Juodkrante (grandparents)

Practical forestry under
climatic change: where are
the bottlenecks for genetic
diversity?,
Darius Danusevicius and Virgilijus Baliuckas
Aleksandras Stulginskis University, Forest Research
Institute, Lithuania,
Problems
Management effects:
Spatial genetic structure
Effect of thinning (after effects in situ)
Effect of thinning retrospectively
When are the homozygotes lost? (Ho and D association)
Sample size
Effects of regeneration method
Dormancy
Build up (effects of fluctuations)
Released (what is the critical situation)
Hyperspectral imaging
Diversity detection
Tolerance issues (dormancy, drought)
Silvicultural treatments of 19th century
Problem: how to secure genetic diversity in future forests
(each box could be a separate thesis)
Gene migration
-Post glacial
-Gene flow
-Co-migration of trees
and birds
Genetics of a climax
species
-Juniperus
(problems related to
climate change)
Functioning
of pine stand
on molecular
level: clusters
of relatives,
their shape;
Modeling climatic change
effects in provenance
trials: microorganisms,
phenology
Climate change
Silviculture
Seed storage
effect on gene
diversity
Optimal
breeding
strategy
What
Artificial
diversity is
populations sufficient
& human Diversity of
stands from
impact
seed orchard
Wild gene
diversity
progeny
Creation of “gene diversity”
measuring tool:
-Diversity at species level
-Region level
-Stand (natural / artificial)
-Trees within stand
Seed orchard mating
patterns and diversity
Paternity analysis: diversity
and breeding efficiency
Seed gene diversity within
tree and within cone
Mating disequilibria
Effect of silvicultural
treatments on gene
diversity
Possible aftereffects of management:
cpSSRs at 4 loci Scots pine
cpDNR genetinės
įvairovės pasiskirstymas
(Nei indeksas)
Why?
There could be a long term
consequence ..
18th century British fregata, french spying
ASU, Faculty of Forest Science and Ecology, Kaunas Lithuania. Issued: January 20, 2015
Origin of white oak in
Lithuania?
A line of English oaks at the 8th teaching building of ASU,
where the first tree to the left (marked with an arrow) is of
H4 haplotype (German line) and the test are of H5-7
haplotype from the refugium in the Balkans. DNA
extracted rom buds.
Authors: Darius Danusevicius, Virgilijus Baliuckas, Jurata Buchovska, Vilma
Kerpauskaite, Darius Kavaliauskas, e-mail: darius.danusevicius@asu.lt
Message: high genetic diversity of oaks in the Baltics
Alksniakiemio sėklinis medynas, Išlaužo gija, Prienų MU
2 k pusbrolis
Brolis
brother
half brother
bother
uncle
brother
1. Spatial genetic structure
Possible structure in “naturally” regenerated and planted
stand
Naturally regenerated may form
clusters, the parents may be
related … .
Planted for SO seed: higher
diversity, no spatial clusters =
more genetically sound
9 natural stands (=self-regenerated), ca 300 trees per stand,
sampled systematically by 8 x 8 m. grid from area of 1 ha ;
tree coordinates from on-land geodesy
(Ramunauskas ir kt.
1975)
For correct estimate –
age >100
Vertinama
stiebo dalis
nuo 1 iki 5 m
aukščio nuo
žemes
1b) Deep craks among flakes
Deep craks among flakes
(žiauberys su giliais palciais plyšiais), (wider) , žievė labai stora, tarpai
tarp žiauberio plokščių platūs)
žievė stora, žiauberio plokštės
plačios ilgos
1a)
3a) Shallow flakes
(turtle)žiauberys be gilių plyšių),
1-5 m
2) Long irregular flakes, palgomis
kamuotomis žiauberio plokštėmis
3b) Shallow flakes with a border,
žievė stora = „vėžlio kiautas“ tik gilesni
4) Spruce-likeSraigiažievė (egliažievė/ atpleišėjusi)
19
For correc
age
1-5 m
Gubriuotoji : zieve labai stora, 1b) Gubriuotoji : žievė stora, tarpai
tarp žiauberio plokščių platūs)
tarpai tarp ziauberio ploksciu
platus
1a)
3a) Ploksteline (Plate) žiauberys be 3b) Ploksteline , zieve stora
gilių plyšių), žievė plona= „vėžlio
2) Gubriuotoja, palgomis siauromis
žiauberio plokštėmis
Vertinama
stiebo dalis
nuo 1 iki 5 m
aukščio nuo
žemes
4) Spruce-likeSraigiažievė (egliažievė/ atpleišėjusi)
Methods
• 12 loci (4 PSYL from cDNA, 7 polymorphic such
as SPAC series)
• 3 multiplexes, DNA sequencer, work at ASP.
• Bayesian clustering with STRUCTURE (no loc
priors, 100 000 iterations & burn ins, 15 reps.)
