Francesc Piferrer
Seminari Departament d’Ecologia, UB. 19 de febrer de 2016
Determinació per factors ambientals de la proporció de sexes
a les poblacions: Mecanismes, contribució de l’epigenètica
i aspectes adpatatius en un context de canvi global
1.- Sex ratio and sex determination. Environmental factors
Outline
2.- Epigenetics. Mechanisms and analytical methods
3.- Application of epigenetics to research in ecology
4.- Epigenomics in natural populations
5.- Adpative aspects in a global change scenario
“Applied” aspects
– Reproductive capacity
- Fisheries
– Sex control
- Aquaculture
- Invasive species
– Monitoring
- Chemical pollution
- Climate change
“Basic” aspects– Mating competition and sexual selection
– Evolution of sex determining mechanims
– Sex allocation theory
– Speciation
Penman and Piferrer (2008). Rev. Fish. Sci. 16 (Suppl. 1): 4–34
Major sex
factors
Minor
sex
factors
Environmental
differences
monofactorial
system
polyfactorial
system
environmental
sex
determination
GSD
ESD
Sex Determination
– Genetic sex determination (GSD)
GSD ESD
– Environmental sex determination (ESD)
Environmental Sex Determination (ESD)
TSD TSD
TSD GSD
Warner and Shine (2008). Nature 451, 566-568
Temperature (TSD)
pH
Population density
Photoperiod
Hypoxia
Salinity
…
ESD
Species with TSD = Natural system in which populations exhibit a
direct unambiguous fitness response to thermal fluctuations
Sex Ratio Response to Temperature in Fish
T. min T. max. Nat. Range Temp. Nat. Range Temp. during Develop.
Temperature
Pro
po
rtio
n o
f m
ale
s
GSD
GSD
+ Temp.
Effects
TSD
RTD
Ospina-Álvarez & Piferrer (2008) PLoS One 3(7): e2837
1. Less TSD species
2. One response pattern
3. Possible effects of cl. change
The European Sea Bass Temperature and Sex Enigma
Sex ratio analysis
Fertilization
0 50 100 150 200 250 300 350 TSP
Formation of gonadal
ridges 35 dpf
Increase in PGC number
60-90 dpf
First sex-related differences in
cyp19a expression 120 dpf
Sex differentiation
First observable signs of morphological
sex differentition 150 dpf
Piferrer et al. (2005). Gen.
Comp. Endocrinol., 142: 102-110
Dicentrarchus labrax (F. Moronidae).
Polyfactorial SD system w. temp. influence
G x E interactions.
Epigenetics
“The study of phenomena and mechanisms that cause chromosome-
bound, mitotically and/or meiotically heritable changes to gene
expression or phenotype that are not dependent on changes to
DNA sequence”
Epigenetic Mechanisms
Small RNAs (sncRNA)
Long RNAs (lncRNAs) Regulatory RNA
Xist, …
Monoallelic gene expression
miRNA (21-22 nt) Translational repression
3. Non-coding RNAs
1. DNA methylation 2. Histone modifications & Histone variants
H3K9me3
repression
H3K4me3
activation
ON Me Me Me
OFF CGATTCGAACG
CGATTCGAACG
Cell membrane
Nucleus
2. Epigenetic initiators - DNA binding factors
- ncRNAs
3. Epigenetic maintainers - DNA modifying enzymes
- Histone modifying enzymmes
- Histone variants
Environment 1. Epigenators - Temperature
- Differentiation signals
- Metabolites
Signaling
(epigenator)
pathway
ncRNA
DNMT
HAT
DNA-BP
HDAC
Cytoplasm
Genotype
Short-term, transient outcomes
- transcription
- DNA replication & repair
Long-term outcomes
- chromatin conformation
- heritable markers
Phenotype
Establishment of Epigenetic Marks
Genotype + Environment = Phenotype
Epigenetic Inheritance
Cell-cell transfer (mitosis)
Organism-organism transfer (meiosis) Multi- vs. transgenerational effects
P F1 F2Multigenerational effects
Transgenerational effects
F3
P F1 F2
Multigenerational Transgenerational Shao C, et al. (2014) Genome Res. 24:604-15
Inheritance of sexual reversal
in tongue sole
(ZW/ZZ sex det. system)
F1 ZW fish exclusively derived
from the paternal pseudo-males
ZW offspring develop as pseudo-
males without temp. treatment
R. Margalef
(1919–2004)
Margalef, 1955.Temperatura, dimensiones y evolución. Publ Inst Biol Apl 19: 13–94
“Merece especial interés la coincidencia entre los carácteres resultantes de
una selección (e incluso producción directa: determinación del sexo,
poliploidía) de variaciones del genotipo y los que consisten en simples
modificaciones o somaciones no hereditarias.
