On the Close Relatedness of Two Rice-Parasitic Root-Knot Nematode Species and the Recent Expansion of Meloidogyne graminicola in Southeast Asia

Abstract

Meloidogyne graminicola is a facultative meiotic parthenogenetic root-knot nematode (RKN) that seriously threatens agriculture worldwide. We have little understanding of its origin, genomic structure, and intraspecific diversity. Such information would offer better knowledge of how this nematode successfully damages rice in many different environments. Previous studies on nuclear ribosomal DNA (nrDNA) suggested a close phylogenetic relationship between M. graminicola and Meloidogyne oryzae, despite their different modes of reproduction and geographical distribution. In order to clarify the evolutionary history of these two species and explore their molecular intraspecific diversity, we sequenced the genome of 12 M. graminicola isolates, representing populations of worldwide origins, and two South American isolates of M. oryzae. k-mer analysis of their nuclear genome and the detection of divergent homologous genomic sequences indicate that both species show a high proportion of heterozygous sites (ca. 1⁻2%), which had never been previously reported in facultative meiotic parthenogenetic RKNs. These analyses also point to a distinct ploidy level in each species, compatible with a diploid M. graminicola and a triploid M. oryzae. Phylogenetic analyses of mitochondrial genomes and three nuclear genomic sequences confirm close relationships between these two species, with M. graminicola being a putative parent of M. oryzae. In addition, comparative mitogenomics of those 12 M. graminicola isolates with a Chinese published isolate reveal only 15 polymorphisms that are phylogenetically non-informative. Eight mitotypes are distinguished, the most common one being shared by distant populations from Asia and America. This low intraspecific diversity, coupled with a lack of phylogeographic signal, suggests a recent worldwide expansion of M. graminicola.

Keywords: Meloidogyne; biological invasion; heterozygous genome; mitogenome; ribosomal DNA; root-knot nematode.

Figure 1

Maximum likelihood (ML) phylogenetic tree of Meloidogyne spp. based on seven mitochondrial genes (cox1-rrnS-cox3-nad4L-nad3-nad4-nad5) from nine root-knot nematode species. The tree was rooted with Pratylenchus vulnus as an outgroup. GenBank (https://www.ncbi.nlm.nih.gov/genbank/) mitogenome reference sequences are indicated in parenthesis (with sequence from this study in bold), except for M. hapla and M. floridensis (*) for which accession numbers corresponding to the different genes used in the phylogeny are given in Table S2. The numbers beside branches represent ML bootstrap support values >50%. Scale bar represents substitutions per nucleotide position.

Figure 2

Reduced-median network of Meloidogyne graminicola mitochondrial haplotypes. The network was reconstructed with Network v.5 [30], using the 13 available mitogenome sequences, excluding the 111R region. Code names of distinct populations are indicated in the circles and geographic origin is displayed by different colors. The number of mutations is shown on the branches with slashes and black squares that respectively indicate SNPs, and indels or inversion sites. See Figure S4 for the network considering heteroplasmic sites.

Figure 3

Maximum likelihood phylogenetic reconstructions of Meloidogyne species belonging to Clade III, based on (A) D2–D3 nrDNA sequence, (B) the whole nrDNA cluster, (C) the ACC6 genomic region, and (D) the TAA6 genomic region, using either the GTR + G (A, B, D) or GTR + I (C) models. The size of each nucleotide alignment is given in parenthesis. Bootstrap values greater than 50% are indicated on nodes of each phylogenetictree. GenBank sequences are indicated in parenthesis, and sequences from this study are in bold font. The phylogeny (A) is rooted with Pratylenchus penetrans as an outgroup. Scale bar represents substitution per nucleotide position. For increasing the readability of panels (B–D), names of M. graminicola and M. oryzae are distinguished by green and blue, respectively.

Figure 4

The 25-mer distributions of M. graminicola (red) and M. oryzae (blue) genomes. The dashed vertical lines indicate the possible positions of hetero- and homo-peaks.