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 Koutsovoulos
Mark Blaxter
Sujai Kumar

COMPLEX HYBRID ORIGINS
OF ROOT KNOT NEMATODES
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

COMPLEX HYBRID
ORIGINS OF ROOT
KNOT NEMATODES
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

COMPLEX HYBRID ORIGINS OF ROOT KNOT NEMATODES
JD Eisenback
JD Eisenback
juveniles
enter
root tip
WHAT’S IN A
GENOME & WHY?
mostly transposons,
repeats, & sequences
of incertae sedis
In eukaryotes its

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?

JD Eisenback
JD Eisenback
juveniles
enter
root tip
WHAT’S IN A
GENOME & WHY?
Evolutionary Forces:
Selection
Gene Flow
Mutation
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 F
sexualasexual
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
A B C D E F
sexualasexual
origin of
asexuality
asexual
MJD White ‘Animal Cytology and Evolution’ 1st ed 1945, p283

A B C D E F
sexualasexual
origin of
asexuality
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’
alleles
by
recom
bination
m
eiosis
hom
ogenizes

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

• 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

M. ﬂoridensis draft genome raw data SRA ERP001338
• 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

• 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

• 100Mb assembly ~100x genomic
coverage
• 15.3k predicted proteins
• similar to published Meloidogyne
genomes
• Suitable for comparative analyses

Comparative genomics questions
• 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 regions
• 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

• 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

• 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• 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 Size
• 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 Hypotheses
• 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
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

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
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 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

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

Testing by Phylogenomics
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
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

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

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

Comparative genomics questions
• 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 Work
• 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 presentation to Nottingham UKNGS 2013

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