Transcriptomic and phenotypic data from common gardens reveal adaptive genetic variation in a Mediterranean alpine plant

Lara-Romero C, Zemp N, García-Fernández A, Morente-Lopez J, Rubio ML,

Widmer A, Iriondo JM

ConGenOmics

Rey Juan Carlos University Madrid

Marginal populations:• grow under suboptimal environmental conditions• great fluctuations and high probability of extinction

Soulé (1973)

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

• Genetically impoverished populations

• Inbreeding depression• Maladaptation

• Not necessarily depauperate for variation in ecologically relevant traits.

• Locally adapted

Marginal populations:• grow under suboptimal environmental conditions• great fluctuations and high probability of extinction

Soulé (1973)

?

Kawecki, T. J. 2008. Annu. Rev. Ecol. Evol. Syst. Soulé M. 1973. Annu. Rev. Ecol. Sys.

Lande R. 1994. EvolutionWhitlock MC. 2003. Genetics

Lande (1994), Whitlock (2003) Kawecki (2008)

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Mediterranean alpine environments: highly vulnerable to global warming

Marginal populations

Central populations

temperature rainfall

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Nogués-Brano et al 2007. Global Environmental Change Paulí et al 2012. Science

Experimental gene flow between populations:– assessment of inbreeding depression and geneflow of

adaptive/maladaptive value– management tool to assist marginal populations

Marginal populations

Central populations

Holt RD, Gomulkiewicz R. 1997. Am NatKirkpatrick M, Barton NH. 1997. Am Nat

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Experimental gene flow between populations:– assessment of inbreeding depression and geneflow of

adaptive/maladaptive value– management tool to assist marginal populations

Marginal populations

Central populations

central – marginal geneflow:• Genetic diversity (Holt & Gomulkiewicz, 1997)• Maladaptive alleles or gene combinations

(Kirkpatrick & Barton, 1997)

Holt RD, Gomulkiewicz R. 1997. Am NatKirkpatrick M, Barton NH. 1997. Am Nat

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Experimental gene flow between populations:– assessment of inbreeding depression and geneflow of

adaptive/maladaptive value– management tool to assist marginal populations

Marginal populations

Central populations

marginal-marginal geneflow• Genetic diversity & adaptive alleles or gene combinations (Sexton et al. 2011)

Sexton JP, Strauss SY, Rice KJ. 2011. PNAS

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Aim: • To assess whether marginal populations at the lowest elevation of

Mediterranean alpine plants are locally adapted/maladapted to the environmental conditions that will prevail with global warming

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

• Circum-mediterranean alpine chamaephyte• Central System at the lowest latitude of the

distibution range:– Sierra de Béjar– Sierra de Gredos– Sierra de Guadarrama

• Elevation range: 1900 – 2500 m

Silene ciliata Pourret

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

• Circum-mediterranean alpine chamaephyte• Central System at the lowest latitude of the

distibution range:– Sierra de Béjar– Sierra de Gredos– Sierra de Guadarrama

• Elevation range: 1900 – 2500 m

Silene ciliata Pourret

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

• Central population

• Marginal populations

Central vs. marginal populations

(Giménez-Benavides et al. 2011)

Giménez-Benavides, L., Albert, M.J., Iriondo, J.M. & Escudero, A. Ecography (2011)

GuadarramaGredosBéjar

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

• Central population

• Marginal populations

Central vs. marginal populations

(Giménez-Benavides et al. 2011)

Giménez-Benavides, L., Albert, M.J., Iriondo, J.M. & Escudero, A. Ecography (2011)

GuadarramaGredosBéjar

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Seeds obtained in common garden conditions from artificial crossings simulating different types of geneflow

Gene flow simulation experiment

Central population from same mountain range

Marginal population

Marginal population from same mountain range

X 6 marginal populations

F1

F2

F3

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Sowing experiment at the locations of the 6 marginal populations (mother plant x type of cross x block x pop. = 24 000 seeds)

Gene flow simulation experiment

Marginal populations

Central populations

F1F2

F3

x 2 marginal populationsx 3 mountains

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Sexton JP, Strauss SY, Rice KJ. 2011. PNAS

Evidence of adaptive geneflow between marginal populations (F1 < F2): Mimulus laciniatus (Sexton et al., 2011)

Marginal populations

Central populations

F1F2

F3

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Gene flow simulation experiment

Survival

Treatment

Sur

viva

l pro

porti

on

F1 F2 F3

0.0

0.2

0.4

Seedling survival

Gene flow

Surv

ival

(%)

0

60

40

No evidence of maladaptive gene flow from central populations (F1 ≤ F3)

Little evidence of inbreeding load (F1 < F2; F1 ≤ F3)

Marginal populations

Central populations

F1F2

F3

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Gene flow simulation experiment

Survival

Treatment

Sur

viva

l pro

porti

on

F1 F2 F3

0.0

0.2

0.4

Seedling survival

Gene flow

Surv

ival

(%)

0

60

40

García-Fernández A, Iriondo JM & Escudero A. 2012 Oikos

Reciprocal sowing experiments among central and marginal populations to test for evidence of local adaptation(mother plant x type of cross x block x pop. = 7 250 seeds)

Reciprocal sowing experiments

Marginal populations

Central populations

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

x 3 mountains

On-going research but…

Reciprocal sowing experiments

Marginal populations

Central populations

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

…previous reciprocal sowing experiments found evidence of local adaptation in seedling survival and growth in central and marginal populations

Reciprocal sowing experiments

Giménez-Benavides L, Escudero A & Iriondo JM 2007. Annals of Botany

Marginal populations

Central populations

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Transcriptome analyses

Massive sequencing of the transcriptome of seedlings from central and marginal populations grown under controlled conditions.

