Dave Lunt presentation to Nottingham UKNGS 2013

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Dave Lunt presentation to Nottingham UKNGS 2013

  1. 1. COMPLEX HYBRID ORIGINS OF ROOT KNOT NEMATODES SEM Meloidogyne female Dave Lunt JD Eisenback JD Eisenback juveniles enter root tip Evolutionary Biology Group, University of Hull Institute of Evolutionary Biology, University of Edinburgh Georgios Koutsovoulos Mark Blaxter Sujai Kumar
  2. 2. COMPLEX HYBRID ORIGINS OF ROOT KNOT NEMATODES SEM Meloidogyne female Dave Lunt JD Eisenback JD Eisenback juveniles enter root tip davelunt.net @davelunt dave.lunt@gmail.com @EvoHull +EvoHull +davelunt Institute of Evolutionary Biology, University of Edinburgh Mark Blaxter nematodes.org Evolutionary Biology Group, University of Hull mark.blaxter@ed.ac.uk http://www.slideshare.net/davelunt/lunt-nottingham
  3. 3. COMPLEX HYBRID ORIGINS OF ROOT KNOT NEMATODES SEM Meloidogyne female Acknowledgements JD Eisenback JD Eisenback juveniles enter root tip Africa Gómez, Richard Ennos,Amir Szitenberg, Karim Gharbi, Chris Mitchell, Steve Moss,Tom Powers, Janete Brito, Etienne Danchin, Marian Thomson & GenePool Funding NERC, BBSRC,Yorkshire Agricultural Society, Nuffield Foundation, University of Hull, University of Edinburgh
  4. 4. COMPLEX HYBRID ORIGINS OF ROOT KNOT NEMATODES SEM Meloidogyne female JD Eisenback JD Eisenback juveniles enter root tip WHAT’S IN A GENOME & WHY? mostly transposons, repeats, & sequences of incertae sedis In eukaryotes its
  5. 5. COMPLEX HYBRID ORIGINS OF ROOT KNOT NEMATODES SEM Meloidogyne female JD Eisenback JD Eisenback juveniles enter root tip WHAT’S IN A GENOME & WHY? mostly transposons, repeats, & sequences of incertae sedis In eukaryotes its But Why?
  6. 6. COMPLEX HYBRID ORIGINS OF ROOT KNOT NEMATODES SEM Meloidogyne female JD Eisenback JD Eisenback juveniles enter root tip WHAT’S IN A GENOME & WHY? Evolutionary Forces: Selection Gene Flow Mutation Drift Recombination
  7. 7. COMPLEX HYBRID ORIGINS OF ROOT KNOT NEMATODES SEM Meloidogyne female JD Eisenback JD Eisenback juveniles enter root tip WHAT’S IN A GENOME & WHY? Evolutionary Forces: Selection Gene Flow Mutation Drift Recombination
  8. 8. COMPLEX HYBRID ORIGINS OF ROOT KNOT NEMATODES Recombination and asexuality • Recombination shapes the genome • We can study its action in species that have lost meiotic recombination- asexuals • Reproduction solely by mitosis has consequences for the genome e.g. • Extreme ‘Allelic’ Sequence Divergence • Decay of genes specific to meiosis, gametes, sexual dimorphism A B C D E F sexualasexual origin of asexuality asexual
  9. 9. RECOMBINATION AND ASEXUALITY Extreme Allelic Sequence Divergence • "If we suppose an ameiotic form evolving for a very long period of time we might imagine its two chromosome sets becoming completely unlike, so that it could no longer be considered as a diploid either in a genetical or cytological sense." • Sometimes called Meselson effect, similar to paralogous loci A B C D E F sexualasexual origin of asexuality asexual MJD White ‘Animal Cytology and Evolution’ 1st ed 1945, p283
  10. 10. RECOMBINATION AND ASEXUALITY Extreme Allelic Sequence Divergence A B C D E F sexualasexual origin of asexuality asexual
  11. 11. RECOMBINATION AND ASEXUALITY loss of meiosis A B C D E F Extreme Allelic Sequence Divergence alleles taxon Recent Ancient 1 2 3 asexual sexualasexual Redrawn after Birky 1996 Divergence between sexual species alleles Divergence between asexual ‘alleles’ alleles by recom bination m eiosis hom ogenizes
  12. 12. THE MELOIDOGYNE RKN SYSTEM Meloidogyne Root Knot Nematodes • Globally important agricultural species • ~5% loss of world agriculture JD Eisenback RKN juveniles enter root tip infected uninfected
  13. 13. THE MELOIDOGYNE RKN SYSTEM Meloidogyne Reproduction • Wide variety of reproductive modes in a single genus • Mitotic parthenogens (apomics) • Meiotic parthenogens (automicts) • Sexual (amphimicts)
  14. 14. THE MELOIDOGYNE RKN SYSTEM Meloidogyne Reproduction • Wide variety of reproductive modes in a single genus • Many species are mitotic parthenogens without chromosome pairs • Incapable of meiosis • Could be ‘ancient’ asexuals • 17 million years without meiosis?
