Talk given UK Population Genetics Group at Popgroup47 in Bath January 2014

1. COMPLEX HYBRID
ORIGINS OF ROOT
KNOT NEMATODES
Dave Lunt
Evolutionary Biology Group, University of Hull
Sujai Kumar
Georgios Koutsovoulos
Mark Blaxter
Institute of Evolutionary Biology, University of Edinburgh

2. COMPLEX HYBRID
ORIGINS 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

3. COMPLEX HYBRID ORIGINS
OF ROOT KNOT NEMATODES
Dave Lunt
Evolutionary Biology Group, University of Hull
@EvoHull
+EvoHull
davelunt.net
dave.lunt@gmail.com
@davelunt
+davelunt
http://www.slideshare.net/davelunt/presentations
http://www.github.com/davelunt

4. THE MELOIDOGYNE RKN SYSTEM
Meloidogyne Root Knot Nematodes
• Globally
important agricultural pest species
• Enormous
• parasitise
• ~5%
plant host range
all main crop plants
loss of world agriculture
RKN
juveniles
enter root tip
infected
JD Eisenback
uninfected
SEM Meloidogyne female
JD Eisenback

5. THE MELOIDOGYNE RKN SYSTEM
Meloidogyne Reproduction
Wide variety of reproductive modes in
a single genus
Asexuals
• Many
species are mitotic parthenogens
without chromosome pairs
Sexuals
• Other
species are meiotic parthenogens
• automixis
• Some
species are obligatory outbreeding
sexuals with males & females
• amphimixis

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

7. MELOIDOGYNE HYBRIDIZATION
Hybrid Speciation
Once thought that hybrid
speciation was rare and
inconsequential in animals
Genome biology is revealing a
very different view

8. MELOIDOGYNE HYBRIDIZATION
Hybrid Speciation in Meloidogyne?
Have the mitotic parthenogen
root knot nematodes arisen by
interspecific hybridization?

9. MELOIDOGYNE HYBRIDIZATION GENOMICS
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 hybridization?
parental species
M.floridensis
x
M. ???
automict
M. incognita
M. arenaria
M. javanica
apomicts

10. MELOIDOGYNE HYBRIDIZATION GENOMICS
Meloidogyne comparative genomics
100MB, 100x coverage, 15.3k protein coding loci
We have sequenced M. floridensis
genome and are able to compare to 2
other Meloidogyne genomes published
by other groups
asexual, hybrid?
parental species
M.floridensis
x
M. ???
automict
sexual, parental?
M. incognita
M. arenaria
M. javanica
apomicts
sexual, outgroup

11. MELOIDOGYNE HYBRIDIZATION GENOMICS
Is M. floridensis the parent of the asexuals?
Investigated using whole genome
sequences and 2 distinct approaches;
1: Intra-genomic diversity
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
2: Phylogenomics

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

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

14. 1. INTRA-GENOMIC ANALYSES
Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
Divergence of protein-coding alleles
Self identity comparisons
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

15. 1. INTRA-GENOMIC ANALYSES
Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
Divergence of protein-coding alleles
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 (nonself) 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

16. 1. INTRA-GENOMIC ANALYSES
Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
Divergence of protein-coding alleles
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

17. 1. INTRA-GENOMIC ANALYSES
Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
Divergence of protein-coding alleles
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
This is exactly the
pattern expected for
hybrid genomes

18. MELOIDOGYNE HYBRIDIZATION GENOMICS
Is M. floridensis the parent of the asexuals?
2: Phylogenomics
1: Intra-genomic diversity
!
look at phylogenetic relationships of all
genes to study origins and parents

19. 2. PHYLOGENOMIC ANALYSES
Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
M. incognita
M. hapla
Z
Z
M. incognita
X
M. floridensis
Z+Z
X
X+Z
X
M. hapla
M. incognita
X
M. hapla
M. floridensis
A Scenario 1 & 2
Z
B
Z
M. incognita
X
M. floridensis
Z
X+Z
X+Y
M. hapla
X
X
B Scenario 3
M. incognita
Z+Z
M. floridensis
M. incognita
X
M. hapla
M. floridensis
A Scenario 1 & 2
M. hapla
Z+Z
A
We have selected a broad range of
possibilities informed by prior knowledge
We have tested their predictions
phylogenetically
X
X
There are very many ways species could
hybridize, duplicate genes, lose genes
M. floridensis
Hybridization Hypotheses
Y+Z
X
Y
Z
C
X
M. floridensis
Y
Z
(X+Y)+Z
X+Y
X
D
M. incognita
Z
Y+Z
M. hapla
X
X+Y
X+Y
M. incognita
X+Z
C Scenario 4
M. floridensis
Z
X
M. hapla
X
M. incognita
Z+Z
B Scenario 3
M. floridensis
M. incognita
X
M. hapla
M. floridensis
M. hapla
A Scenario 1 & 2
Y
Z

