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Zlatko Zlatko ŠŠatoviatoviććUniversity of Zagreb, Faculty of AgricultureUniversity of Zagreb, Faculty of Agriculture,, Zagreb, CroatiaZagreb, Croatia
Centre of Excellence for Biodiversity and Molecular Plant BreediCentre of Excellence for Biodiversity and Molecular Plant Breeding, ng, Zagreb, CroatiaZagreb, Croatia
EE--mail: zsatovic@agr.hrmail: zsatovic@agr.hr
GENETIC DIVERSITY OFGENETIC DIVERSITY OFWILD WILD vvs. s. CCULTIVATEDULTIVATED//NATURALIZED NATURALIZED POPULATIONS OF DALMATIAN SAGEPOPULATIONS OF DALMATIAN SAGE
(1) Dalmatian sage (Salvia officinalis L.)
(2) Genetic diversity and relationships
(3) Genetic structure
(4) Ecological niche modelling
(5) Demographic history
PLANPLAN
(1) Dalmatian sage (Salvia officinalis L.)
(2) Genetic diversity and relationships
(3) Genetic structure
(4) Ecological niche modelling
(5) Demographic history
PLANPLAN
DALMATIAN SAGEDALMATIAN SAGE
- Salvia officinalis L.
- outcrossing, insect-pollinated
- perennial subshrub- widely used since ancient times
for medicinal, culinary and ornamental purposes
- natural distribution:coastal region of the western Balkan and central and southern ApenninePeninsulas
- cultivation:throughout the Mediterranean region, in Australia,Germany, USA etc.
- naturalized populations:plants that have escaped from earlier cultivation
NATURAL DISTRIBUTION OF DALMATIAN SAGENATURAL DISTRIBUTION OF DALMATIAN SAGE
SVNHRV
BIH
MNE
MKDALB
GRC
ITA
KOS
SRB
ROM
MDA
BLG
HUN
(1) Dalmatian sage (Salvia officinalis L.)
(2) Genetic diversity and relationships
(3) Genetic structure
(4) Ecological niche modelling
(5) Demographic history
PLANPLAN
GGENETIC DIVERSITYENETIC DIVERSITY
(1) Collecting
- leaf tissue for DNA extraction
- 30 populations / 709 samples (20 to 25 per populations) - origin:
11 Balkan countries
- status:23 wild populations
7 cultivated/naturalized populations
(2) DNA extraction
(3) Molecular marker analysis
- 8 microsatellite markers developed for Dalmatian sage
(simple sequence repeats; SSRs)
- 165 alleles
COLLECTINGCOLLECTING
Populations:
wild
cultivated
(naturalized)
KomiKomižža, a, iisland of sland of VVisis
CCaavtatvtat
(A) Isolation and characterization of microsatellites- construction of microsatellite libraries from genomic
DNA for GA, GT, AGA and ACA repeats- primer pairs flanking microsatellite repeats were
designed for 29 clones- 29 microsatellites were tested using 25 plants(B) Microsatellite marker analysis- eight most polymorphic markers: 165 alleles
MOLECULAR MAMOLECULAR MARRKER ANALYSISKER ANALYSIS
sequencing scoring
microsatellite region
ind2
ind3
ind1130100
172
142130
130
MOLECULAR DATAMOLECULAR DATA
Population Individual SoUZ001 SoUZ001 SoUZ002 SoUZ002 ... SoUZ019 SoUZ019
P01 S001 173 189 183 185 135 150
P01 S002 165 199 185 185 150 153
P01 S003 183 185 195 197 150 159
P01 S004 163 187 189 195 150 150
P01 S005 165 167 187 195 135 150
P01 S006 165 183 185 187 135 156
P01 S007 173 183 185 195 150 159
P01 S008 165 167 183 195 135 135
P01 S009 165 171 181 183 147 150
P01 S010 165 173 195 201 150 159
...
