TALES OF SEX, HYBRIDISATION & ADAPTATION Dave Lunt Evolutionary Biology Group, University of Hull COMPARATIVE GENOMICS OF ROOT KNOT NEMATODES @davelunt speakerdeck.com/davelunt slides available davelunt@gmail.com davelunt.net

Dave Lunt Evolutionary Biology Group, University of Hull COMPARATIVE GENOMICS OF ROOT KNOT NEMATODES Institute of Evolutionary Biology, University of Edinburgh Georgios Koutsovoulos Mark Blaxter Sujai Kumar

COMPARATIVE GENOMICS OF ROOT KNOT NEMATODES Acknowledgements 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

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COMPARATIVE GENOMICS OF ROOT KNOT NEMATODES WHAT’S IN A GENOME & WHY? mostly transposons, repeats, & sequences of incertae sedis For many eukaryotes: but why?

COMPARATIVE GENOMICS OF ROOT KNOT NEMATODES WHAT’S IN A GENOME & WHY? Evolutionary Forces: Selection! Mutation! Drift! Gene Flow! Recombination

THE GENUS MELOIDOGYNE: ROOT KNOT NEMATODES • Globally important agricultural pathogens! • Extreme polyphagy and rapid adaptation! • Hyper-diverse breeding systems! • Ancient asexuals?

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 Wide variety of reproductive modes in a single genus • Many species are mitotic parthenogens (apomixis) without chromosome pairs Asexuals- meiosis absent • Other species are meiotic parthenogens • automixis • Some species are obligatory outbreeding sexuals with males & females • amphimixis Sexuals- meiosis present

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?

THE MELOIDOGYNE RKN SYSTEM Meloidogyne Reproduction Wide variety of reproductive modes in a single genus

MELOIDOGYNE REPRODUCTION Are RKN Ancient Asexuals? Investigations based on multi-species gene sequencing and phylogenetics: ! •testing for changes in molecular evolution pattern of sex-specific loci! •testing for extreme allelic sequence divergence Lunt DH 2008 BMC Evolutionary Biology 8:194

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 sexual asexual origin of asexuality asexual MJD White ‘Animal Cytology and Evolution’ 1st ed 1945, p283

RECOMBINATION AND ASEXUALITY Extreme Allelic Sequence Divergence A B C D E F sexual asexual origin of asexuality asexual

RECOMBINATION AND ASEXUALITY loss of meiosis A B C D E F Extreme Asexual ASD alleles taxon Recent Ancient 1 2 3 asexual sexual asexual Redrawn after Birky 1996 Divergence between alleles of sexual species Divergence between asexual species ‘alleles’ alleles by recom bination m eiosis hom ogenizes

THE SIGNATURES OF ANCIENT ASEXUALITY Extreme allelic sequence divergence Allelic Sequence Divergence is much greater in asexual (apomict) species than sexual species Pi (nucleotide diversity) Sexual Asexual apomicts |

THE SIGNATURES OF ANCIENT ASEXUALITY Extreme allelic sequence divergence ASD can be very large within asexual individuals Species Max intraspecific substitutions Substitutions to closest relative M. incognita 15 0 M. javanica M. javanica 16 0 M. incognita RNA polymerase II Dystrophin Species Max intraspecific substitutions Substitutions to closest relative M. javanica 30 0 M. arenaria M. arenaria 32 0 M. javanica Yet identical alleles can be found between different species

THE SIGNATURES OF ANCIENT ASEXUALITY Extreme allelic sequence divergence ASD can be very large within asexual individuals Species Max intraspecific substitutions Substitutions to closest relative M. incognita 15 0 M. javanica M. javanica 16 0 M. incognita RNA polymerase II Dystrophin Species Max intraspecific substitutions Substitutions to closest relative M. javanica 30 0 M. arenaria M. arenaria 32 0 M. javanica Yet identical alleles can be found between different species This allele sharing is not predicted by ancient asexuality, and suggests interspecific hybridisation

RECOMBINATION AND ASEXUALITY loss of meiosis A B C D E F Extreme Asexual ASD alleles taxon Recent Ancient 1 2 3 asexual sexual asexual Redrawn after Birky 1996 Divergence between alleles of sexual species Divergence between asexual species ‘alleles’ alleles by recom bination m eiosis hom ogenises

RECOMBINATION AND ASEXUALITY A B A D C Extreme Hybrid ASD Alleles Taxa Recent Ancient Sexual parental species Redrawn after Birky 1996 Divergence between alleles of parental species Divergence between hybrid species alleles m eiosis hom ogenises alleles by recom bination A C D Ancestor of sexual parental species Hybridization event meiosis homogenises alleles Sexual parental species hybrid apomict hybrid apomict mitotic

TESTING ANCIENT ASEXUALITY Sex Specific Loci Electron microscope images from Ward lab, http:// www.mcb.arizona.edu/wardlab/ • Nematodes have amoeboid (crawling) sperm! • msp genes only expressed in sperm and spermatocytes! • Structural protein of sperm! • Prediction: msp gene should show signatures of loss of function (pseudogenization) in asexuals Major Sperm Protein

msp intron diversity in asexuals 14 Mutations are not randomly distributed but cluster within the intron, exactly as for functional genes Selection on this gene cannot have been abandoned anciently

msp intron diversity in asexuals 14 ML models of evolution are identical on sexual and asexual branches of tree Selection on this gene cannot have been abandoned anciently

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! Data suggests interspecific hybrid origins Lunt DH 2008 BMC Evolutionary Biology 8:194

HYBRID SPECIATION • Developing methods for investigating the hybrid status of species using genomics! • How has hybridisation influenced Meloidogyne species?! • How do hybrid genomes behave?

