Abstract
Species of the nematode genus Heterorhabditis are important biological control agents against agricultural pests. The taxonomy of this group is still unclear as it currently relies on phylogenetic reconstructions based on a few genetic markers with little resolutive power, specially of closely related species. To fill this knowledge gap, we sequenced several phylogenetically relevant genetic loci and used them to reconstruct phylogenetic trees, to calculate sequence similarity scores, and to determine signatures of species- and population-specific genetic polymorphism. In addition, we revisited the current literature related to the description, synonymisation, and declaration as species inquirendae of Heterorhabditis species to compile taxonomically relevant morphological and morphometric characters, characterized new nematode isolates at the morphological and morphometrical level, and conducted self-crossing and cross-hybridization experiments. The results of this study show that the sequences of the mitochondrial cytochrome C oxidase subunit I (COI) gene provide better phylogenetic resolutive power than the sequences of nuclear rRNA genes and that this gene marker can phylogenetically resolve closely related species and even populations of the same species with high precision. Using this gene marker, we found two new species, Heterorhabditis ruandica n. sp. and Heterorhabditis zacatecana n. sp. A detailed characterization of these species at the morphological and morphometric levels and nematode reproduction assays revealed that the threshold for species delimitation in this genus, using COI sequences, is 97% to 98%. Our study illustrates the importance of rigorous morphological and morphometric characterization and multi-locus sequencing for the description of new species within the genus Heterorhabditis, serves to clarify the phylogenetic relationships of this important group of biological control agents, and can inform future species descriptions to advance our efforts towards developing more tools for sustainable and environmentally friendly agriculture.
Highlights
Ribosomal RNA gene sequences such as internal transcribed spacer (ITS) sequences and the sequences of the D2–D3 expansion segments of the 28S rRNA are traditionally used for identification purposes and for novel taxo nomic status descriptions of the species of the genus Heterorhabditis (Adams et al, 1998; Campos‐ Herrera et al, 2011; Li et al, 2012; Malan et al, 2008; Nguyen et al, 2008; Rana et al, 2020; Spiridonov and Subbotin, 2016)
Our study illustrates the importance of rigorous morphological and morphometric characterization and multi-locus sequencing for the description of new species within the genus Heterorhabditis, serves to clarify the phylogenetic relationships of this important group of biological control agents, and can inform future species descriptions to advance our efforts towards developing more tools for sustainable and environmentally friendly agriculture
Our study illustrates the importance of multi-locus sequencing for the characterization of new species within the genus Heterorhabditis, serves to clarify the phylogenetic relationships of these important biological control agents, and can inform future species descriptions to advance our efforts towards developing more tools for sustainable and environmentally friendly agriculture
Summary
Heterorhabditis nematodes used in this study were collected by us during different nematode collection campaigns carried out in Rwanda, Mexico, and India, or were collected by different collaborators at different locations around the world (Table S1) (Bai et al, 2013; Bhat et al, 2021b; Bruno et al, 2020; Carrera, 2015; Fallet et al, 2020; Mukuka et al, 2010; Rana et al, 2020; Yan et al, 2016). (isolates MEX39, MEX-40, and MEX-41), H. bacteriophora (isolates DE2, DE6, PT1, IT6, EN01, and TT01); H. georgiana Hbb, H. beicherriana H06, H. indica CH7, and H. atacamensis MEX-20 To complete this data set and to obtain genomic sequences of nematodes that belong to all the validly described species of the genus Heterorhabditis, we searched the database of the National Center for Biotechnology Information (NCBI) by the Basic Local Alignment Search Tool (BLAST) using the accession numbers of the sequences obtained previously (Dhakal et al, 2020) (Table S3). The strains were further sub-cultured and maintained on LB agar plates at 28°C To establish their taxo nomic identities, we reconstructed phylogenetic relationships based on whole genome sequences of the isolated bacteria and all the different species/. Whole genome sequence similarities were calculated by the digital DNA-DNA hybridization (dDDH) method using the recommended formula 2 of the genome-togenome distance calculator (GGDC) web service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (Auch et al, 2010a, 2010b; Meier-Kolthoff et al, 2013, 2014)
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