  15. 15. THE MELOIDOGYNE RKN SYSTEM Meloidogyne Reproduction • Wide variety of reproductive modes in a single genus • Many species are mitotic parthenogens without chromosome pairs • Other species are meiotic parthenogens or sexual • automixis or amphimixis • undergo meiosis and syngamy
  16. 16. THE MELOIDOGYNE RKN SYSTEM Meloidogyne Reproduction • Wide variety of reproductive modes in a single genus
  17. 17. MELOIDOGYNE REPRODUCTION Previous Single Gene Sequencing • I can reject ancient asexuality on basis of interspecific allele sharing and identical molecular evolution of sperm protein genes • Although meet ASD expectations of ancient asexuality, other explanations fit better -- ie interspecific hybrid origins Lunt DH 2008 BMC Evolutionary Biology 8:194
  18. 18. MELOIDOGYNE REPRODUCTION Hybrid Speciation • Once thought that hybrid speciation was rare and inconsequential in animals • Genome biology is revealing a different view • We have investigated the origins of Meloidogyne asexuals in this context SEM Meloidogyne female JD Eisenback JD Eisenback RKN juveniles enter root tip
  19. 19. Comparative genomics of hybrid origins • We have a phylogenetic design for investigations • Can map breeding system onto tree • Origins of hybrid genomes can be investigated with whole genome sequences MELOIDOGYNE HYBRIDIZATION GENOMICS
  20. 20. Is M. floridensis the parent of the asexuals? We can investigate this using genome sequences; --look at the within-genome patterns of diversity --look at phylogenetic relationships of all genes MELOIDOGYNE HYBRIDIZATION GENOMICS M.floridensis M. ??? M. incognita M. javanica M. arenaria x apomicts parental species automict
  21. 21. MELOIDOGYNE HYBRIDIZATION GENOMICS Meloidogyne comparative genomics We have sequenced M. floridensis genome and are able to compare to 2 other Meloidogyne genomes published by other groups M.floridensis M. ??? M. incognita M. javanica M. arenaria x apomicts parental species automict asexual hybrid? sexual parental? sexual outgroup
  22. 22. MELOIDOGYNE COMPARATIVE GENOMICS The Meloidogyne floridensis genome • Illumina HiSeq2000 v2 reagents • 100bp paired end • 250bp fragments • 81k scaffolds • N50 3.5k • 30% GC M. floridensis draft genome raw data SRA ERP001338 Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
  23. 23. MELOIDOGYNE COMPARATIVE GENOMICS The Meloidogyne floridensis genome M. floridensis draft genome raw data SRA ERP001338 Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 • DNA isolated from nematodes on plant roots will include many microbial ‘contaminants’ • preliminary assembly of trimmed reads ignoring pairing information • annotate 10k random sampled contigs with taxonomic info determined by megablast • Scatterplot of %GC and read coverage coloured by taxonomy Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
  24. 24. 24 Methodology: Kumar S, Blaxter ML (2012) Simultaneous genome sequencing of symbionts and their hosts. Symbiosis 55: 119–126. doi: 10.1007/s13199-012-0154-6 nematodes
  25. 25. MELOIDOGYNE COMPARATIVE GENOMICS The Meloidogyne floridensis genome • Stringent removal of bacterial sequences • Clusters of bacterial orders Bacillales, Burkholderiales, Pseudomonadales and Rhizobiales • lower coverage and higher %GC clusters excluded • Second round of megablast and hits to bacteria removed Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
  26. 26. MELOIDOGYNE COMPARATIVE GENOMICS The Meloidogyne floridensis genome • 100Mb assembly ~100x genomic coverage • 15.3k predicted proteins • similar to published Meloidogyne genomes • Suitable for comparative analyses Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
  27. 27. MELOIDOGYNE COMPARATIVE GENOMICS Comparative genomics questions Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 • Is there evidence of hybrid origins of asexual species? • Is M. floridensis a parental? • How do offspring and parental genomes differ? • What was the other parent? • Broader implications?
