Mitochondrial COI gene is valid to delimitate Tylenchidae (Nematoda: Tylenchomorpha) species

Morphological analysis

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).

Molecular analysis

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.

Results

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 https://doi.org/10.6084/m9.figshare.12110667.v1). Exceptionally, two newly recovered Lelenchus leptosoma populations (MN542202, MN542203) are placed separately, one population sister to Lelenchus brevislitus KU234167 (PP = 1, BS = 97) while another sister to all Lelenchus species (PP = 1, BS = 100). The morphological and morphometric comparison showed that two L. leptosoma populations were similar (Fig. 2; Supplementary Table 3 in https://doi.org/10.6084/m9.figshare.12110667.v1) with only few differences: e.g. the excretory pore is more anterior in L. leptosoma population 2 than population 1 (57.5-73.8 vs 74.4-77.0 μm), and L. leptosoma population 2 has shorter pharynx than population 1 (66.0-88.6 vs 89.5-100 μm). Moreover, SEM analysis (Fig. 1) suggested that L. leptosoma population 2 has a broader amphidial aperture than population 1. These differences appear in the variation range of L. leptosoma stated in the study of Geraert (2008). With the limited knowledge of this genus and overall problematic taxonomy in Tylenchidae, here we considered these differences as intra-specific variations of L. leptosoma.

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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 https://doi.org/10.6084/m9.figshare.12110667.v1) together with rRNA molecular evidence. We compared compositional bias of COI sequences and the result suggested Tylenchidae has similar GC content to Hoplolaimina (sensu Siddiqi, 2000) in three codon positions but different from Criconematina (sensu Siddiqi, 2000) in GC content of first and third codon position (Table 2). The analysis of genetic distance suggested that most species can be well-separated except for two reciprocally similar genera Aglenchus and Coslenchus (Table 3).

Discussion

In the present study, we recovered two populations of L. leptosoma that similar in morphology but divergent in phylogenetic placements. Such inconsistency is not surprising as similar cases have been reported in genus Malenchus and Labrys (Qing et al., 2017, 2018). Lelenchus leptosoma is the most frequently encountered species in the genus Lelenchus that includes all Lelenchus spp. without distinct incisures. This species shows great variations in morphology, e.g., body length ranges from 470 to 780 μm, tail 145 to 278 μm (Geraert, 2008). We demonstrated that even only with minor morphological variations, two populations can be significantly divergent in genetics. We concur with De Ley (2000) that the extremely small size masks the actual morphological difference in nematodes. Indeed, only a few morphological characters (including SEM) are practically helpful for Tylenchidae diagnosis, and a substantial amount of cryptic species were therefore ignored (Qing and Bert, 2019). Similarly, our two recovered L. leptosoma were likely to contain at least one cryptic species. However current knowledge in Lelenchus is far from sufficient, especially the type of material and molecular data from different reported populations. Consequently, we followed the suggestion given by De Ley (2000) that the key priority for a difficult taxonomic group is to understand major patterns and clades rather than the compilation of a single taxonomic unit.

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. Bursaphelenchus spp. (Kanzakiand and Giblin-Davis, 2012; Ye et al., 2007), Aphelenchoides spp. (Sánchez-Monge et al., 2017; Xu et al., XXXX), Meloidogyne spp. (Kiewnick et al., 2014), Pratylenchus spp. (Janssen et al., 2017; Qing et al., 2019a), and Scutellonema spp. (Van den Berg et al., 2013). However, due to the problematic taxonomic status and a lack of taxonomic attention to the Tylenchidae (Qing and Bert, 2019), COI data are only available for one species (L. brevislitus) (Soleymanzadeh et al., 2016) regardless of its great diversity. Here we added 27 new COI sequences covering 13 species of Tylenchidae. Our result suggested that the overall resolution of COI phylogeny was low and inferred tree topologies failed to reject rRNA phylogenies. Therefore, we demonstrated that apart from less informative 18S and 28S genes (Qing and Bert, 2019; Qing et al., 2017), COI is also inadequate to resolve Tylenchidae, and therefore searching for valid alternative genes is the key to Tylenchidae phylogeny. Although failing to definitively resolve phylogenies, our analysis of inter-specific/generic differences confirms the validity of COI as a barcode for Tylenchidae. Alongside with our high success rate in PCR amplification using universal COI primer pair JB3/JB4.5 (Bowles et al., 1992), we, therefore, acknowledge the COI as suitable options for Tylenchidae diagnosis.

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