Es comprensible que la observación de este paralelismo haya sido tomada
como argumento a favor del lamarckismo, aunque sin dar una explicación
racional que justifique su asimilación”.
Met
hyla
tion
(%)
Low Temp.
High Temp.
a
b
c
c
14
8 9 9
MalesFemales
100
50
0
environment DNA methylation gene expression phenotype
* *
*
cyp19a +SF1
cyp19a +Foxl2
cyp19a +SF1
+Foxl2
Ctrl cyp19aLu
cife
rase
Act
ivity
0
2
4
6
8
10
12
14
16
18
Control Methylated
*
Foxl2 Sf-1 cyp19a
Foxl2 Sf-1
cyp19a
Males > Females
An Epigenetic Switch Linking Temperature and cyp19a Expression
Temperature group
Low High
cyp19a
(R
Q)
0
2
4
6
8
10
Methylation (%)0 20 40 60 80
cyp
19a
(R
Q)
0
5
10
15
20
Females only
a
b
r2 = 0.3 P = 0.01
Navarro-Martín et al. (2011). PLoS Genet 7(12): e1002447
J. Experimental Zoology Aquaculture PLoS Genetics
Science
Time
Patent
Private company
Matsumoto et al. (2013). PLoS ONE 8(6): e63599
Red-eared slider turtle
(Trachemys scripta)
26°C cyp19a1 hyperMe 100% males
31% cyp19a1 hypoMe 100% females
Does a similar mechanism operate in reptiles with TSD?
Thermosensitive period Sex differentiation period
Thermosensitive period
Sex differentiation period
FISH
REPTILES
Individual variation in
ecologically important traits
Evolutionarily important
• Provides the raw material for natural selection
• Conditions the evolutionary trajectory of populations
• Shapes the demographic structure of populations
• Affects the dynamics of populations and communities
Ecologically important
Contribution of Epigenetics to Ecological Research
How Epigenetics Integrates Genetic and Environmental Information
After Bossdorf et al 2008. Ecol Lett.11: 106–115
Alter
ECOLOGICAL
EPIGENETICS
Regulates Epigenetic
variation
Alters
Affects
Gene
expression
Phenotypic
variation
Ecological
interactions
Natural
selection
Genetic
variation
Affects
Affects
Through Alters
ECOLOGICAL
GENETICS
Affects
Morán & Pérez-Figueroa, 2011. BMC Genetics 12:86
Circles – River Tea Triangles – River Ulla
Genetic (AFLP) Epigenetic, liver (MS-AFLP) Epigenetic, gonads (MS-AFLP)
Open – Immature Filled – Mature
Extent and Structure of Epigenetic Variation
within and among Natural Populations
VP = VG + VE + VGE
VG = VA + VD + VI VG = VA + VD + VI + VEpi
Abiotic factors: Thermal stress
Nutrients
Light
Water
What is the relative importance of epigenetic variation in
determining the outcome of abiotic and biotic interactions?
Biotic interactions: Competition
Resistance to predators
Resistance to pathogens
Herrera & Bazaga 2011. Mol Ecol. 20: 1675–1688
Effects of
herbivory on
Viola
cazorlensis
Model 4
Genotype
Phenotype
Epiphenotype
Model 3
Genotype
Epiphenotype
Phenotype
Model 2
Genotype
Phenotype
Epiphenotype
Model 1
Genotype
Epiphenotype
Phenotype
Epigenetics Has a Long Way to Go in Ecology
Topic 22/04/2013 14/02/2016 % increaseCancer 2675 4563 71 Development 2250 4252 89 Genetics 650 1127 73 Evolution 400 719 80 Animal production 60 76 27 Neurobiology 40 61 53 Ecology 30 58 93
0500
100015002000250030003500400045005000
22/04/2013
14/02/2016
Individual variation in
ecologically important traits
Evolutionarily important
• Provides the raw material for natural selection
• Conditions the evolutionary trajectory of populations
• Shapes the demographic structure of populations
• Affects the dynamics of populations and communities
Ecologically important
Individual Variation in Ecologically Important Traits
production
Production
artificial
performance
Production
2. Epigenetics and Animal Production
Growth, Product quality, Health, Welfare, Reproduction
gamete development
fertilization
embryonic development
Post-hatch/birth development. Internal External
External External
Environmental influences
(biotic and abiotic)
Fertilization
Internal
Early develop.
Internal
Effects of early environment in the sea bass
Illes Medes natural reserve
Experimental Aquarium facility
Size of genome (Mb) 675
Genes with
description 25,433
Number of CpG sites 10,325,421
% of CpGs 1.53
% Cs in CG context 7.66
Number of CpG
islands 30,391
Tine et al. (2014). Nature Comm. 5:5770.