P30 S709 161 165 185 195 135 150
GGENETIC DIVERSITYENETIC DIVERSITY
Wild Cultivated
No. 23 7
Nav 8.696 3.821
Nar 7.920 3.672
Range 5.13-10.30 2.71-4.19
P(Nar) P < 0.001
Npr(total per population)
20 0
Npr(wild vs.
cultivated)
115 0
(1) Average no. of alleles per population (Nav)
(2) Allelic richness (Nar)- average no. of alleles per population independent of sample size
(3) No. of private alleles (Npr)- number of allelesdetected in a single population
(or in a group of populations: wild vs. cultivated)
AALLELIC RICHNESSLLELIC RICHNESS
Allelic richness:
< 5
5 - 7
7 - 9
> 9
GRC 1
GRC 2
ALB 2
MNE 2
MKD 1
MKD 2HRV 6
MNE 1
SRB 2
BIH 3BIH 2
BIH 1
ALB 1
BIH 4
HRV 8HRV 7
HRV 5
HRV 3
HRV 4
HRV 2
HRV 1
SVN 2
SVN 1
SRB 1 ROU 2
MDA 1
MDA 2
ROU 1KOS 1
KOS 2
0.01
81
99
99
100
99
72
CultivatedNaturalized
- distance matrix:Cavalli-Sforza and Edwards' chord distance
- tree algorithm:Neighbour-joining
- unrooted tree
- seven cultivated/naturalized populations groupedseparately from the restand formed a well-supported clade (bootstrap support 99%) suggesting the common origin of the cultivated plant material
GENETIC RELATIONSHIPSGENETIC RELATIONSHIPS
Bootstrap support:
(1) Dalmatian sage (Salvia officinalis L.)
(2) Genetic diversity and relationships
(3) Genetic structure
(4) Ecological niche modelling
(5) Demographic history
PLANPLAN
- Bayesian model-based clustering method for inferring population structure
Assumption:
- there are K populations of origin each of which is characterized by a set of allele frequencies at each locus
Goal:
- assign individuals to populations of origin in such a way that within each population the departures from:
(1) Hardy-Weinberg equilibrium (HWE) and
(2) linkage equilibrium (LE)
are minimized
GENETIC STRUCTUREGENETIC STRUCTURE
PROCEDUREPROCEDURE
(1) Estimation of the number of Ks
populations of origin
(1.1) Choose different values of K
(1.2) Compute posterior probabilities for each K
(1.3) Choose the most likely number of populations (K)
(2) Assignment
(2.1) Assign individuals to populations (K)
(2.2) For each individal, estimate the proportion of genome derived from the different population of origin
GGENETIC STRUCTURE AT K = 2ENETIC STRUCTURE AT K = 2
100
75
50
25
0
K = 2
Q
Cluster: A B
P30
P29
P28
P27
P26
P25
P24
P23
P22
P21
P20
P19
P18
P17
P16
P15
P14
P13
P12
P11
P10
P09
P08
P07
P06
P05
P04
P03
P02
P01
BCultivatedpopulations
AWild
populations
- proportions of membership (Q) of each individual plant in each of the two clusters (population of origin):- each individual plant is represented by a single
vertical line divided into colors representing different clusters
- the length of the colored segment shows the individual’s estimated proportion of membership in that cluster
P26Sićevo Gorge
Serbia
SVN HRV BIH MNE ALB MKD GRE KOS SRB ROM MDA
100
75
50
25
0
100
75
50
25
0
K = 3
K = 2
Pro
port
ion o
f m
embers
hip (Q
)
Cluster: A B
Cluster: A1 A2 B
P30
P29
P28
P27
P26
P25
P24
P23
P22
P21
P20
P19
P18
P17
P16
P15
P14
P13
P12
P11
P10
P09
P08
P07
P06
P05
P04
P03
P02
P01
BCultivatedpopulations
A1North-Westpopulations
A2South-Eastpopulations
- the cluster A (Wild populations) is at K = 3 split intotwo clusters according to geographical locationsA1 North-West: Adriatic populationsA2 South-East: Montenegro-Albania-Macedonia-Greece
SVN HRV BIH MNE ALB MKD GRE KOS SRB ROM MDA
GGENETIC STRUCTURE AT K = 3ENETIC STRUCTURE AT K = 3
100
75
50
25
0
100
75
50
25
0
100
75
50
25
0
K = 4
K = 3
K = 2
Pro
port
ion o
f m
embers
hip (Q
)
Cluster: A1 A2 A3 B
Cluster: A B
Cluster: A1 A2 B
P30
P29
P28
P27
P26
P25
P24
P23
P22
P21
P20
P19
P18
P17
P16
P15
P14
P13
P12
P11
P10
P09
P08
P07
P06
P05
P04
P03
P02
P01
BCultivatedpopulations
A1NothernAdriatic
A2SouthernAdriatic
A3MacedoniaGreece
- wild populations split further into three clusters
GENETIC STRUCTURE AT K = 4GENETIC STRUCTURE AT K = 4
A1
A2 A3
B
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
Geographic distance
Genetic d
ista
nce r = 0.47
PMantel = 0.0001
R2 = 0.23
GENETIC STRUCTURE AT K = 4GENETIC STRUCTURE AT K = 4
(1) Dalmatian sage (Salvia officinalis L.)