  28. 28. INTRA-GENOMIC ANALYSES ID of duplicated protein-coding regions Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 • Coding sequences from each of the three target genomes (M. hapla, M. incognita and M. floridensis) were compared to the set of genes from the same species • The percent identity of the best matching (non-self) coding sequence was calculated, and is plotted as a frequency histogram • Both M. incognita and M. floridensis show evidence of presence of many duplicates, while M. hapla does not Self identity comparisons
  29. 29. INTRA-GENOMIC ANALYSES ID of duplicated protein-coding regions Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 • Coding sequences from each of the three target genomes (M. hapla, M. incognita and M. floridensis) were compared to the set of genes from the same species • The percent identity of the best matching (non-self) coding sequence was calculated, and is plotted as a frequency histogram • Both M. incognita and M. floridensis show evidence of presence of many duplicates, while M. hapla does not Self identity comparisons
  30. 30. INTRA-GENOMIC ANALYSES ID of duplicated protein-coding regions Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 • Coding sequences from each of the three target genomes (M. hapla, M. incognita and M. floridensis) were compared to the set of genes from the same species • The percent identity of the best matching (non-self) coding sequence was calculated, and is plotted as a frequency histogram • Both M. incognita and M. floridensis show evidence of presence of many duplicates, while M. hapla does not Self identity comparisons
  31. 31. INTRA-GENOMIC ANALYSES ID of duplicated protein-coding regions Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 Self identity comparisons• We have strong evidence that both M. incognita and M. floridensis contain diverged gene copies. • These loci duplicated at approximately the same point in time. • A ploidy change is not involved. • This is expected pattern for hybrid genomes
  32. 32. COMPARATIVE GENOMICS M. floridensis Genome Size Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 • Assembly size is not haploid genome size for hybrid species • Divergence (4-8%) between homeologous (hybrid) copies will preclude assembly • Our assembly of 100Mb is ~2x 50-54Mb genome size of M. hapla
  33. 33. HYBRIDIZATION HYPOTHESES Hybridization Hypotheses Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 • There are very many ways species could hybridize, 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)+ZM.hapla X Z M.floridensis M.incognita X X+Z M.hapla X Z M.floridensis M.incognita X Z+Z X+Y D
  34. 34. 34 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 DM.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 & 2Hybridization hypotheses A B C D
  35. 35. M.hapla X M.floridensis X B Scenario M.hapla X Z M.floridensis M.incognita X Z+Z A Scenario 1 & 2 (A) Whole genome duplication(s)
  36. 36. 36 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
  37. 37. M.hapla X Y ZM.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
  38. 38. 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
  39. 39. HYBRIDIZATION HYPOTHESES 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 • Coding sequences from 3 genomes were placed into orthologous groups and trees constructed • InParanoid algorithm, ML trees constructed with RAxML • Found 4018 clusters of orthologs that included all 3 species • We retained just those that had a single copy in the outgroup M. hapla and resolved the relationships between Mi and Mf gene copies • Trees were parsed and pooled to represent frequencies of different relationships
  40. 40. 40 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
  41. 41. HYBRIDIZATION HYPOTHESES 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
  42. 42. HYBRIDIZATION HYPOTHESES Testing by Phylogenomics Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 M.hapla X Y Z M.floridensis M.incognitaX+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 • The genome data supports both M. incognita and M. floridensis as interspecific hybrids • M. floridensis is a parental species of M. incognita with other parent unknown • Complex hybridization may be a feature of this genus? M.hapla X Y ZM.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
  43. 43. MELOIDOGYNE COMPARATIVE GENOMICS Comparative genomics questions Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 • Is there evidence of hybrid origins of asexual species? • Yes, complex hybrid origins are clear • Is M. floridensis a parental? • Yes, identified by phylogenomics and sequence identity • How do offspring and parental genomes differ? • Broader implications?
  44. 44. MELOIDOGYNE COMPARATIVE GENOMICS Ongoing Work Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 • 19 genomes in a phylogenetic design • Testing effect of breeding system on genome change • hybrids, inbred, outbred, loss of meiosis • TEs, mutational patterns, gene families Current NERC grant on breeding system and Meloidogyne genome evolution
  45. 45. COMPLEX HYBRID ORIGINS OF ROOT KNOT NEMATODES SEM Meloidogyne female Dave Lunt JD Eisenback JD Eisenback juveniles enter root tip Evolutionary Biology Group, University of Hull Institute of Evolutionary Biology, University of Edinburgh Georgios Koutsovoulos Mark Blaxter Sujai Kumar
  46. 46. COMPLEX HYBRID ORIGINS OF ROOT KNOT NEMATODES SEM Meloidogyne female Dave Lunt JD Eisenback JD Eisenback juveniles enter root tip davelunt.net @davelunt dave.lunt@gmail.com @EvoHull +EvoHull +davelunt Institute of Evolutionary Biology, University of Edinburgh Mark Blaxter nematodes.org Evolutionary Biology Group, University of Hull mark.blaxter@ed.ac.uk http://www.slideshare.net/davelunt/lunt-nottingham

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