Paraphyletic genus Ditylenchus Filipjev (Nematoda, Tylenchida), corresponding to the D. triformis-group and the D. dipsaci-group scheme

Abstract The genus Ditylenchus has been divided into 2 groups: the Ditylenchus triformis-group, and the Ditylenchus dipsaci-group based on morphological and biological characters. A total of 18 populations belong to 5 species of Ditylenchus was studied: Ditylenchus africanus, Ditylenchus destructor, Ditylenchus myceliophagus and dipsaci, Ditylenchus weischeri, the first 3 belong to the Ditylenchus triformis-group, the last 2 the Ditylenchus dipsaci-group. The species of Ditylenchus triformis-group were cultured on fungi, while the species from Ditylenchus dispaci-group cultured on excised roots of plant hosts in petri dish. DNA sequences of regions of the nuclear ribosomal first internal transcribed spacer (ITS1) and the small subunit 18S were PCR amplified, sequenced and the phylogenetic analyses also including the sequences of the closely related species from the GenBank. The randomly amplified polymorphisms of genomic DNA (RAPD) were also generated. Two clusters or clades corresponding to the 2 groups were consistently observed with significant statistical support from the 3 datasets. The phylogenetic analysis also revealed that the genus is paraphyletic, separating the 2 groups by species of Anguina and Subanguina.


Introduction
The genus Ditylenchus Filipjev (1936) consists of 80-90 accepted species (Brzeski 1991) of either mycophagous, entomophlic or plant parasitic species. The genus includes some of the most destructive nematode pests, e.g. the mushroom spawn nematode D. myceliophagus Goodey 1958, the potato rot nematode D. destructor Thorne 1945, and the stem and bulb nematode D. dipsaci (Kühn, 1857) Filipjev 1936, the latter two are also internationally quarantined. As the climate change intensifies and international trade increases, invasive alien species such as nematode species are increasingly becoming serious problems, as demonstrated by the recent outbreak of the stem and bulb nematode in central Canada and the neighboring states of USA, (Yu et al. 2010, Qiao et al. 2013, and the recent finding of potato rot nematode in Ontario (Yu et al. 2012), which was the first finding on the continental Canada for the pest.
Taxonomy of the genus both above and below the rank has been confusing. The genus was first placed in the family Tylenchidae of Tylenchina (Filipjev 1936), moved to Anguillulina Schneider (1939) and moved again to Anguinidae ( Paramonov 1970). The family has been moved between Hexatylina and Tylenchina (Siddiqi 1986, and2000). Within the genus, species delimitation based on morphology has been rather arbitrary, since many morphometrical characters are highly variable and only a few were constant enough to be used for taxonomic purposes (Fortuner 1982). The species complex of D. dipsaci (Sturhan & Brzeski, 1991) makes this situation even more confusing. Recently applications of molecular methods have provided new tools for researchers to better understand the biology and taxonomy of the genus. For example, D. weischeri Chizhov, Borisov & Subbotin (2010) has been separated as a valid species from the D. dipsaci species complex, D. gigas Vovlas (2011) from the giant race of D. dipsaci, and D. africanus Wendt (1995) from D. destructor. Recent phylogenetic studies of ribosomal DNA indicated that the genus may be paraphyletic (Holterman et al. 2009;Giblin-Davis et al. 2010).
Two groups of the genus were recognized: the D. triformis-group and D. dipsacigroup (Siddiqi 1980). The D. triformis-group includes species with a rounded tail tip, lateral fields of six lines, and having mycophagous life cycle such as D. destructor and D. myceliophagous, while the D. dipsaci-group includes obligate plant parasites with a sharp-pointed tail tip and lateral fields of four lines. Those entomophlic species such as D. halictus are also mycophagous; belong to the D. triformis-group (Giblin-Davis et al. 2010).
The objective of the study was to use three molecular datasets, namely ITS1 and 18S fragment sequences of ribosomal DNA and RAPD polymorphisms of genomic DNA, to determine the phylogenetic relationships of the two groups of Ditylenchus species.

Nematode population
Live nematodes of eight populations of D. destructor, six populations of D. dipsaci, one of each D. africanus, D. weischeri and D. myceliophagus from different regions of three countries were collected (Table 1). Species identifications were confirmed using morphological and molecular methods.