(2) Genetic diversity and relationships
(3) Genetic structure
(4) Ecological niche modelling
(5) Demographic history
PLANPLAN
ECOLOGICAL NICHE MODELLINGECOLOGICAL NICHE MODELLING
- species distribution modelling (SDMs)- estimate the relationship between species records at
sites and the environmental characteristics of those sites
- predict the suitability of sites for occupation or persistence of the species
- produce a modelled distribution of the species(= identify species' suitable environmental space)
- input data:(1) geographic distribution
68 data on occurrence (evenly distributed)(2) environmental characteristics of the sites
19 bioclimatic variables (WorldClim database)- 11 temperature- and 8 precipitation-related- representing the annual trends, seasonal variations and extremes in temperature and precipitation
0.26 - 0.50
0.51 - 0.75
0.76 - 0.94
Suitability
EENM: PRESENT DAY CONDITIONSNM: PRESENT DAY CONDITIONS
0.26 - 0.500.51 - 0.750.76 - 0.94
Suitability
Apennine Peninsula
Adriatic Sea
Balkan Peninsula
0.26 - 0.50
0.51 - 0.75
0.76 - 0.94
Suitability
EENM: PRESENT DAY CONDITIONSNM: PRESENT DAY CONDITIONS
0.26 - 0.500.51 - 0.750.76 - 0.94
Suitability
Apennine Peninsula
Adriatic Sea
Balkan Peninsula
A1
A2A3
PAST DISTPAST DISTRRIBUTIONIBUTION
- to model potential species distribution during the
Last Glacial Maximum (LGM; ~21,000 years BP)
- to identify putative glacial refugia of the species
(= region which made possible the survival of the species and allowed a post-glacial re-colonization)
- input:
(1) the present model
(2) past environmental characteristics of the sites
19 bioclimatic variables
bioclimatic data for the LGM developed by
Paleoclimate Modelling Intercomparison Project Phase II
- two models:
CCSM (Community Climate System Model; USA)
MIROC (Model for Interdisciplinary Research on Climate; Japan)
EENNMM: LAST GLACIAL MAXIMUM: LAST GLACIAL MAXIMUM CCCSM CONDITIONSCSM CONDITIONS
Balkan PeninsulaApennine Peninsula
0.26 - 0.500.51 - 0.750.76 - 0.94
Suitability
A3
Adriatic Sea
- CCSM: severe temperature decline
EENNMM: LAST GLACIAL MAXIMUM : LAST GLACIAL MAXIMUM MMIROCIROC CONDITIONSCONDITIONS
Suitability0.26 - 0.50
0.51 - 0.75
0.76 - 0.84
0.26 - 0.500.51 - 0.750.76 - 0.94
Suitability
Apennine Peninsula
Balkan Peninsula
A3
A1
A2
Adriatic Sea
- MIROC: moderate temperature decline
(1) Dalmatian sage (Salvia officinalis L.)
