THE PHYLOGEOGRAPHY OF IBERIAN ORGANISMS
Nuno Ferrand
CIBIO
Centro de Investigação em Biodiversidade e Recursos Genéticos
Departamento de Biologia, Faculdade de Ciências,
Universidade do Porto, Portugal
Outline of the talk
• Phylogeographic patterns observed in a southern
European refugium using amphibians and reptiles
as model organisms
• Investigating the processes that determined
present-day patterns of genetic diversity in a
model species, the European rabbit (Oryctolagus
cuniculus)
The heterogeneity of the biological
processes in the last 2.5 my
• Population contractions,
fragmentations and long
periods of isolation
• Speciation and endemism
Post-glacial expansions
• The establishment of
hybrid zones
• General appreciation of
refugia
Amphibians and Reptiles as model species
• Why amphibians and reptiles
• Focus: An analysis of types of evolutionary processes that
generate Iberian diversity using selected organisms, not a
descriptive study of species.
Strategy
1. Multiple types of molecular markers including
- mtDNA
- slow- and fast-evolving nuclear markers
(SNPs, nuclear sequences, allozymes and microsatellites)
2. New developments of population genetics (e.g.
the coalescent)
3. More powerful methods of analysis
Our questions
• Is there evidence for multiple divergent lineages within
Iberian amphibians and reptiles?
• Is there evidence for secondary contact between divergent
genetic entities within the Iberian Peninsula? What are the
patterns of admixture?
• Is there evidence for a genetic imprint of both geographical
and demographic expansions in Iberian species?
Highly divergent lineages (5-7 My)
Podarcis spp.
Lissotriton boscai
Podarcis spp. mtDNA analysis
Pinho et al., BMC Evol Biol 2008
Podarcis spp. nuclear genealogies
β-fibint7
Pinho et al., BMC Evol Biol 2008
L. boscai mtDNA analysis
MB
Martinez-Solano et al., Mol Ecol 2006
L. boscai nuclear genealogies
β-fibint7
Teixeira, 2007
Comparison of both nuclear genealogies
β-fibint7
Podarcis spp.
L. boscai
Other examples
Salamandra salamandra
Steinfartz et al. 2000
Alytes obstetricans
Gonçalves 2007
Blanus cinereus
Albert et al. 2007
Vipera latastei
Brito et al. unpublished
Moderately divergent lineages (1-3 My)
Lacerta schreiberi
Chioglossa lusitanica
Lacerta schreiberi mtDNA analysis
Lineage A
Lineage B
B1
A1
27
A
B
27
A2
B2
Paulo et al. Proc Royal Soc B (2001)
L. schreiberi nuclear genealogies
β-fibint7
β-fibint7
J
A
I
B
G
D
H
C
F
E
Other examples
Euproctus asper
Pelodytes punctatus
Carranza & Amat 2005
Pleurodeles waltl
Tejedo et al. unpublished
Carranza & Arnold 2004
Low divergent lineages (< 1 My)
Alytes cisternasii
Distribution of mtDNA lineages in Alytes cisternasii
Gonçalves et al., Mol Ecol 2009
Other examples
Discoglossus galganoi
Lacerta vivipara
Rana iberica
Martinez-Solano 2004
Surget-Groba et al. 2001
Teixeira 2007
Hybrid Zones in Iberia
Triturus marmoratus / pygmaeus
mtDNA evidence for the contact zone between
T. marmoratus and T. pygmaeus
A
T.m.marmoratus
T.m.pygmeus
B
Network of β-fibint7 haplotypes in T. marmoratus
A
A
432
525
244
J
C
463
19 posições
H
T. cristatus
164
526
177
527
B
B
306
389
247
466
487
244
405
419
4 54
306
517
26
49
233
306
307
336
463
307
G
244
80
80
F
D
E
244
225
C
Detection and reconstruction of the
recombinant haplotype
2 3 3 4 4 4
2 4 8 3 2 3 0 1 5
6 9 0 3 7 6 5 9 4
Distribution of β-fibint7 T. marmoratus haplotypes
in Portugal
Geographical and demographical expansion
Other examples are abundant and under current
study in our laboratories
Podarcis spp.
Salamandra salamandra
Garcia-Paris et al. 2003
Chioglossa lusitanica
Pinho et al., 2008
Lacerta schreiberi.
