Root Knot Nematode Genomes JD Eisenback RKN juveniles enter root tip SEM Meloidogyne female JD Eisenback Understanding their evolution, diversity and threat through comparative genomics Amir Szitenberg Georgios Koutsovoulos Mark Blaxter Sujai Kumar Evolutionary Biology Group, University of Hull Institute of Evolutionary Biology, University of Edinburgh @davelunt speakerdeck.com/davelunt slides available Dave Lunt
THE MELOIDOGYNE RKN SYSTEM Meloidogyne Root Knot Nematodes • Globally important agricultural pest species • ~5% loss of world agriculture • Enormous plant host range • parasitise all main crop plants JD Eisenback RKN juveniles enter root tip infected uninfected SEM Meloidogyne female JD Eisenback
THE MELOIDOGYNE RKN SYSTEM Meloidogyne Reproduction • Mitotic parthenogens (apomixis) without chromosome pairs. Asexuals- meiosis absent • Meiotic parthenogens (automixis) • Obligatory outbreeding sexuals with males & females (amphimixis) Sexuals- meiosis present Wide variety of reproductive modes in a single genus
MELOIDOGYNE HYBRIDISATION Hybrid Speciation Once thought that hybrid speciation was rare and inconsequential in animals Genome biology is revealing a very different view Heliconius butterflies Lak Malawi cichlids Polar and brown bears
MELOIDOGYNE HYBRIDISATION Hybrid Speciation in Meloidogyne? Previous work suggests interspecific hybridisation may be involved with Meloidogyne asexual species Heliconius butterflies Lake Malawi cichlids Root knot nematodes?
Is M. floridensis the parent of the asexuals? M. floridensis is found within the phylogenetic diversity of asexual species It reproduces sexually by automixis MELOIDOGYNE HYBRIDISATION GENOMICS M.floridensis M. ??? M. incognita M. javanica M. arenaria x apomicts parental species automict apomict apomict automict
M.floridensis M. ??? M. incognita M. javanica M. arenaria x apomicts parental species automict Is M. floridensis the parent of the asexuals? Could it be a parent of the asexual lineages via interspecific hybridisation? MELOIDOGYNE HYBRIDISATION GENOMICS apomict apomict automict What can that tell us about the origins of crop pathogenicity?
MELOIDOGYNE COMPARATIVE GENOMICS From Genomics to Biology Genomics and bioinformatics can reveal complex biological stories, and suggest novel approaches to agricultural problems
MELOIDOGYNE HYBRIDISATION GENOMICS Meloidogyne comparative genomics We have sequenced M. floridensis genome and compare to 2 other published Meloidogyne genomes M.floridensis M. ??? M. incognita M. javanica M. arenaria x apomicts parental species automict asexual, hybrid? sexual, parental? sexual, outgroup 100MB, 100x coverage, 15.3k protein coding loci
Is M. floridensis the parent of the asexuals? 1. look at the within-genome patterns of diversity to determine hybrid nature of genomes 2. look at phylogenetic relationships of all genes to study origins and parents MELOIDOGYNE HYBRIDISATION GENOMICS 1: Intra-genomic diversity 2: Phylogenomics Investigated using whole genome sequences and 2 distinct approaches;
1. INTRA-GENOMIC ANALYSES Divergence of protein-coding alleles Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 Coding sequences from each species were compared to loci in the same species The percent identity of the best match was plotted Self identity comparisons Both M. incognita and M. floridensis show evidence of presence of many duplicates, while M. hapla does not
1. INTRA-GENOMIC ANALYSES Divergence of protein-coding alleles Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 Self identity comparisons Both M. incognita and M. floridensis show evidence of presence of many duplicates, while M. hapla does not This is exactly the pattern expected for hybrid genomes
Is M. floridensis the parent of the asexuals? look at phylogenetic relationships of all genes to study origins and parents MELOIDOGYNE HYBRIDISATION GENOMICS 1: Intra-genomic diversity 2: Phylogenomics
Hybridisation Hypotheses Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 There are very many ways species could hybridise, duplicate genes, lose genes We have selected a broad range of possibilities informed by prior knowledge We have tested their predictions phylogenetically M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 A M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 B M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z C Scenario 4 M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 C M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z M. hapla X Z M. floridensis M. incognita X X+Z M. hapla X Z M. floridensis M. incognita X Z+Z X+Y D 2. PHYLOGENOMIC ANALYSES
17 M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z C Scenario 4 M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z D Scenario 5 M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 Z M. incognita Z+Z 1 & 2 X+Y M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z C Scenario 4 M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z D Scenario 5 Z M. incognita +Z X+Y M. hapla X Y M. floridensis X+Y C Scenario 4 M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z C Scenario 4 M. hapla D M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 Hybridisation hypotheses A B C D We have selected a broad range of possibilities informed by prior knowledge We have tested their predictions phylogenetically
19 M. hapla X M. floridensis X+Y C Scena M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. incognita Z (B) M. incognita is an interspecific hybrid with M. floridensis as one parent
M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z C Scenario 4 M. hapla X Y M. florid X+Y D Scenario X+Y (C) M. incognita and M. floridensis are independent hybrids sharing one parent
