Tylenchidae is a widely distributed soil-inhabiting nematode family characterized by a weak stylet, an undifferentiated non-muscular pharyngeal corpus, and a filiform tail. Currently, it comprises 412 nominal species belongs to 44 genera and estimated species number ranged from 2,000 to 10,000 species (Qing and Bert, 2019). Regardless of their abundance, the delimitation of taxa in this group remains poorly documented and highly uncertain. Consequently, there is no consensus regarding their classification from species level up to family level (Andrássy, 2007; Brzeski, 1998; Qing and Bert, 2019; Siddiqi, 2000).
With the improved availability of genetic sequencing, molecular sequences in species diagnosis and phylogeny analysis have consolidated them as one of the most powerful tools in current taxonomy. Among marker genes, the ribosomal RNA (rRNA) genes are being used as the standard barcode for almost all animals and successfully resolved several groups in Nematoda (Bert et al., 2008; Holterman et al., 2006; Subbotin et al., 2006). However, rRNA genes are problematic in Tylenchidae phylogeny and the unresolved status is unlikely to be improved by intensive species sampling (Qing et al., 2017; Qing and Bert, 2019). Therefore, finding a proper molecular marker gene is crucial for the Tylenchidae study. In this study we examined the mitochondrial Cytochrome Oxidase I gene (COI) of 12 species belong to Tylenchidae (
Soil samples were collected in China from 2018 to 2019. The details on sampling locations and habitats were given in Table 1. The nematodes were extracted from soil samples by Baermann tray and subsequently collected by a 400 mesh sieve (37 μm opening) after 24 hr of incubation. For morphological analysis, the extracted nematodes were manually picked up, fixed with 4% formalin, rinsed several times with deionized water and then transferred to anhydrous glycerin, following the protocol of Seinhorst (1962) and Sohlenius and Sandor (1987).
List species examined in this study and their corresponding sampling locations.
Species | GPS coordinates | Al. | Vegetation environment |
---|---|---|---|
|
26°04´52.9˝N,119°14´26.7˝E | 28 | Scrubland soil with ferns and bamboo |
|
26°08´57.6˝N,119°17´34.4˝E | 107 | Rhizosphere of |
|
26°05´08.2˝N,119°14´10.0˝E | 27 | Swamp soil |
|
26°05´00.9˝N, 119°14´32.6˝E | 25 | Rhizosphere soil of bamboo |
|
26°08´57.6˝N,119°17´34.4˝E | 107 | Rhizosphere soil of |
|
26°09´09.2˝N,119°17´35.7˝E | 88 | Rhizosphere soil of grass near the bamboo |
|
26°09´56.3˝N,117°55´34.2˝E | 644 | Rhizosphere soil of peanut |
|
26°05´00.9˝N,119°14´32.6˝E | 25. | Rhizosphere soil of bamboo |
|
26°05´00.9˝N,119°14´32.6˝E | 25. | Rhizosphere soil of bamboo |
|
26°08´57.3˝N,119°17´34.1˝E | 107 | Rhizosphere soil of |
|
43°48´53.1˝N,125°24´40.3˝E | 225 | Rhizosphere soil of aspen |
|
26°05´23.9˝N,119°14´00.3˝E | 12 | Rhizosphere soil of locust tree |
|
26°05´09.4˝N,119°13´50.2˝E | 7 | Rhizosphere soil of grass |
Measurements and photography were made from slides using Nikon Eclipse Ni-U 931609 Microscope (Nikon Corporation, Tokyo, Japan). Illustrations were prepared manually based on light microscope drawings and edited with Adobe Illustrator CS3 and Adobe Photoshop CS3.
For scanning electron microscopy (SEM), the samples were fixed by formalin, gradually washed with water and post-fixed with 2% PFA + 2.5% glutaraldehyde in 0.1M Sorensen buffer, then washed and dehydrated in ethanol solutions and subsequently critical point dried with CO2. After mounting on stubs, the samples were coated with gold by JFC-1200 and observed with a JSM-3680 (JEOL, Tokyo, Japan).
The fresh nematodes were directly used for DNA extraction. The single nematode was placed in the 10 μl worm lysis buffer (50 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl2, 0.45% NP40, 4.5% Tween 20, pH = 8.3) on a glass slide. The nematode cuticle was broken by a needle and subsequently transferred to a 200 μl Eppendorf tube. After 1 min for freezing in liquid nitrogen, 1 μl proteinase K (1.0 mg/ml) was added and incubated for 1 h at 65˚C and 10 min at 95˚C.
The 18S rRNA was amplified with primers 1096F (5´-GGT AAT TCT GGA GCT AAT AC-3´), 988F (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´) (Holterman et al., 2006). The D2-D3 domains of 28S rRNA (28S) were amplified with primers D2A (5´-ACA AGT ACC GTG AGG GAA AGT-3´), D3B (5´-TCG GAA GGA ACC AGC TAC TA-3´) (Nunn, 1992). The cytochrome c oxidase subunit 1 (COI) gene fragment was amplified using JB3 (5´-TTT TTT GGG CAT CCT GAG GTT TAT-3´) and JB4.5 (5´-TAA AGA AAG AAC ATA ATG AAA ATG-3´) (Bowles et al., 1992). The PCR products were sent for sequencing at BioSune Ltd. (Shanghai, China). The newly obtained sequences were deposited in GenBank (accession numbers MN542198-MN542210 for 18S, MN542185-MN542197 for D2-D3 of 28S, MN577595-MN577621 for COI).
