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

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).

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, Hirschmanniella mucronata (KR819278) was placed as a sister to Basiria aberrans (MN577605, MN577606). Such placement was contrary to its morphological assignment and rRNA-based phylogeny (Bert et al., 2008). Since this standalone sequence was not supported by morphology, and other related species (e.g. Pratylenchus spp.) were properly placed, we considered likely that this sequence had been mislabeled. On the basis of this assumption, the monophyly of Tylenchidae was moderately supported (BS = 83) by ML analysis but not supported by BI analysis (split into three clusters, Figs. 4, 5). In all analyses, individuals of the same population were clustered together, either in a fully supported clade in BI (PP=1) or weakly supported clade in ML (BS from 43 to 72).

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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) Boleodorus thylactus (MN577607, MN577608) was not sister to B. aberrans (MN577605, MN577606); (ii) genus Coslenchus is not monophyletic, with Coslenchus rafiqi (MN577601, MN577602) more close to Aglenchus geraerti (MN577603, MN577604) while Coslenchus costatus (MN577611, MN577612) was placed more divergently; (iii) genus Lelenchus is not monophyletic; (iv) Tylenchus arcuatus (MN577620, MN577621) is not sister to Filenchus vulgaris (MN577613, MN577614); (v) Labrys fuzhouensis (MN577599, MN577600) is closer to two populations of L. leptosoma (MN577595, MN577596, MN577615, MN577616) than to Labrys fujianensis (MN577697, MN577698); (vi) two L. leptosoma populations were clustered together not as sister of Lelenchus brevislitus; (vii) Psilenchus hilarulus (MN577609, MN577610) or B. thylactus was placed as outgroup of all other Tylenchidae species. Aside from Tylenchidae, species from other taxa were in general agreement with rRNA-based phylogeny (Figs. 6, 7).

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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