Phylogeography of the western
Mediterranean Cymbalaria and
Systematic implications
Pau Carnicero Campmany
Western Mediterranean Islands
Thompson, 2005
Endemisms
How?
Helicodicerus muscivorus
LDD?
30 Ma, Mansion et al., 2008
Palaeoendemics?
High habitat diversity: cliffs
Cymbalaria
Cymbalaria: Western taxa
C. aequitriloba subsp.
insularis
Linaria hepaticifolia
subsp. majorica
Cymbalaria aequitriloba
EXTREMELY VARIABLE!!
Cymbalaria: Western taxa
Cymbalaria hepaticifolia
Cymbalaria fragilis
species or subspecies?
distribution?
Cymbalaria muelleri
Phylogeny
nrDNA phylogeny
Bayesian dating
Ancestral area
reconstruction
Cymbalaria
Monophyly
origin ~7.5 Ma
E-W colonization
Western species
Monophyly
origin: yesterday (~2 Ma)
polyploid group 6-8x
...but (of course)
Incongruities
nrDNA
Hybridization
Incomplete Lineage Sorting
...
low resolution!
cpDNA
Phylogeography and
consequences on present
species?
1- Systematics
What is the best taxonomic
classification according to
genetic and morphological data?
2- Phylogeography
Where is the origin of the western
species and what is the pattern
of colonization among islands?
3- Gene flow
Are there genetic evidences
of present or ancient
hybridization?
?
Methods: Lab work
“Wax on wax off”
Karate kid, Avildsen 1984
Methods: AFLPs (1)
Material
•4 species (?) + 3 outgroups
Restriction
•43 populations
•8-10 inds/population
Ligation
•2 datasets:
• 4 species
• C. aequitriloba
Preselective PCR
Selective PCR:
1. Primer selection (12 primer pairs, 7 inds.)
2. PCR with three primers
http://www.ncbi.nlm.nih.gov/genome/probe/doc/TechAFLP.shtml
Vos et al., 1995
Methods: AFLPs (2)
Peack inspection
(Genographer)
Peack counting & scoring
(150-500 pb, Peack Scanner 1.0, RawGeno 2.0-1)
0.3
11
1111111
1111
1 1 111111111111 111 111
11 1
1 1 11 0.2
1 11 1 1
11 1
1
11 1111111 1
1 1
1
Distance matrix
0.1
-0.4
43 4
3333 33 4 4
4444
3
33
33 3
33 333 3 33333 3
33
3
33
3
3333
3 -0.3
44
4 4
4 44 4
444 4 4
4 4
4
-0.2
0
0.1
-0.1
1 11 11212
22 1 1
1 22 2
11 2 22222 222 22
222
2222 2 2 2
2 2 2222222
2
222
2 2 222
2 2 22
222
-0.2
-0.3
-0.4
AMOVA
--------------------------------------------------------------------Source of
Sum of
Variance
Percentage
variation
d.f.
squares components
of variation
--------------------------------------------------------------------Among
groups
3
6275.551
28.57538 Va
28.77
(species)
Among
populations
within
groups
39
11088.111
36.21828 Vb
Within
populations 257
8873.145
34.52586 Vc
--------------------------------------------------------------------Total
299
26236.807
99.31952
---------------------------------------------------------------------
36.47
34.76
111
11
111
1
1 11
1111111 1111 11
1
111111
1 1 1 1
1 1 1111
1
1 1111
1 1
0
-0.1
1
11
11 11 11 1111 111
1
1 1 1111 1 1
1 1 1 111 11
1
1
0.2
0.3
0.4
Methods: Haplotypes
Material
• 4 species (?) + 2 outgroups
• 43 populations
DNA amplification
(2 primers rpl32, 4 primers ndhF)
Purification
• 3 inds/population
• 2 cpDNA regions: rpl32-trnL(UAG) and ndhF-607R
Analyses
• Parsimony network (TCS 1.21)
• Maximum Parsimony and Bootstrap
(PAUP 4.0b10)
• Bayesian Inference + Ancestral Area
reconstruction (BEAST2)
Sanger sequencing
Methods: Morphometric analyses
• Field (flowers) and lab measures
(Image J, microscope)
• Analyses: MorphoTools (PCA, CDA,
R 3.0.2)
Phylogeography and
consequences on present
species?