(2) Genetic diversity and relationships
(3) Genetic structure
(4) Ecological niche modelling
(5) Demographic history
PLANPLAN
DEMOGRAPHIC HISTORYDEMOGRAPHIC HISTORY
- inference on demographic history of a species based on approximate Bayesian computation (ABC)
- input:- molecular data
- scenarios describing demographic history
- procedure:- simulate (a large number of) datasets for each scenario
- compare simulated and observed datasets
- the most similar simulated dataset is the most likely
- goal:- compare competing scenarios
posterior probability of each scenario
- estimate parameters for one or more scenarios
effective population size; time of divergence
PPOPULATIONSOPULATIONS
A1
A2A3
Allelic richness:
A1 7.97
A2 8.49
A3 6.25
ABC analysis:
A1: 92 inds from 7 pops
A2: 91 inds from 10 pops
A3: 82 inds from 4 pops
FFIVE SIMPLE IVE SIMPLE HHISTORIC ISTORIC SSCENARIOSCENARIOS
Scenario 1
Population A1 is derived from population A2,
itself derived from population A3
Scenario 2
Population A3 is derived from population A2,
itself derived from population A1
A1
A2
A3
A3 A2 A1
A1 A2 A3
A1
A2
A3
Scenario 3
Both populations A1 and A3 derived independently from
population A2
Scenario 4
Population A2 was generated by admixture of populationsA1 and A3
A1
A2
A3
A3 A2 A1
A1 A2 A3
A1
A2
A3
FFIVE SIMPLE IVE SIMPLE HHISTORIC ISTORIC SSCENARIOSCENARIOS
Scenario 5
All three populations
diverged at the same time
A2 A1 A3
A1
A2
A3
FFIVE SIMPLE IVE SIMPLE HHISTORIC ISTORIC SSCENARIOSCENARIOS
Scenario Posterior probability (PP)
1 0.166
2 0.193
3 0.075
4 0.126
5 0.440
RESULTS:RESULTS:
HISTORICAL RECONSTRUCTION: ANCIENT PASTHISTORICAL RECONSTRUCTION: ANCIENT PAST
?
Vegetation map of Europeduring the last glacial period
Distibution of Dalmatian sage before the last glaciation ?
ABC Effective population sizes:Ancestral 8,550 inds
A1 4,330 indsA2 7,190 indsA3 2,440 inds
Glacial refugia:1. Iberian Peninsula2. Apennine Peninsula3. Balkan Peninsula
Source: http://www.metatech.org/07/ice_age_global_warming.html
Suitability0.26 - 0.50
0.51 - 0.75
0.76 - 0.84
0.26 - 0.500.51 - 0.750.76 - 0.94
Suitability
Apennine peninsula
Balkan peninsula
A3
A1
A2
Adriatic sea
Suitability0.26 - 0.50
0.51 - 0.75
0.76 - 0.84
0.26 - 0.500.51 - 0.750.76 - 0.94
Suitability0.26 - 0.500.51 - 0.750.76 - 0.94
Suitability
Apennine peninsula
Balkan peninsula
A3
A1
A2
Adriatic sea
Distribution of Dalmatian sage during Distribution of Dalmatian sage during the Last Glacial Maximumthe Last Glacial Maximum
A2A3
A1
Map of the 52 putative refugia within the Mediterranean region
ABC Time of divergence:572 generations ago(95% CI: 157-1,540)
Generation time (life span):up to 300 years
HISTORICAL RECONSTRUCTION: HISTORICAL RECONSTRUCTION: DDIVERGENCEIVERGENCE
A2 A1 A3
Source: http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2699.2008.02051.x/abstract
A1
A2 A3
B
HISTORICAL RECONSTRUCTION: PRESENT TIMEHISTORICAL RECONSTRUCTION: PRESENT TIME
Rešetnik, I., Baričević, D., Batîr Rusu, D., Carović-Stanko, K., Chatzopoulou, P., Dajić-Stevanović, Z., Gonceariuc, M., Grdiša, M., Greguraš, D., Ibraliu, A., Jug-Dujaković, M., Krasniqi, E., Liber, Z., Murtić, S., Pećanac, D., Radosavljević, I., Stefkov, Gj., Stešević, D., Šoštarić, I., Šatović, Z. 2016. Genetic Diversity and Demographic History of Wild and Cultivated/Naturalised Plant Populations: Evidence from Dalmatian Sage (Salvia officinalis L., Lamiaceae). PLOS One 11(7): e0159545 (doi: 10.1371/journal.pone.0159545)
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