Nematode culturing
Ditylenchus destructor, D. myceliophagus and D. africanus were cultured on Fusarium oxysporium on 10% potato dextrose agar (PDA). Ditylenchus dipsaci and D. weischeri were cultured on yellow pea and soybean excised roots on White's medium (White 1939) respectively but attempts were also made to culture D. dipsaic, and D. weischeri on F. oxysporium.

Sample preparation
PDA with fungus media and roots infested with nematodes were cut into small pieces and nematodes extracted using the Baermann funnel method (Baermann 1917).

Sequencing and alignment of ITS1 and 18S regions of nuclear rRNA
A region of the internal transcribed spacer 1 (ITS1) gene was amplified using the primers ITS-F (5'-TTGATTACGTCCCTGCCCTTT-3'), ITS-R (5'-ACGAGC-CGAGTGATCCACCG-3'). The amplification protocol was: initial denaturation at 94 °C for 3 min, followed by 40 cycles of denaturation (30 s at 94 °C), annealing (45 s at 58 °C), and extension (2 min at 72 °C), with a final extension for 10 min at 72 °C. A region of the small subunit (SSU) 18S rRNA gene (18S) was amplified using the primers 18S-F (5'-TTGGATAACTGTGGTTTAACTAG-3') and 18S-R (5'-ATTTCACCTCTCACGCAACA-3'). The amplification condition was: 95 °C for 3 min, followed by 40 cycles of 30 s at 95 °C, 45 s at 60 °C and 2 min at 72 °C, with final extension of 10 min at 72 °C. All PCR reactions were performed in 25 ul volumes including 10 ng DNA, 2.5 μl 10×PCR buffer, 1.5 μl 2.5 mM dNTPs, 0.2 ul 10 μM primers and 0.25 μl Titanium Taq DNA polymerase (supplier). The ITS and 18S fragments were sequenced in-house with an ABI Prism 377 sequencer (Perkin Elmer) in both directions and unambiguous consensus sequences obtained. The sequences were deposited into the genBank database. DNA sequences were aligned by Clustal W (http://workbench.sdsc.edu, Bioinformatics and Computational Biology group, Dept. Bioengineering, UC San Diego, CA). The sequences were compared with those of the other nematode species available at the genBank sequence database using the BLAST homology search program. The model of base substitution was evaluated using MODELTEST (Posada and Crandall 1998;Huelsenbeck and Ronquist 2001). The Akaike-supported model, the base frequencies, the proportion of invariable sites and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 (Huelsenbeck and Ronquist 2001) running the chain for 1 × 106 generations and setting the "burnin" at 1,000. We used the Markov Chain Monte Carlo (MCMC) method within a Bayesian framework to estimate the posterior probabilities of the phylogenetic trees (Larget and Simon 1999) using 50% majority rule.

RAPD (randomly amplified polymorphic DNA) and data analysis
Twenty seven random primers were used for RAPD analysis. These primers were previously shown to be suitable for inter-species comparison of Ditylenchus (Digby and Kempton 1987;Zouhar et al. 2007). All PCR reactions were performed in 25 μl volumes consisting of 1 μL of genomic DNA prepared earlier as described above, 2.5 μl of 10×PCR buffer, 1.25 μl of 2.5 mM dNTPs, and 0.25 μl of Titanium Taq DNA polymerase (Clontech Lab Inc.). Amplification conditions were as follows: an initial denaturation at 94 °C for 1 min, followed by 40 cycles of denaturation at 94 °C for 1min, annealing/extension at 72 °C for 1min and a final extension at 72 °C for 10 min. The PCR products were separated by electrophoresis (100V, 1h) in 2.0% agarose gels in TAE buffer with 180-200 ng DNA. The gels were stained with ethidium bromide, visualized and photographed under UV-light (Bio-rad DX, USA). All reactions were repeated twice for clear and stable banding patterns. The presence or absence of DNA fragments was scored as one or zero, respectively, in the binary matrix. Simple matching coefficients (SM) (Digby and Kempton 1987) and hierarchical cluster analysis were performed with NTSYS2.1 (Exeter Software, Setauket, NY). Cluster analysis, by the un-weighted pair method with arithmetic mean (UPGMA), was performed with the SAHN (sequential, agglomerative, hierarchical and nested clustering method). The robustness of the dendrogram was tested with 1000 bootstrap replicates using PAUP software (Swofford 2003).