The obtained sequences were analyzed with other relevant reference sequences available in the PPNID database (Qing et al., 2020). Multiple alignments of rRNA genes were made using the Q-INS-I algorithm of MAFFT v. 7.205 (Katoh and Standley, 2013) and the COI gene was aligned using TranslatorX (Abascal et al., 2010) under the invertebrate mitochondrial genetic code. The best-fitting substitution model was estimated using AIC in jModelTest v. 2.1.2 (Darriba et al., 2012). Maximum likelihood (ML) and Bayesian inference (BI) was performed at the CIPRES Science Gateway (Miller et al., 2010) using RAxML 8.1.11 (Stamatakis et al., 2008) and MrBayes 3.2.3 (Ronquist et al., 2012), respectively. ML analysis included 1,000 bootstrap (BS) replicates under the GTRCAT model. Bayesian phylogenetic analysis was carried out using the GTR + I + G model, analyses were run for 5 × 106 generations and Markov chains were sampled every 100 generations and 25% of the converged runs were regarded as burn-in. Gaps were treated as missing data for all phylogenetic analysis. ML bootstrap values and posterior probabilities (PP) were plotted on Bayesian 50% majority rule consensus trees using Tree View v. 1.6.6 (Page, 1996) and Illustrator CS3.
To evaluate the validation and robustness of COI phylogeny in comparison to well-established rRNA phylogeny, we newly sequenced corresponding 28S and 18S rRNA of analyzed Tylenchidae species. Our results concur with previous studies that both regions show serious limitations: phylogenies are poorly resolved and support values do not agree with each other (Qing et al., 2017, 2018). In general, the newly sequenced species are placed in the same cluster or closely related to their corresponding species in GenBank (the morphology details are given in Figs. 1-3 and Supplementary Tables 1-4 in
We obtain 27 newly generated COI sequences from 12 species with lengths ranging from 436 bp to 445 bp. The identification of our representatives was confirmed by their key morphological features (Supplementary Figs. 1-15 in
The compositional bias (GC content) and 1st, 2nd, and 3rd codon position nucleotide alignments.
Taxa | |||
---|---|---|---|
Nucleotide composition | Tylenchidae | Criconematina | Hoplolaimina |
GC | 28.72 | 22.54 | 29.42 |
GC 1st | 39.80 | 28.07 | 38.82 |
GC 2nd | 35.14 | 35.07 | 36.61 |
GC 3rd | 11.23 | 4.48 | 12.84 |
The p-distance of COI gene between studied Tylenchidae species.
LFJ | LFZ | CR | AG | BA | BT | PH | CC | FV | LL1 | LL2 | MB | TA | LB | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
LFJ | 99.5 | |||||||||||||
LFZ | 78.9 | 99.5 | ||||||||||||
CR | 83.0 | 81.4 | 100 | |||||||||||
AG | 83.2 | 81.3 | 99.8 | 99.5 | ||||||||||
BA | 79.8 | 76.2 | 81.5 | 81.3 | 99.5 | |||||||||
BT | 78.7 | 76.6 | 82.4 | 82.3 | 79.4 | 99.8 | ||||||||
PH | 72.5 | 73.5 | 77. 8 | 77.7 | 73.2 | 77.2 | 100 | |||||||
CC | 80.5 | 78.2 | 87.5 | 87.3 | 79.8 | 80.8 | 75.7 | 98.6 | ||||||
FV | 82.4 | 79.5 | 83.3 | 83.2 | 80.9 | 82.5 | 76.3 | 82.4 | 100 | |||||
LL1 | 76.8 | 76.8 | 81.0 | 81.1 | 71.7 | 73.9 | 70.8 | 77.6 | 75.1 | 96.0 | ||||
LL2 | 79.2 | 81.2 | 84.4 | 84.2 | 75.5 | 79.0 | 75.1 | 82.8 | 80.1 | 86.9 | 97.7 | |||
MB | 81.1 | 78.8 | 82.6 | 82.5 | 78.1 | 81.8 | 74.8 | 81.0 | 82.7 | 74.0 | 79.6 | 99.7 | ||
TA | 82.4 | 79.2 | 88. 9 | 88.8 | 81.8 | 85.6 | 78.5 | 83.7 | 83.8 | 76.7 | 81.3 | 84.7 | 100 | |
LB | 84.1 | 81.2 | 86.6 | 87.0 | 81.5 | 81.5 | 72.9 | 87.2 | 84.3 | 77.9 | 84.6 | 84.0 | 84.9 | 0 |
A total of 52 species in Tylenchomorpha and outgroups (alignment of 1,581 characters) were used for COI phylogeny analysis. The resulting ML and BI trees are largely divergent in topologies, and therefore their phylogenies were presented separately. In both ML and BI analyses,
Although COI phylogeny was unable to reject rRNA phylogenies with full confidence, several COI placements were incongruent with rRNA phylogenies with moderate support in ML analyses: (i)
In the present study, we recovered two populations of
The mitochondrial COI gene is one of the most important standard barcoding genes that has been used for almost all animals (Hebert et al., 2004). Its higher mutation rate provides a better differentiation of closely related species and is particularly useful for the identification and description of hybrid or cryptic species (Palomares-Rius et al., 2014; Powers, 2004; Shaw et al., 2013). Although it has only been explored for a limited number of nematode species compared to rRNA (Palomares-Rius et al., 2014), the COI gene has recently received increasing attention for nematode barcoding and phylogeny. In plant-parasitic species, COI data were already available for several important taxa, e.g.