20. Z
M. incognita
1&2
X
X
Z
Y+Z
Y
X
C Scenario 3 4
B Scenario
X+Z
Z
Z
X
X+Y
X+Y
X
Y
20
D Scenario
C Scenario 4 5
Z
X+Y
Y+Z
Y
Z
X
Z
(X+Y)+Z
X
M. floridensis
X+Z
X
Y
C
D
D Scenario 5
X+Y
Y+Z
X Z
A
X+Y
(X+Y)+Z
X+Y
Z
Z
Y
Z
X
M. floridensis
M. hapla
M. hapla
M. incognita
M. incognita
M. hapla
M. floridensis
X+Z
M. incognita
X
M. floridensis
Z+Z
M. hapla
M. incognita
M. incognita
M. floridensis
M. floridensis
M. hapla
X
M. hapla
X
M. incognita
Z
M. hapla
M. incognita
X
X
M. incognita
M. floridensis
M. hapla
Z+Z
M. floridensis
M. hapla
M. hapla
B
A Scenario 2 3
B Scenario C Scenario 4
3
A Scenario 1 2
B Scenario
Hybridization 1hypotheses
M. floridensis
M. incognita
X+Y
M. incognita
M. floridensis
M. floridensis
M. hapla
Z+Z
M. hapla
M. incognita
+Z
X
X+Y
Y
C Scenario 4
D

21. X
Z
A Scenario 1 2
M. floridensis
M. incognita
Z+Z
M. hapla
M. floridensis
M. hapla
X
X
X
(A)
Whole genome
duplication(s)
B Scenario

22. X+Z
X
Z
B Scenario 3
22
M. floridensis
M. incognita
X
M. hapla
M. floridensis
M. hapla
M. incognita
Z
X+Y
X
(B)
M. incognita is an
interspecific hybrid with
M. floridensis as one
parent C Scena

23. X
M. florid
M. incognita
Y+Z
Y
C Scenario 4
M. hapla
M. floridensis
M. hapla
X+Y
X+Y
Z
X+Y
X
Y
(C)
M. incognita and M.
floridensis are
independent hybrids
sharing one parent
D Scenario

24. M. floridensis
M. hapla
Z
M. incognita
X+Y
(X+Y)+Z
X+Y
X
Y
Z
(D)
M. floridensis is a hybrid
and M. incognita is a
secondary hybrid
between M. floridensis and
a 3rd parent

25. 2. PHYLOGENOMIC ANALYSES
Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
X
Z
X
A Scenario 1 2
A Scenario 1 2
X
Z
X+Z
Z
X
B 3
B Scenario Scenario 3
•
Phylogenies of relationships between Mi and
Mf gene copies (RAxML)
•
Trees were parsed and pooled to represent
frequencies of different relationships
M. floridensis
M. hapla
M. incognita
M. incognita
M. floridensis
M. incognita
M. floridensis
M. incognita
Y+Z
Y
X
(X+Y)+Z
X+Y
Y+Z
X+Y
B Scenario 3
B Scenario 3
X+Y
X+Y
Z
Y
(X+Y)+Z
X+Y
X
Z
Y
X
Z
Y
D
C
C Scenario 4
C Scenario 4
D Scenario
D Scenario 5 5
Z
M. hapla
M. incognita
M. incognita
M. floridensis
M. floridensis
M. hapla
M. incognita
M. hapla
M. incognita
M. floridensis
M. floridensis
X+Y
X
Z
Z
Z
B
M. floridensis
M. hapla
M. incognita
X+Z
X+Z
X+Z
X
X
A Scenario 1 21 2
A Scenario
X
X
X
M. hapla
We retained those with a single copy in the
outgroup M. hapla
Z
Z
A
M. incognita
M. floridensis
X
Z+Z
Z+Z
X
M. hapla
Z+Z
M.
M. hapla floridensis
M. hapla
Z+Z
X
M. incognita
M. incognita
M. floridensis
X
M. hapla
•
4018 ortholog clusters included all 3 species
M. floridensis
•
X
X
X
Coding sequences from 3 genomes were
placed into orthologous groups (InParanoid)
M. hapla
•
M. hapla
M. hapla
Testing by Phylogenomics