Wild sea bass vs.
Famed sea bass (NT or HT)
DNA methylation – RRBS
Histone modification – ChIP
Gene expression – RNA-seq
Tissues
Early
environment
Genetic
variation
Epigenetic
modifications
Gene
expression Phenotype
Temperature
Pollutants
Stress
Farming
AFLP
SNPs
MSAP
RRBS
ChIP
Bis-PCR2
qPCR
RNA-seq
Fitness
Performance
Pr. quality
H-O Pörtner & Peck 2011 Encyclopedia of Reproduction.
• Changing water temperatures
• Hypoxia events
• Ocean acidification
• Shifts in hydrodynamics
• Sea level rise
Effects of Climate Change
Organismal Population Community/EcosystemMolecular/Cellular
Sex differentiation
Behavioral responses
Alterations in developmental time
Changes in productivity Changes in food-web interactions
DNA methylation Gene expression Metabolism
Decrements in aerobic performance
Larval dispersal
ncRNA H3K9MeT
H3K9DMe Polycomb
Epigenetic modification-
related genes
Sex differentiation-
related genes
LT HT
Long-lasting temperature effects on gene expression
Common Temperature
Gene expression is altered in 170 dpf sea bass treated with high
temperature (HT) during the thermosensitive period (TSP)
0 50 100 150 200 250 300 350
TSP Sex differentiation
LT HT
Age
(dpf)
HT LT
Díaz & Piferrer. 2015. BMC Genomics 16:679
Genes upregulated transgenerationally:
- Metabolic genes shifts in energy production.
- Immune- and stress-responsive genes the second generation of fish to better cope with elevated temperatures.
- Genes involved with tissue development and transcriptional regulation
- Similar suite of dierentially expressed genes among developmental and transgenerational inheritance
- Heat-shock protein genes were surprisingly unresponsive
Species
Pivotal temp.
(ºC)
Percent of sexes (:) at
pivotal temp. + 1.5ºC
Percent of sexes (: )
at pivotal temp. + 4ºC
Apistogramma spp. (n = 33 sp) 25.3 62 : 38 81 : 19
Hoplosternum littorale 18.8 56 : 44 65 : 35
Limia melanogaster 25.8 57 : 43 68 : 32
Menidia menidia 14.5 61 : 39 75 : 25
Menidia peninsulae 26.6 57 : 43 69 : 31
Odontesthes argentinensis 25.7 60 : 40 76 : 24
Odontesthes bonariensis 24.2 73 : 27 98 : 2
Poeciliopsis lucida 25.6 68 : 32 92 : 8
Pivotal temp. (mean ± S.E.M.) 23.3 ± 1.5 - -
Percent males (mean ± S.E.M.) 50.0 61.7 ± 2.1 78.0 ± 4.1
Pivotal temperature in fish species with TSD
and predicted sex ratio shifts with temperature increases
Ospina-Álvarez & Piferrer (2008) PLoS ONE 3(7): e2837
Summary/Take home messages
- In a global change scenario, epigenetics provide organisms with a source
of phenotypic plasticity that can allow adaption to a new environment
- The study of variation in natural populations can be greatly benefited
if research in ecology integrates epigenetics
- The sex ratio is a fundamental parameter in population demography
and in many cases is determined by a combination of
genetic and environmental factors
- Epigenetic mechanisms provide organisms the capability to integrate
genomic and environmental information to produce a given phenotype
Laia Ribas
Laia Navarro
Natalia Ospina
Noèlia Díaz
Dafni Anastasiadi
Alejandro Valdivieso
Susanna Pla
Silvia Joly
Gemma Fuster
Núria Sánchez
Paula Gómez
Acknowledgments
Collaborators:
Christopher Bock, Univ. of Vienna
Marco Álvarez, Univ Andrés Bello,Chile
Luciano di Croce, CRG, Barcelona
Paloma Morán, Univ. Vigo
0.285 Project
Project Epigen-Aqua
Program I-LINK
differentiation
eeppiiggeenneettiiccss
LLaammaarrcckk selection
environment
phenotypic
plasticity
heritability
hybridisation
induction Arabidopsis
speciation
Darw
in
chromatin global change
apomixis
development
mem
ory
natural populations
DNA sequence paramutation
phenotype
ecology epigenome
DNA methylation conservation
miRNA
evolution epialleles imprinting variation
inheritance
maternal effects histones
stress
interactions
transgenerational
animal production
genotype
aquaculture
cloning
tarantin
o
Waddington
non-lin
eal n
arra
tive