Dave Lunt presentation to Nottingham UKNGS 2013

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 KoutsovoulosMark Blaxter

Sujai Kumar



Dave Lunt

JD Eisenback

JD Eisenback


davelunt.net

@davelunt

[email protected]

@EvoHull +EvoHull

+davelunt

Institute of Evolutionary Biology, University of EdinburghMark Blaxter

nematodes.org


[email protected]

http://www.slideshare.net/davelunt/lunt-nottingham

mailto:[email protected]








Acknowledgements

JD Eisenback

JD Eisenback


Africa Gómez, Richard Ennos, Amir Szitenberg, Karim Gharbi, Chris Mitchell, Steve Moss, Tom Powers, Janete Brito, Etienne Danchin, Marian

Thomson & GenePool

FundingNERC, BBSRC, Yorkshire Agricultural Society,

Nuffield Foundation, University of Hull, University of Edinburgh



JD Eisenback

JD Eisenback


WHAT’S IN A GENOME & WHY?

mostly transposons, repeats, & sequences of incertae sedis

In eukaryotes its



JD Eisenback

JD Eisenback



mostly transposons, repeats, & sequences of incertae sedis

In eukaryotes its

But Why?



JD Eisenback

JD Eisenback



Evolutionary Forces:Selection

Gene FlowMutation

Drift Recombination



JD Eisenback

JD Eisenback



Evolutionary Forces:Selection

Gene FlowMutation

Drift

Recombination


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 Fsexualasexual

origin of asexuality

asexual

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



asexual

MJD White ‘Animal Cytology and Evolution’ 1st ed 1945, p283





asexual


loss of meiosis

A B C D E F


alleles

taxon

Recent

Ancient

1 2 3

asexual sexualasexual

Redrawn after Birky 1996

Divergence between sexual species alleles

Divergence between asexual ‘alleles’

allele

s by r

ecom

binati

on

meiosis

homog

enize

s

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


Meloidogyne Reproduction•Wide variety of reproductive modes in a

single genus

•Mitotic parthenogens (apomics)

•Meiotic parthenogens (automicts)

• Sexual (amphimicts)



single genus

•Many species are mitotic parthenogens without chromosome pairs• Incapable of meiosis

• Could be ‘ancient’ asexuals• 17 million years without meiosis?



single genus•Many species are mitotic parthenogens

without chromosome pairs

•Other species are meiotic parthenogens or sexual• automixis or amphimixis

• undergo meiosis and syngamy


Meloidogyne Reproduction• Wide variety of reproductive modes in a single genus

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

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


JD Eisenback

JD Eisenback

RKN juveniles enter root tip

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

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


M.floridensis M. ???

M. incognita

M. javanica

M. arenaria

x

apomicts

parental species

automict


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

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

http://arxiv.org/abs/1306.6163



The Meloidogyne floridensis genome

M. floridensis draft genome raw data SRA ERP001338Lunt 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






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


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





The Meloidogyne floridensis genome• 100Mb assembly ~100x genomic

coverage

• 15.3k predicted proteins

• similar to published Meloidogyne genomes

• Suitable for comparative analyses





Comparative genomics questionsLunt 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?



INTRA-GENOMIC ANALYSES

ID of duplicated protein-coding regionsLunt 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





















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



COMPARATIVE GENOMICS

M. floridensis Genome SizeLunt 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



HYBRIDIZATION HYPOTHESES

Hybridization HypothesesLunt 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. h

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34

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. flo

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Hybridization hypothesesA B

C D

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(A)Whole genome duplication(s)

36

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(B)M. incognita is an

interspecific hybrid with M. floridensis as one

parent

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(C)M. incognita and M.

floridensis are independent hybrids sharing one parent

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(D)M. floridensis is a hybrid

and M. incognita is a secondary hybrid

between M. floridensis and a 3rd parent


Testing by PhylogenomicsLunt et al arXiv 2013 http://arxiv.org/abs/1306.6163

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• 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

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



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•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





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s

M. i

ncog

nita

X Z+Z

A Scenario 1 & 2

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. h

apla

X Y ZM

. flo

riden

sis

M. i

ncog

nita

X+Y Y+Z

C Scenario 4M

. hap

la

X Y Z

M. f

lorid

ensi

s

M. i

ncog

nita

X+Y

(X+Y)+Z

D Scenario 5

M. h

apla

X Z

M. f

lorid

ensi

s

M. i

ncog

nita

X X+Z

B Scenario 3

M. h

apla

X Z

M. f

lorid

ensi

s

M. i

ncog

nita

X Z+Z

A Scenario 1 & 2

X+Y

Hypothesis D




Comparative genomics questionsLunt 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?




Ongoing WorkLunt 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





Dave Lunt

JD Eisenback

JD Eisenback



Institute of Evolutionary Biology, University of Edinburgh

Georgios KoutsovoulosMark Blaxter

Sujai Kumar



Dave Lunt

JD Eisenback

JD Eisenback


davelunt.net

@davelunt

[email protected]

@EvoHull +EvoHull

+davelunt

Institute of Evolutionary Biology, University of EdinburghMark Blaxter

nematodes.org


[email protected]








Technology

Dave Lunt presentation to Nottingham UKNGS 2013