Sequeira et al. 2005, 2008
Geographic and demographic expansions:
Mismatch distributions and patterns of genetic
diversity at multiple loci
Distribution of mtDNA lineages in Rana iberica
Teixeira, 2007
Mismatch analysis in Rana iberica
0.5
Relative frequency
0.45
Lineage A
Sub-lineage A1
0.4
Sub-lineage A2
0.35
Lineage B
0.3
Lineage B South of Douro
Lineage B North of Douro
0.25
0.2
0.15
0.1
0.05
0
0
1
2
3
4
5
Pairwise differences
6
7
8
9
Out of Iberia
and more…
Into Iberia
and more…
Conclusions
Different and well-defined genetic entities in
multiple Iberian taxa
- strongly support the “refugia within refugia” model
- have possible taxonomic implications:
- at the species level
e.g. Podarcis spp.
Triturus marmoratus
Lissotriton boscai
- at the subspecies level e.g. C. lusitanica
Conclusions
A remarkable set of old Hybrid Zones that may have
been in place across multiple Ice Ages
- Evidence for more than one nuclear cline
- Evidence for multiple contacts in the past
- Suggest multiple instances of ongoing early stages of
speciation
- Support for the “melting pot” idea
Conclusions
A crossroad of geographical expansions
- Multiple evidences for expansions within the Iberian
Peninsula, originating in divergent lineages: a combination of
clines and clusters best describe the genetic structure of
species
- The Iberian Peninsula as a source
- The Iberian Peninsula as a receptor
Conclusions
Multiple recent evolutionary processes
e.g. fragmentation, expansion, hybridization, admixture,
speciation, invasions
have been sculpting the genetic architecture of Iberian
amphibians and reptiles, resulting in a complex and fascinating
diversity.
- Future studies
- Conservation implications
- Insights for other studies in different refugia of biological
diversity (e.g. the Brasilian Atlantic Forest or the Amazon)
The European rabbit (Oryctolagus cuniculus) as a
model species in evolutionary biology: new insights
into the investigation of incipient speciation
A brief description of the European rabbit geographical expansion
Ferrand & Branco (2007) Springer
Co-evolution with myxomatosis and haemorhagic viral disease
Curiosity of man and the singularity of rabbit domestication
A huge diversity of rabbit domestic breeds
mtDNA data: evidence for two divergent lineages
53
SWIP
NEIP+FR+DOM
Ferrand (Unpublished data)
mtDNA: a clear contact zone running NW-SE
mtDNA A
mtDNA B
Branco et al 2002 (Evolution)
Two divergent lineages on the Y-chromosome
• Identification of SNPs [ ] in the SRY gene region
HMG
SRY
-500
1
500
1000
1500
• Two lineages (A and B) separated by seven mutations
• Large-scale genotyping of two diagnostic SNPs [ ]
Geraldes & Ferrand 2006 (Genetics, Selection, Evolution)
Y-chromosome data: similar contact zone but… a sharper cline!
Lineage A
Lineage B
Geraldes et al., 2008 (Molecular Ecology)
An example of gene genealogies in different rabbit autosomes
C
T
Carneiro, Ferrand & Nachman 2009 (Genetics)
Using molecular markers from the X-chromosome
• Easy: single-copy in males
• Contrary to mtDNA and the Y-chromosome, the X is a
source of hundreds of independent realizations of the
evolutionary process
• It may have an important role in the early stages of
reproductive isolation
Examples of networks obtained for centromeric (MSN e SMCX)
and telomeric (PHKA2 e HPRT) loci in the X-chromosome
B
A
B
R
A
MSN
PHKA2
A
A
B
R
B
SMCX
HPRT
R
Geraldes, Ferrand & Nachman 2006 (Genetics)
Evidence from the X: absence of introgression in centromeric
markers
SMCX
MSN
7
Lineage A for genes SMCX and MSN
Lineage B for genes SMCX and MSN
Geraldes, Ferrand & Nachman 2006 (Genetics)
Evidence from the X: complete introgression in telomeric
markers
PHKA2
HPRT
7
Lineage A for genes PHKA2 and HPRT
Lineage B for genes PHKA2 and HPRT
Geraldes, Ferrand & Nachman 2006 (Genetics)