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? Have the mitotic parthenogen root knot nematodes arisen by interspecific hybridisation? Heliconius butterflies Lak 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! Could it be a parent of the asexual lineages via interspecific hybridisation? MELOIDOGYNE HYBRIDISATION GENOMICS M.floridensis M. ??? M. incognita M. javanica M. arenaria x apomicts parental species automict apomict apomict automict

MELOIDOGYNE HYBRIDISATION 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 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;

Extreme Hybrid Allelic Sequence Divergence 1. INTRA-GENOMIC ANALYSES: ALLELIC SEQUENCE DIVERGENCE 1: Intra-genomic diversity look at the within-genome patterns of diversity to determine hybrid nature of genomes A B A D C Alleles Taxa Recent Ancient Sexual parental species Divergence between alleles of parental species Divergence between hybrid species alleles A C D Ancestor of sexual parental species Hybridization event Sexual parental species hybrid apomict hybrid apomict mitotic

Extreme Hybrid Allelic Sequence Divergence 1. INTRA-GENOMIC ANALYSES: ALLELIC SEQUENCE DIVERGENCE ‘Alleles’ (homeologues) may date to the divergence of the parental species which hybridized A B A D C Alleles Taxa Recent Ancient Sexual parental species Divergence between alleles of parental species Divergence between hybrid species alleles A C D Ancestor of sexual parental species Hybridization event Sexual parental species hybrid apomict hybrid apomict mitotic

1. INTRA-GENOMIC ANALYSES Divergence of protein-coding alleles Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163 Coding sequences from each of the three target genomes 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

1. INTRA-GENOMIC ANALYSES Divergence of protein-coding alleles 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

1. INTRA-GENOMIC ANALYSES Divergence of protein-coding alleles 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

1. INTRA-GENOMIC ANALYSES Divergence of protein-coding alleles 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! This is exactly the pattern expected for hybrid genomes Self identity comparisons

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 1: Intra-genomic diversity 2: Phylogenomics

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

!40 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 Hybridization hypotheses A B C D

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)

!42 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 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 • Coding sequences from 3 genomes were placed into orthologous groups (InParanoid)! • 4018 ortholog clusters included all 3 species! • We retained those with a single copy in the outgroup M. hapla ! • Phylogenies of relationships between Mi and Mf gene copies (RAxML)! • Trees were parsed and pooled to represent frequencies of different relationships

!46 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

MELOIDOGYNE COMPARATIVE GENOMICS Meloidogyne hybrid species formation • Suggestions that hybrid speciation may be common in Meloidogyne! • Do asexual agricultural pathogens have a single (hybrid) origin! • What are the common features of hybrid genome architecture?! • Ongoing work...

Ongoing work! ! (and wild speculation)

MELOIDOGYNE COMPARATIVE GENOMICS Ongoing Work • ~20 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 Current NERC grant on Meloidogyne breeding system and genome evolution

COMPARATIVE GENOMICS AND MOLECULAR EVOLUTION Phylogenetic models of genome change

COMPARATIVE GENOMICS AND MOLECULAR EVOLUTION Phylogenetic models of genome change

COMPARATIVE GENOMICS AND MOLECULAR EVOLUTION Phylogenetic models of genome change Sexual Sexual Sexual Asexual Species What happens here? Understanding the mutational and selective changes in replicated phylogenetic design

COMPARATIVE GENOMICS AND MOLECULAR EVOLUTION Phylogenetic models of genome change Understanding the mutational and selective changes in replicated phylogenetic design Do transposons increase or decrease with loss of recombination?

COMPARATIVE GENOMICS AND MOLECULAR EVOLUTION Phylogenetic models of genome change Understanding the mutational and selective changes in replicated phylogenetic design What mechanisms promote variation in obligatory inbreeding versus outbreeding species?

COMPARATIVE GENOMICS AND MOLECULAR EVOLUTION Phylogenetic models of genome change Understanding the mutational and selective changes in replicated phylogenetic design How does meiosis affect adaptation?! • gene duplication and origin of new genes! • transgressive segregation

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’

Conclusions RKN are not ancient asexuals despite showing some of the classic signatures instead some are interspecific hybrids

Conclusions M. floridensis is a hybrid and a parent of the double-hybrid M. incognita genomic tests for hybrids, whether recent or ancient, can be sensitive and powerful

Conclusions Root knot nematodes are amazingly successful and have adapted to agriculture very rapidly hybridisation and transgressive segregation may be involved in this rapid adaptation

COMPARATIVE GENOMICS OF ROOT KNOT NEMATODES speakerdeck.com/davelunt Slides available davelunt.net @davelunt davelunt@gmail.com Dave Lunt Evolutionary ! Biology Group! University of Hull @EvoHull EvoHull.org