DNA sequences:
Ribosomal DNA fragments of the internal transcribed spacer 1 (404 bp) and fragments of the 18S ribosomal RNA gene (902 bp) were amplified and sequenced and sequences deposited in GenBank (www.ncbi.nlm.nih.gov/genbank). GenBank accession numbers are listed in Table 1. Phylogeny: Phylogenetic trees based on the ITS1 and 18S sequences of rDNA are shown in Figures 1 and 2 respectively. The results are consistent for both ITS and 18S with species separating into two clusters, one cluster comprising D. destructor, D. africanus and D. myceliophagus, and the second comprising D. dipsaci, D. weischeri and D. gigas, with the groupings corresponding well with the tail endings. The 2 clusters were separated by species of Anguina. RAPD analysis: Among the 27 primers (excepting RAPD2, RAPD3, RAPD5, RAPD7, OPA17 and OPB16 which amplified no visible bands) 21 random primers produced clear and reproducible bands. A total of 212 bands ranging from 100-2000 bp in size were produced by the 21 primers. 121 and 42 polymorphic bands were obtained for D. destructor and D. dipsaci respectively, which suggests higher genetic variation among populations of the D. destructor than those of D. dipsaci. Figure 3 presents the RAPD profiles obtained from primers OPG-05 to exemplify the banding patterns observed.  The RAPD binary data matrix and resulting simple matching coefficient (SM) are presented in Table 2. Figure 4 shows the dendrogram indicating the relationships among all collections. Species of Ditylenchus separated into two clusters consistent with the phylogenetic results based on the ITS1 and 18S sequences. D. destructor, D. africanus, and D. myceliophagus comprised one cluster and D. dipsaci and D. weischeri the second cluster. All D. destructor populations were in one cluster with similarity of 74.2%, and all six populations of D. dipsaci in the other cluster with a higher degree of genetic similarity (87%).

Conclusions
All three molecular data supports morphological schemes for this genus to be divided into two groups: D. triformis-group and D. dipsaci-group, and that the genus is paraphyletic dividing along the group line by Anguina and Subanguina.

Discussion
The results of the study provide strong evidence for divide the genus into 2 groups, one for D. triformis-group and D. dipsaci-group, and genus is paraphyletic. Paraphyletic and polyphyletic taxa are nothing new to biosystematics, even in nematoda several taxa have been found either paraphyletic or polyphyletic: such as Hoplolaimus is paraphyletic (Bae et al. 2008, Ma et al. 2011 and Aphelenchoididae polyphyletic (Kanzaki et al. 2009). It is debateable whether non-monophyletic taxa should be accepted. However as taxonomy advances from traditional to phylogenetic; however, more and more researchers would reject paraphyletic or polyphyletic taxa since they are inconsistent with evolution.
When the genus Ditylenchus was established by Filipjev (1936) by synonymizing Tylenchus dipasci to D. dipsaci it was placed in the family Tylenchidae (Nematoda: Tylenchida) as the sister genus to Tylenchus. Even today differences between species of the two genera are primarily morphometric, although now the genus is placed in the family of Anguinidae. There is some molecular evidence suggesting that one of the evolutionary paths of plant parasitism in nematodes is from algae-feeding nematodes Tylenchus to Ditylenchus (Holterman et al. 2009), which may be true for the obligate plant parasitic Ditylenchus species since the sharp-pointed tail tip is a feature in common for the two genera. Morphologically, the D. triformis-group is closely related with Safianema, and there is also molecular evidence (Giblin-Davis 2010) that they belong to one clade, that the species of D. triformis-group should be synonymized into Safinema, and there are also molecular evidences that Safianema and D. triformis-group are closely related to Neotylenchidae (suborder Hexatylina) than to Tylenchidae (suborder: Tylenchina) (Robin-Davis 2010), and a rounded tail tip (shared characteristic for both D. triformis-group and Safianema) and is a shared character in Hexatylina. To resolve the synonymization and the eventual high rank placement of the putatively synonymized Safinema, more studies are needed.