Z M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z D Scenario 5 X+Y (D) M. floridensis is a hybrid and M. incognita is a secondary hybrid between M. floridensis and a 3rd parent
2. PHYLOGENOMIC ANALYSES Testing by Phylogenomics Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 M. hapla M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 A M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 B M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z C Scenario 4 M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z D Scenario 5 M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 X+Y C M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z C Scenario 4 M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z D Scenario 5 M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 X+Y D • Recover all genes from 3 genomes • CDS orthologues determined by InParanoid • 4018 ortholog clusters included all 3 species • Retained those with a single copy in the outgroup M. hapla • ML Phylogenies of relationships between Mi and Mf gene copies • Trees parsed and pooled to represent frequencies of different relationships
23 Each tree contains a single M. hapla sequence as outgroup (black square) Grey square indicates relative frequency of those topologies Trees are pooled within squares into different patterns of relationships Grid squares represent different numbers of gene copies
2. PHYLOGENOMIC ANALYSES Testing by Phylogenomics Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 M. hapla M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 A M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 B M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z C Scenario 4 M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z D Scenario 5 M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 X+Y C M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z C Scenario 4 M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z D Scenario 5 M. hapla X Z M. floridensis M. incognita X X+Z B Scenario 3 M. hapla X Z M. floridensis M. incognita X Z+Z A Scenario 1 & 2 X+Y D We assess the fit of the tree topologies to our hypotheses • Five out of seven cluster sets, and 95% of all trees, support hybrid origins for both M. floridensis and M. incognita • ie exclude hypotheses A and B • Hypothesis C best explains 17 trees • Hypothesis D best explains 1335 trees
2. PHYLOGENOMIC ANALYSES Testing by Phylogenomics Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z M. hapla X Z M. floridensis M. incognita X X+Z M. hapla X Z M. floridensis M. incognita X Z+Z X+Y A M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z M. hapla X Z M. floridensis M. incognita X X+Z M. hapla X Z M. floridensis M. incognita X Z+Z X+Y B M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z M. hapla X Z M. floridensis M. incognita X X+Z M. hapla X Z M. floridensis M. incognita X Z+Z X+Y C M. floridensis is a parental species of “double hybrid” M. incognita with other parent unknown M. hapla X Y Z M. floridensis M. incognita X+Y Y+Z C Scenario 4 M. hapla X Y Z M. floridensis M. incognita X+Y (X+Y)+Z D Scenario 5 X Z M. floridensis M. incognita X X+Z B Scenario 3 X+Y Hypothesis D Conclusion:
2. PHYLOGENOMIC ANALYSES Testing by Phylogenomics Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 Pooled analysis of ~1000 gene loci M. hapla M. incognita M. floridensis M. incognita Two topologies are present ‘Conflict’ is evidence for hybridisation
MELOIDOGYNE COMPARATIVE GENOMICS From Genomics to Biology Hybridisation seems common in this group Genomics reveals that both species are hybrids and M. floridensis is a parent of M. incognita
MELOIDOGYNE COMPARATIVE GENOMICS 1. Ongoing Work Genomes in a phylogenetic design Testing effect of recombination & breeding system on genome change • hybrids, inbred, outbred, loss of meiosis, TEs, mutational patterns, gene families
MELOIDOGYNE COMPARATIVE GENOMICS 1. Ongoing Work • Diverse sampling of most important species • Intraspecific diversity and interspecific divergence • Polymorphisms and gene loci linked to resistance change Meloidogyne diversity and adaptation to crop parasitism
MELOIDOGYNE COMPARATIVE GENOMICS 2. Future work • Increase sampling of Meloidogyne species • fully represent this genus • determine intraspecific variation • detect parental species Dave Lunt Evolutionary Biology Group, University of Hull [email protected] davelunt.net I’m happy to collaborate
MELOIDOGYNE COMPARATIVE GENOMICS 2. Future work • Functional Meloidogyne genomics • what is the genomic basis of resistance breaking lines? • can pathogenicity be predicted from genotype? • diagnostic tools for functional genomics? Dave Lunt Evolutionary Biology Group, University of Hull [email protected] davelunt.net
MELOIDOGYNE COMPARATIVE GENOMICS 2. Future work • Increase sampling of Meloidogyne species • What genes are associated with pathogenicity? • Adaptation to crops through transgressive segregation? • The ‘hybrid threat’ • Other nematode species Dave Lunt Evolutionary Biology Group, University of Hull [email protected] davelunt.net
ADAPTATION TO THE AGRICULTURAL ENVIRONMENT Transgressive segregation Transgressive segregation is when the absolute values of traits in some hybrids exceed the trait variation shown by either parental lineage Hybrid Parent 1 Parent 2 Arrows represent ‘phenotypic range’
MELOIDOGYNE COMPARATIVE GENOMICS 2. Future work Dave Lunt Evolutionary Biology Group, University of Hull [email protected] davelunt.net Question If hybridisation can lead to extreme polyphagy and crop pathogenicity, do we need to monitor the “hybrid threat”? speakerdeck.com/davelunt slides available
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