Research Article |
Corresponding author: Qing Yu ( qing.yu@agr.gc.ca ) Academic editor: Hans-Peter Fagerholm
© 2016 Yuejing Qiao, Qing Yu, Ahmed Badiss, Mohsin Zaidi, Ekaterina Ponomareva, Yuegao Hu, Weimin Ye.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Qiao Y, Yu Q, Badiss A, Zaidi MA, Ponomareva E, Hu Y, Ye W (2016) Paraphyletic genus Ditylenchus Filipjev (Nematoda, Tylenchida), corresponding to the D. triformis-group and the D. dipsaci-group scheme. ZooKeys 568: 1-12. https://doi.org/10.3897/zookeys.568.5965
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The genus Ditylenchus has been divided into 2 groups: the D. triformis-group, and the D. dipsaci-group based on morphological and biological characters. A total of 18 populations belong to 5 species of Ditylenchus was studied: D. africanus, D. destructor, D. myceliophagus and dipsaci, D. weischeri, the first 3 belong to the D. triformis-group, the last 2 the D. dipsaci-group. The species of D. triformis-group were cultured on fungi, while the species from D. 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.
Ditylenchus , ITS, 18S ribosomal DNA, RAPD, genetic variations, mycophagous, plant parasitic nematodes, phylogeny
The genus Ditylenchus
Taxonomy of the genus both above and below the rank has been confusing. The genus was first placed in the family Tylenchidae of Tylenchina (
Two groups of the genus were recognized: the D. triformis-group and D. dipsaci-group (
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.
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
Origins, hosts and access numbers of Ditylenchus species and populations used in this study
Code | Species | Location | Host | Accession No. | |
---|---|---|---|---|---|
ITS | 18S | ||||
CH01 | D. destructor | Inner Mongolia, China | Sweet potato | KJ567140 | KJ492926 |
CH02 | D. destructor | Jilin, China | Sweet potato | KJ567141 | KJ492927 |
CH03 | D. destructor | Henan, China | Sweet potato | KJ567142 | KJ492928 |
CH04 | D. destructor | Shandong, China | Sweet potato | KJ567143 | KJ492929 |
CH05 | D. destructor | Jiangsu, China | Sweet potato | KJ567144 | KJ492930 |
CH06 | D. destructor | Hebei, China | Sweet potato | KJ567145 | KJ492931 |
CA01 | D. destructor | Ontario, Canada | Sweet potato | KJ567146 | KJ492932 |
CU01 | D. destructor | Clemson University, USA | Sweet potato | KJ567147 | KJ492933 |
CA02 | D. dipsaci | Ontario, Canada | Onion | KJ567148 | KJ492934 |
CU02 | D. dipsaci | Clemson University, USA | Garlic | KJ567149 | KJ492935 |
CA03 | D. dipsaci | Ontario, Canada | Garlic | KJ567150 | KJ492936 |
CA04 | D. dipsaci | Ontario, Canada | Garlic | KJ567151 | KJ492937 |
CA05 | D. dipsaci | Ontario, Canada | Garlic | KJ567152 | KJ492938 |
CA06 | D. dipsaci | Ontario, Canada | Garlic | KJ567153 | KJ492939 |
DA | D. africanus | South Africa | Peanut | KJ567154 | KJ492940 |
DW | D. weischeri | Manitoba, Canada | Canada thistle | KJ567155 | KJ492941 |
DM | D. myceliophagus | Ontario, Canada | Grass | KJ567156 | KJ492942 |
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 (
PDA with fungus media and roots infested with nematodes were cut into small pieces and nematodes extracted using the Baermann funnel method (Baermann 1917).
One or two extracted nematodes were subjected to DNA extraction. The nematodes were crushed in microtubes containing 40 μL 10×PCR buffer (100 mM Tris-HCl, pH 9.0 at 25 °C, 500 mM KCl, 15 mM MgCl2), 10 μL Proteinase K (1 mg/mL), 50 μL distilled water. The microtubes were incubated for 1.5 h at 65°C followed by 15 min at 95 °C and stored at -20 °C. DNA templates were quantified using a NanoDrop ND-1000 Spectrophotometer (Wilmington, DE, USA).
A region of the internal transcribed spacer 1 (ITS1) gene was amplified using the primers ITS-F (5’-TTGATTACGTCCCTGCCCTTT-3’), ITS-R (5’-ACGAGCCGAGTGATCCACCG-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 (
Twenty seven random primers were used for RAPD analysis. These primers were previously shown to be suitable for inter-species comparison of Ditylenchus (
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
Phylogeny: Phylogenetic trees based on the ITS1 and 18S sequences of rDNA are shown in Figures
The 10001st Bayesian likelihood tree inferred from ITS sequences under GTR+I+G model (lnL = 9697.1895; freqA = 0.2646; freqC = 0.2062; freqG = 0.2602; freqT = 0.269; R(a) = 0.9399; R(b) = 3.4936; R(c) = 2.4954; R(d) = 0.5528; R(e) = 5.2698; R(f) = 1; Pinva = 0.4389; Shape = 0.7862). Posterior probability values exceeding 50% are given on appropriate clades.
The 10001st Bayesian likelihood tree inferred from 18S sequences under GTR+I+G model (lnL = 9697.1895; freqA = 0.2646; freqC = 0.2062; freqG = 0.2602; freqT = 0.269; R(a) = 0.9399; R(b) = 3.4936; R(c) = 2.4954; R(d) = 0.5528; R(e) = 5.2698; R(f) = 1; Pinva = 0.4389; Shape = 0.7862). Posterior probability values exceeding 50% are given on appropriate clades.
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
The RAPD binary data matrix and resulting simple matching coefficient (SM) are presented in Table
Similarity matrix (Simple Matching Coefficient) among all Ditylenchus species obtained with 21 primers and based on shared DNA fragments.
CH01 | CH02 | CH03 | CH04 | CH05 | CH06 | CA01 | CU01 | CA02 | CU02 | CA03 | CA04 | CA05 | CA06 | DA | DW | DM | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
CH01 | 1.000 | ||||||||||||||||
CH02 | 0.909 | 1.000 | |||||||||||||||
CH03 | 0.909 | 0.818 | 1.000 | ||||||||||||||
CH04 | 0.681 | 0.681 | 0.681 | 1.000 | |||||||||||||
CH05 | 0.773 | 0.773 | 0.773 | 0.909 | 1.000 | ||||||||||||
CH06 | 0.773 | 0.773 | 0.773 | 0.909 | 0.818 | 1.000 | |||||||||||
CA01 | 0.727 | 0.727 | 0.727 | 0.772 | 0.773 | 0.864 | 1.000 | ||||||||||
CU01 | 0.773 | 0.773 | 0.773 | 0.818 | 0.818 | 0.909 | 0.955 | 1.000 | |||||||||
CA02 | 0.409 | 0.409 | 0.409 | 0.455 | 0.455 | 0.455 | 0.500 | 0.455 | 1.000 | ||||||||
CU02 | 0.682 | 0.591 | 0.682 | 0.455 | 0.455 | 0.455 | 0.591 | 0.545 | 0.909 | 1.000 | |||||||
CA03 | 0.500 | 0.500 | 0.500 | 0.545 | 0.545 | 0.545 | 0.591 | 0.545 | 0.909 | 0.818 | 1.000 | ||||||
CA04 | 0.500 | 0.500 | 0.500 | 0.545 | 0.545 | 0.545 | 0.591 | 0.545 | 0.909 | 0.818 | 1.000 | 1.000 | |||||
CA05 | 0.500 | 0.500 | 0.500 | 0.545 | 0.545 | 0.545 | 0.591 | 0.545 | 0.909 | 0.818 | 1.000 | 1.000 | 1.000 | ||||
CA06 | 0.500 | 0.500 | 0.500 | 0.545 | 0.545 | 0.545 | 0.591 | 0.545 | 0.909 | 0.818 | 1.000 | 1.000 | 1.000 | 1.000 | |||
DA | 0.591 | 0.591 | 0.591 | 0.727 | 0.727 | 0.636 | 0.591 | 0.636 | 0.545 | 0.545 | 0.636 | 0.636 | 0.636 | 0.636 | 1.000 | ||
DW | 0.591 | 0.500 | 0.591 | 0.455 | 0.545 | 0.455 | 0.500 | 0.455 | 0.818 | 0.727 | 0.818 | 0.818 | 0.818 | 0.818 | 0.545 | 1.000 | |
DM | 0.591 | 0.591 | 0.591 | 0.727 | 0.727 | 0.727 | 0.773 | 0.727 | 0.636 | 0.727 | 0.636 | 0.636 | 0.636 | 0.636 | 0.636 | 0.545 | 1.000 |
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.
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 (
When the genus Ditylenchus was established by
We thank the Chinese Scholarship Council for granting of a scholarship to the senior author to work in the laboratory of Dr. Qing Yu for this study. This study is one part of the doctoral thesis for the senior author. Thanks also go to Dr. Mario Tenuta, University of Manitoba for providing D. weischeri, Dr. Tom Prior, the Food and Environment Research Agency, UK for re-measuring the stylet length of D. myceliophagous, and to Dr. Sonia Steenkamp, ARC-Grain Crops Institute, South Africa for providing D. africanus. We also wish to thank Dr. John Bissett for reviewing the manuscript.