1- Systematics
What is the best taxonomic
classification according to
genetic and morphological data?
Systematics (1)
AFLPs
Distances network
• 4 monophyletic
species
Systematics (2)
Congruence with morphology
PCA
CDA
C. fragilis
C. aequitriloba
C. hepaticifolia
Systematics (3)
C. fragilis
Seed
surface:
not so
important!
• endemism of
Menorca… and
Cabrera!
• rank: species
Systematics (4)
AFLPs
Distances tree
Cymbalaria aequitriloba
Balearic
Islands
Sardinia
Corsica
C. aequitriloba
BS = 100%
One morphological variable species
2- Phylogeography
Where is the origin of the western
species and what is the pattern
of colonization among islands?
Phylogeography (1)
Haplotypes
Eastern origin
Sardinia &
Corsica
Phylogeography (2)
Colonization of the Balearic Islands:
• 1st: C. fragilis origin
• 2nd: C. aequitriloba range expansion
Gene diversity
C. fragilis
Balearic Islands
C. aequitriloba
Balearic Islands
Phylogeography (3)
1st: C. fragilis
2nd: C. aequitriloba
Coast line during glaciations
Phylogeography (4)
C. aequitriloba genetic groups (BAPS):
• Sardinian origin
• Dispersal origin: NW Sardinia
3- Gene flow
Are there genetic evidences
of present or ancient
hybridization?
?
Gene flow (1)
AFLPs
INCONGRUITIES
• AFLPs: No present gene
flow
• Haplotypes: Ancient, past
hybridization
Haplotypes
Gene flow (2)
Examples:
C. aequitriloba x C. muralis
• C. muralis naturalized in Sardinia
• Same haplotype: recent
hybridization
0
35
70
140
1/99
H26
1/100
Sardinia
C. aequitriloba x C. fragilis Balearic
Islands
•
C. muralis
1/64
0.74/-
H16
1/88
Corsica
H17
0
1/85
Balearic Islands
45
90
180
H22 - H23
Sicily
0.52/-
2
0.13/-
2 possibilities:
H5 - H10
C. aequitriloba
1
3
C. fragilis
C. hepaticifolia
4
1. Chloroplast capture
C. muelleri
1/71
9
5
6
8
2. Same haplotype origin
10
0.86/-
•
13
0.75/-
11
7
Ancient
12
H1 - H4
1/-
15
14
*
19
16
17
18
0.95/70
22
Other in contact zones...
24
25
20
0.75/-
H18 - H19
1/73
0.18/-
1/100
0.21/1/100
1/91
1/55
0.77/-
0.002
21
H13
H12
0.75/0.99/-
C. pubescens
H24 - H25
H20 - H21
H11
1/94
23
H14 - H15
26
Gene flow (3)
...but also ancestral polymorphism!
Conclusions (1)
• 4 monophyletic species: C. aequitriloba, C.
fragilis, C. hepaticifolia and C. muelleri.
Conclusions (2)
C. fragilis is a species that shows a disjunct
distribution in Menorca and Cabrera. Seed
morphology is not a taxonomic important
character.
Conclusions (4)
• C aequitriloba is a single morphologically
variable species with its diversification center
in Sardinia.
Conclusions (3)
• Origin of Western Cymbalaria species in the
East (Corsica-Sardinia).
Conclusions (5)
• 2 colonizations of the Balearic Islands by long
distance dispersal
1st: C. fragilis
2nd: C. aequitriloba
Conclusions (6)
• Reproductive isolation among species
effective at present, but hybridization ocurred
in the past.
past
present
Acknowledgements
To my directors:
M. Galbany, L. Sáez i N. Garcia
The Evolutionary Systematics group of the Institute of Botany (Innsbruck):
M. Magauer, D. Pirkebner, B. Frajman & P. Schönswetter
... i a vosaltres, gràcies!