Epigenetic vs. genetic diversity of stenoendemic short‐toothed sage (Salvia brachyodon Vandas)

Biruš, I., Liber, Z., Radosavljević, I., Bogdanović, S., Jug‐Dujaković, M., Zoldoš, V., Šatović, Z.

Balkan Botanical Congress 2015. 

ShortShort‐‐toothed sage toothed sage ((S. brachyodon S. brachyodon Vandas)Vandas)

‐ three known localities:1. Sv. Ilija, Pelješac2. Velji do, Konavle3. Vrbanje, Mt. Orjen, 

BH/Monte Negro border

- Illyrian‐Adriaticstenoendemic species 

Introduction

‐ very narrow distribution range

ShortShort‐‐toothed sage toothed sage ((S. brachyodon S. brachyodon Vandas)Vandas)

Introduction

‐ propagation germinatively and vegetatively ‐ via stolons (field observation)

Could epigenetic modifications be the molecular mechanism enabling evolutionary 

persistence and phenotypic plasticity?

Could epigenetic modifications be the molecular mechanism enabling evolutionary 

persistence and phenotypic plasticity?

narrow endemic species, clonality limited genetic diversity?

‐ in CRO near threatened (NT), in MN endangered species (EN)

• To study the relationship between epigenetic and genetic variation in narrow endemic and clonally propagated species

1. compare the levels of clonal, genetic and epigenetic diversity among populations2. determine the relationship between epigenetic and genetic distance among individuals3. determine the amount of epigenetic variation found among genetically identical organisms

Aims of the Study

(2) Epigenetic markers a) MSAP‐Methylation‐Sensitive Amplified Polymorphism (MSAP) 

– 4 primer combinations, > 3000 loci

(1) Genetic markersa) Microsatellites‐ Simple Sequence Repeats (SSRs) 

– 8 microsatellites, total of 63 markers

Methods

‐ 3 populations/ 25 individuals per population

• MSAP (methylation sensitive amplified polymorphism) – modification of AFLP  based on differential sensitivity of restriction enzymes (isoshizomeres) such as MspI/HpaIIto methylation (5’‐CCGG‐3’)

Methods - Analysis of global genome methylation

0.05

53

50

71

69

Population:P01 Pelješac, HRVP02 Cavtat, HRVP03 Mt. Orjen, MNE

Results: TREE BASED ON GENETIC DISTANCES

Unrooted  Neighbour‐Joining  tree  of  short‐toothed  sage  samples  from  3  populations based on DPSAM genetic distance. The size of the circles at the branch tips is proportional to size (no. of individual plant samples) of the clonal lineage.

6

4

2

1

Parameter P01 P02 P03

Pgen 1.24 × 10-11 - 5.55 × 10-7 9.03 × 10-10 - 3.36 × 10-6 8.55 × 10-9 - 9.31 × 10-4

Pgen(Fis) 3.80 × 10-11 - 7.99 × 10-7 1.39 × 10-9 - 3.30 × 10-6 4.71 × 10-9 - 7.49 × 10-4

Psex (1 reencounter) 3.66 × 10-8 - 1.39 × 10-5 2.26 × 10-8 - 8.39 × 10-5 3.38 × 10-7 - 2.30 × 10-2

Psex(Fis) (1

reencounter)7.41 × 10-8 - 2.00 × 10-5 3.47 × 10-8 - 8.24 × 10-5 2.97 × 10-7 - 1.86 × 10-2

Range of P-values across different multi-locus genotypes (MLGs)

Results: PROOF OF CLONALITY

P < 0.05 in all cases

Our marker set has enough power to discriminate among clones: identical MLGs can be considered as pertaining to the same clone!

1612 11

0

2

4

6

8

10

12

14

16

18

P01 P02 P03

0.625

0.4580.417

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

P01 P02 P03

0.950 0.927 0.903

0.00

0.20

0.40

0.60

0.80

1.00

P01 P02 P03

Number of genets Genotypic richness Simpson's complement index

D*G R

Results: CLONAL DIVERSITY

High clonal diversity!

No of ramets = 25

5.34 4.873.88

0.00

1.00

2.00

3.00

4.00

5.00

6.00

P01 P02 P03

Nar

Allelic richness Observed heterozyogsity Expected heterozygosity

0.750.66 0.58

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

P01 P02 P03

HO

0.73 0.660.55

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

P01 P02 P03

HE

Results: GENETIC DIVERSITY

No. of genets = 16 (P01), 12 (P02), 11 (P03)

High genetic diversity!All populations in Hardy‐Weinberg Equilibrium!No signs of recent bottleneck events!

PBottleneckP1: 0.19P2: 0.47P3: 0.77

PFISP01: 0.32P02: 0.59P03: 0.22

not significant

not significant

0.458 0.446 0.469 0.4580.442 0.426 0.4300.4690.464 0.441

0.4810.468

0.00

0.10

0.20

0.30

0.40

0.50

MSAP Total u‐subepiloci h‐subepiloci m‐subepiloci

Shannon's information index

H

Population:P01P02P03

Results: EPIGENETIC DIVERSITY

Similar levels of epigenetic diversityacross populations and across subepiloci!

Excluding within‐clonecomparisons:r = 0.280P < 0.01R2 = 0.079

0.00

0.20

0.40

0.60

0.80

1.00

0.00 0.20 0.40 0.60 0.80 1.00

Genetic distance (Dpsa)

Epigen

etic distance (Dice’s)

All data:r = 0.421P < 0.01R2 = 0.178

Results: GENETIC VS. EPIGENETIC DISTANCES

Significant but weak correlation between genetic and epigenetic distances!Genetic variability explains between 8 and 18% of epigenetic variability!

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

P01 P02 P03 P01/P02P01/P03

P02/P03

Between clones

P01 P02 P03

Between populationsWithin clones

Gen

etic/Epigene

tic distance

Marker:SSRMSAP

WITHIN CLONES, BETWEEN CLONES AND BETWEEN POPULATIONSResults: GENETIC VS. EPIGENETIC DISTANCES

Average epigenetic distance among genetically identical plants only slightly lower than those among genetically different plants!

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

Between clones Between populationsWithin clones

Epigen

etic distance (Dice’s )

MSAP markers:Totalu‐subepilocih‐subepilocim‐subepiloci

WITHIN CLONES, BETWEEN CLONES AND BETWEEN POPULATIONS

Results: EPIGENETIC DISTANCES

Similar levels of epigenetic distancesacross subepiloci!

• narrow endemic and clonaly propagated species– high both clonal and genetic diversity– in HWE; no bottleneck

• epigenetic diversity was more even across populations than clonal and genetic diversity

• significant but weak correlation between epigenetic and genetic distances

• relatively high epigenetic distances even among genetically identical individuals

• epigenetic mechanisms contribute in the evolutionary persistence of these populations

Conclusion:

Thank you for your kind attention!!