A fine-scale analysis of the X-chromosome using multiple
genealogies
STS (7M)
GLRA2 (15M)
SH3KBP1 (20M)
POLA1(25M)
DMD (32M)
CYBB (37M)
MAOA (43M)
Pseudo-autosomal region
PHKA2 (19M)
CNB3(49M)
ALAS2 (55M)
MTMR8 (63M)
FAAH2 (57M)
EDA (68M)
SMCX (53M)
CENTROMERE
MSN (65M)
ATRX (76M)
DACH2(85M)
POU3F4 (83M)
PABPC5 (90M)
DIAPH2 (95M)
PLP1 (103M)
TRPC5 (110M)
WDR44 (117M)
BIRC4 (123M)
ENOX2(129M)
HPRT (133M)
SOX3 (139M)
FMR1 (147M)
G6PD (155M)
Pseudo-autosomal region
An example of the diversity of X-chromosome genealogies
Carneiro, Ferrand & Nachman 2010 (Evolution)
60
FST (%)
40
FST
HPRT
80
PHKA2
100
MSN
SMCX
Distribution of Fst values in the X-chromosome
20
0
Carneiro, Ferrand & Nachman 2010 (Evolution)
A plausible scenario for the recent evolution of rabbit populations
in southern Europe
A good approximation: divergence with gene flow
Present
Past
Hey & Nielsen 2004 (Genetics)
Estimating relevant population parameters
Tempo (anos)
Ne (1000 indivíduos)
Taxa de migração
Carneiro, Ferrand & Nachman 2009 (Genetics)
A simulated evolution of Fst values for a set of molecular
markers in allopatry, followed by secondary contact and
admixture between two diverging genetic entities
The bimodality of the European rabbit genome
14
Autosomes
X-Chromosome
mtDNA
12
Número de loci
Y-Chromosome
10
8
6
4
2
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Fst
Geraldes et al ., 2008 (Molecular Ecology)
A fine-scale and bi-dimensional analysis of the European rabbit
hybrid zone
Carneiro et al . (submitted)
Clinal variation for representative loci and the hybrid index
obtained for the European rabbit hybrid zone
Carneiro et al . (submitted)
Clinal variation for representative loci obtained in the European
rabbit hybrid zone
Carneiro et al . (submitted)
The asymmetry of gene flow patterns between parapatric
population groups
0  Fst  1
Ayala & Coluzzi 2005 (PNAS)
The Dobzhansky-Muller model
New mutation
Ancestral
genotype
Fixation of
new mutation
A
B
a
B
a
B
a
B
Hybrid
A
B
a
B
A
B
A
b
A
b
A
b
A
B
A
b
The experimental analysis of the early stages of reproductive
isolation in the European rabbit
Cross A
♀A
x
♂
A(AC)
x
AC F 1
Cross C
♂C
♀
x
(AC)C
♀C
x
♂
C(CA)
x
CA F 1
♂A
♀
x
(CA)C
The questions we are addressing:
• Is there evidence of hybrid sterility or hybrid breakdown
in experimental crosses between the two subspecies?
• Do we find similar contrasting patterns with a more
dense marker coverage of the X-chromosome?
• Do we find support for a X-chromosome crucial role in
the early stages of reproductive isolation, as recently
experimentally described for Mus (sub)species (Oka et al.,
2007)?
• Are there similar regions in the autosomes that
apparently show reduced levels of gene flow?
• Can we find genetic interactions between autosomes
and the X-chromosome causing reproductive isolation
between the two subspecies?
Very preliminary data on experimental crosses are exciting!
Hybrid males
Hybrid females
14
12
10
Mean
Mean±SE
8
6
4
Number of kittens
2
0
-2
aa/ac
cc/ca
ca/aa
ca/cc
BACKCROSSES TYPES (xx/xy)
Concluding remarks
• The European rabbit genome is a mosaic of two divergent
entities due to very permeable genomic boundaries
• Gene flow has probably occurred during multiple pulses in
previous inter-glacial periods
• Islands of differentiation still persist and are apparently
associated with regions of low recombination or overrepresented in sex-chromosomes
• These islands of differentiation may be asociated to the early
stages of incipient isolation between nascent species
• These patterns are likely to be found in numerous non-model
species, not only in Iberia, but also in many other refugia of
biological diversity
CIBIO - Centro de Investigação
em Biodiversidade e Recursos
Genéticos
[email protected]
Universidade do Porto