26. Each tree
contains a
single M. hapla
sequence as
outgroup
(black square)
Grid squares
represent
different
numbers of
gene copies
Trees are
pooled within
squares into
different
patterns of
relationships
Grey square
indicates
relative
frequency of
those
topologies
26

27. 2. PHYLOGENOMIC ANALYSES
Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
X
• ie
Z
X
exclude hypotheses A and B
X
Z
C best explains 17 trees
• Hypothesis
D best explains 1335 trees
M. incognita
M. incognita
(X+Y)+Z
X+Y
X+Y
Z
Y
(X+Y)+Z
X+Y
X
Z
Y
X
Z
Y
D
C Scenario 4
C Scenario 4
D Scenario
D Scenario 5 5
Z
M. hapla
M. incognita
M. incognita
M. floridensis
M. floridensis
M. floridensis
M. hapla
Y+Z
Y
X
M. incognita
Y+Z
X+Y
M. floridensis
M. incognita
M. floridensis
M. floridensis
X+Y
X
Z
Z
Z
B Scenario 3
B Scenario 3
M. hapla
X+Z
X
X
X+Z
X+Z
X+Y
C
B 3
B Scenario Scenario 3
M. hapla
M. incognita
M. hapla
Z
Z
X
X
B
M. hapla
M. hapla
M. incognita
M. incognita
M. floridensis
X
A Scenario 1 21 2
A Scenario
Z
X
• Hypothesis
A Scenario 1 2
A Scenario 1 2
X+Z
X
Z+Z
Z+Z
A
M. incognita
X
Z+Z
M. floridensis
M.
M. hapla floridensis
M. hapla
Z+Z
M. incognita
M. incognita
M. floridensis
M. hapla
M. floridensis
M. hapla
out of seven cluster sets, and 95% of all
trees, support hybrid origins for both M.
floridensis and M. incognita
X
X
X
X
• Five
X
M. floridensis
assess the fit of the tree topologies to
our hypotheses
M. hapla
• We
M. hapla
Testing by Phylogenomics

28. B Scenario 3
X
A
M. floridensis
X
XX
Z+Z
Z+Z
Z+Z
XX
Z
Z
Z
X
X
B
X+Z
X+Z
X+Z
X
X
Z
ZZ
M. incognita
X
X
C Scenario 4
C
X+Y X+Y Y+Z Y+Z
X+Y
Y+Z
XX X
Y
Z
Z
(X+Y)+Z
X+Y
(X+Y)+Z
X+Y
Z
X
X
X
X+Y
Y
Z
M. incognita
M. incognita
M. incognita
M. floridensis
M. floridensis
M. hapla
M. floridensis
M. incognita
Y+Z
M. hapla
M. floridensis
YY
M. hapla
M. hapla
M. incognita
Y
M.M. incognita
incognita
M. floridensis
M. hapla
M. floridensis is a parental species
of “double hybrid” M. incognita
with other parent unknown
X+Y
M. floridensis
M. floridensis
M. floridensis
M. hapla
M. hapla
X
M. hapla
M. incognita
Z
M. incognita
M. floridensis
M. floridensis
M. floridensis
M. hapla
M. hapla
M. hapla
M. incognita
M. incognita
M. incognita
M. floridensis
X+Z
X
M. floridensis
M. incognita
X
M. hapla
M. hapla
M. hapla
M. floridensis
X
M. incognita
2. PHYLOGENOMIC ANALYSES
Lunt et al arXiv 2013 http://arxiv.org/abs/1306.6163
Testing by Phylogenomics
X+Y
(X+Y)+Z
X+Y
Y
(X+Y)+Z
X+Y
X+Y X+Y
Y Y Z Z Z
D Scenario 5
Hypothesis D
Z

29. 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...

30. COMPLEX HYBRID ORIGINS
OF ROOT KNOT NEMATODES
Dave Lunt
Evolutionary Biology Group, University of Hull
@EvoHull
+EvoHull
davelunt.net
dave.lunt@gmail.com
@davelunt
+davelunt
http://www.slideshare.net/davelunt/presentations
http://www.github.com/davelunt