Identification of polymorphisms and differential expression levels in candidate genes between seedlings from central and marginal populations.

T G T C G G T C TT G T C G G T C T

T G T C A G T C TT G T C A G T C T

Single Nucleotide Polymorphism (SNP)

Central

Marginal

Differential expression

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Transcriptome analyses

Massive sequencing of the transcriptome of seedlings from central and marginal populations grown under controlled conditions.

Identification of polymorphisms and differential expression levels in candidate genes between seedlings from central and marginal populations.

Functional annotation & Enrichment analysis

We expect to find some candidate genes codifying proteins involved in responses to abiotic stimulus, particularly drought stress.

T G T C G G T C TT G T C G G T C T

T G T C A G T C TT G T C A G T C T

Single Nucleotide Polymorphism (SNP)

Central

Marginal

Differential expression

RPKM (Reads per kilobase per million mapped reads)

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Comparison of expression levels (RPKM) between central and marginal populations

Differential expression analysisCentral

Marginal

Differential expression

RPKM (Reads per kilobase per million mapped reads)

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Comparison of expression levels (RPKM) between central and marginal populations

129 contigs differentially expressed

GO term & Enrichment analysis

• 114 contigs annotated (i.e., protein-coding genes)

• Response to extracellular stimulus (n=9) & external stimulus (n=19) overrepresented

Central

Marginal

Differential expression

Differential expression analysis

Selection of candidate genes

Contig: pieces of DNA representing overlapping regions of a particular chromosome

7 reads needed to infer genotypeDeletion of paralogous SNPsBiallelic SNPs with no missing data

Depth of coverage and posterior probability did not affect outlier detection

147 118 SNPs & 12 688 contigs (mean x contig =13.7)

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

SNP calling & outlier detection

SNP calling (Software Reads2SNP)

SNP: a nucleotide site (base pair) in a DNAsequence that is polymorphic in a population

[1] Contingency table and Pearson’s Chi-square test (X2)

[2] Dispersal parameter (m, Muller et al 2010 Evolutionary Applications)

[3] Allelic frequency differentials (AFDs)

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Alternative strategies for selection of outlier SNPs

Selection of candidate genes

SNP calling & outlier detection

Muller et al 2010 Evolutionary Applications, Turner et al 2010 Nature, Stölting et al 2015 New Phytologist

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

SNP calling & outlier detection

Dispersal param. Allele freq.

AFD

336 606

275

20

131246

Muller et al 2010 Evolutionary Applications, Turner et al 2010 Nature, Stölting et al 2015 New Phytologist

Selection of candidate genes

[1] Contingency table and Pearson’s Chi-square test (X2)

[2] Dispersal parameter (m, Muller et al 2010 Evolutionary Applications)

[3] Allelic frequency differentials (AFDs)

Alternative strategies for selection of outlier SNPs

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

6 genes overlapped among three approaches GO TERM: response to stress & metabolic process

Dispersal param. Allele freq.

AFD

336 606

275

20

13124 6

SNP calling & outlier detection

Muller et al 2010 Evolutionary Applications, Turner et al 2010 Nature, Stölting et al 2015 New Phytologist

Selection of candidate genes

[1] Contingency table and Pearson’s Chi-square test (X2)

[2] Dispersal parameter (m, Muller et al 2010 Evolutionary Applications)

[3] Allelic frequency differentials (AFDs)

Alternative strategies for selection of outlier SNPs

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

6 genes overlapped among three approaches GO TERM: response to stress & metabolic process

163 genes overlapped among two approaches• 143 annotated genes• Enrichment analysis (before FDR correction)

- Response to abiotic stimulus (n = 53)- Response to stress (n = 59)- Several additional terms related to metabolic processes and response to stimulus

SNP calling & outlier detection

Dispersal param. Allele freq.

AFD

336 606

275

20

131246

Muller et al 2010 Evolutionary Applications, Turner et al 2010 Nature, Stölting et al 2015 New Phytologist

Selection of candidate genes

[1] Contingency table and Pearson’s Chi-square test (X2)

[2] Dispersal parameter (m, Muller et al 2010 Evolutionary Applications)

[3] Allelic frequency differentials (AFDs)

Alternative strategies for selection of outlier SNPs

1. Adaptive geneflow between marginal populations at the seedling stage.

2. No maladaptive geneflow between central and marginal populations at the seedling stage.

3. Some evidence of inbreeding load in marginal populations.

4. Genes involved in stress responses might play an important role in the adaptation to marginal environments.

5. Marginal populations might be of high importance to assist central populations as they can provide alleles or gene combinations adapted to the environmental conditions that will prevail with global warming.

Introduction Aims In situ experiments Transcriptomic experiment Conclusions

Conclusions

Introduction Past studies Current research Future prospectsAcknowledgements:

• C. Diaz, G. Escribano, S. Prieto, P. Tabares, S. Eleazar, L. Cano, L. Martinez, S. Fior, M. Roumet

• Sierra de Guadarrama National Park• Sierra de Gredos Regional Park• Sierras de Béjar y Francia Biosphere Reserve

• AdAptA Project CGL2012-33528, Spanish National R&D&I Plan

• ESF networking programma ConGenOmics

Funding: