ingrowths. The diagnosis of Sarisodera is emended to exclude cysts, which do not form in the type species, S. hydrophila. Afenestrata africana differs from S. hydrophila by the formation of cysts, the dorsal position of the anus in females, the shorter stylet, and a pore-like phasmid opening in second-stage juveniles. In addition, the lip pattern of males and juveniles is characterized by a greater degree of fusion of lip parts, the host response is a syncytium (versus a single uninucleate giant cell in S. hydrophila), and the cuticle is thinner and lacks a D layer. Unlike Heterodera, the cyst of Afenestrata lacks fenestrae.
Six geographic isolates of Heterodera avenae, including two isolates each from Sweden, Australia, and the United States, were compared on the basis of 2-D PAGE protein patterns and the complete DNA sequence for the two internal transcribed ribosomal DNA spacers (rDNA ITS1 and ITS2) and the 5.8S rRNA gene. The protein pattern data and rDNA ITS sequence data both indicated that the Swedish Gotland strain of H. avenae differed markedly from the rest of the isolates. Protein patterns for the Australia isolates differed more from a Swedish strict H. avenae isolate and isolates from Oregon and Idaho, than the two U.S. isolates and the Swedish strict H. avenae isolate differed from each other. Except for the Gotland strain isolate, the rDNA ITS sequences were highly conserved among all of the H. avenae isolates, just as we earlier found them to be conserved among species of the schachtii group of Heterodera.
Ribosomal DNA (rDNA) sequence data were compared for five species of Globodera, including G. rostochiensis, G. pallida, G. virginiae, and two undescribed Globodera isolates from Mexico collected from weed species and maintained on Solanum dulcamara. The rDNA comparisons included both internal transcribed spacers (ITS1 and ITS2), the 5.8S rRNA gene, and small portions of the 3' end of the 18S gene and the 5' end of the 28S gene. Phylogenetic analysis of the rDNA sequence data indicated that the two potato cyst nematodes, G. pallida and especially G. rostochiensis, are closely related to the Mexican isolates, whereas G. virginiae is relatively dissimilar to the others and more distantly related. The data are consistent with the thesis that Mexico is the center of origin for the potato cyst nematodes.
The process of multiple sequence alignment provides homology statements for the phylogenetic analysis of molecular data. Unfortunately, multiple alignments are frequently nonunique. Two sources of these multiple alignments are analysis based on different sets of alignment parameter values (gap:change cost ratios) and nonunique equally costly alignments based on a single set of alignment parameters. By "eliding" these individual alignments into a single grand alignment, phylogeny that is weighted toward those positions that align more consistently can be reconstructed. Positions that show greater variation among alignments will be relatively downweighted. The technique results in a weighting procedure that is a posteriori and based on the evidence established from the original sequence alignments.
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
We used nucleotide sequences of the large subunit ribosomal genes (26S rDNA) to examine evolutionary relationships among species of the genus Pratylenchus (Order: Tylenchida, Family: Pratylenchidae), commonly known as root-lesion nematodes. Ten species of Pratylenchus were studied including, P. penetrans, P. crenatus, P. minyus, P. vulnus, P. thornei, P. musicola, P. coffeae, P. hexincisus, P. scribneri, and P. brachyurus. The species Hirschmanniella belli, Meloidogyne javanica, Heterorhabditis bacteriophora, Nacobbus aberrans, Radopholus similis, and Xiphinema index were used as outgroups. Based on parsimony analyses of approximately 307 aligned nucleotides of the D3 expansion region of the 26S rDNA, it is clear that species of Pratylenchus are a paraphyletic assemblage. The outgroup taxon H. belli shares a common ancestor with the clade that includes P. vulnus and P. crenatus while N. aberrans and R. similis share a common ancestor with 5 other species included in this study.