• Followed by Harvest and the deltaK to decide
on optimum number of clusters
• Spatial Autocorrelation in GeneAlex
Results
Old unmanaged forests
Cluster 2
Cluster 1
Modeling K= likelihood decay
Azvintis reserve forest
Punia reserve forest
IN the middle of large tract, 1 ha = fine scale structure
1 km
Azvintis Vaisniunai (GR)
•
•
•
•
Natural, middle of large tract,
High stocking (670/ha), no precommercial thinning,
Sampled 400 trees (ca 4 x4 grid).
even-aged 100, typical for pine (Nb)
sandy soil, mirtilosum vegetation
10 km
Ažvintis sengirė (Vaisniunai) (4 groups)
0.20-0.28-0.52
81
108
116
100
Ažvintis sengirė (Vaisniunai) (3 groups)
0.10-0.30-0.60
134
144
128
Ažvinčio sengirė (A2 neretintas)
Spatial genetic structure in Punia (age ca
150, self regenerated, nature reserve for
80 years)
= no spatially continuous grouops
Cluster 2
Cluster 1
Bark morphotype was associated with the
STRUCTURE clusters in Punia (K= 5)
Cluster 3 (green
triangles: turtle oval
flakes, deep bark
cracks, straw color
(trees # 1, 19, 32,
36, 41, 44, 46
Cluster 5 (blue
x’s: long narrow
flakes, shallow
bark cracks,
reddish color
(largest group)
Cluster 2 (red
squares: long
narrow flakes,
shallow bark
cracks, NO reddish
color (trees # 4, 6,
9, 13, 24, 27, 35,
39)
Cluster 1
(tendency for
bumpy stem (trees
# 5 (not bumpy, 28
(bumpy)
Juodkrante (grandparents)
10 meters
Goodness of fit
0.00
0.14
0.86
Was the old forest
in Juodkrante
established with
seeds from
Germany?
nDNR 12 lokusų
Juodkrante structure of relatives (colony)
Maternal hsib fams (n=46 fams)
Selfs
Paternal hsib fams (n=44 fams)
Juodkrante structure of relatives (colony)
Maternal hsib fams (n=46 fams)
Paternal hsib fams (n=44 fams)
Dzukija D1 Marcinkonys
Goodness of fit
0.01 0.33 0.67
Dzukija D2 Margionys (pazeistas)
Goodness of fit
0.01
0.62
0.37
What are these groups?
• Non random mating (gene flow, nat
selection= phenology)
• Founder effects
Founder effects
Būtent ši zona randasi š.p. 50°- 55° N ir rytų ilg. 35°- 45° E
Didžiausia B koncentracija ten kur galėjo išlikti augmenija
LGM metu rodo B mitotipo poledynmetinio prieglobsčio
zoną
300 bp ilgio mtDNR fragmentas rodo
universalųjį A mitotipą ir retesnis B 295 bp
ilgio mtDNR fragmentas rodo B mitotipą (iš
šiaurinės prieglobsčio zonos).
Rytų Lietuvoje daugiau
B mitotipų iš šiaurinės
prieglobsčio zonos
32
A – universalusis mitotipas
Gražtės PGD (Zarasų r.)
B – šiaurinis mitotipas
Gražtės PGD (Zarasų r.)
33
Is the seed tree regeneration
method genetically sound?
Spatial genetic structure
Jonava 2 (111/13)
Planted & seed tree regenerated
in 1935
• 4 groups
• 3 more or less spatial
continuous (groups 1 to 3)
• One intermixed (group 4)=
perhaps represents the
refilled trees to support
natural regeneration
• Such spatial clusters may
form after natural
regeneration
100
meters
100
meters
Colony: group 2 are
half sibs
Some association with bark morphotype in JV2
stand
JV2 continued
Higher diversity
of cluster 4
(refiling)
Cluster 2
regenerated
100
meters
Number of trees
1
2
3
30
52
31
4
35
100
meters
Conclusion
Conclusions
• There are 2-4 discrete genetic groups in 1 ha area of natural
stands.
• The genetic kinship of these groups uncertain ( work with
Colony for family structure)
• In natural self-regenerating stands the spatial genetic
structure is random, where the genetic groups are intermixed
(= lower inbreeding in the progeny).
• After seed tree regeneration system spatially continues
groups may form, which may increase inbreeding in the
progeny (= need for genetic enrichment planting)
Effect of establishment method
Higher inbreeding in natural
regeneration
Complex spatial genetic structure in artificial
stands (seed orchard, Saliskes)
Effect of tending
• Overall (A1 versus A2)
• Retrospective modelling:
– Tending (juvenile stage)
– Thinning premature stage (commercial/tending)
Managed versus unmanaged
• Seemingly similar at age of 100
Managed versus unmanaged
Managed (less groups)
1 group lost in managed
stand?
Less rare alleles in
managed stand
Unmaged (less inbreeding)
Retrospective modeling of thinning
Ho and morphotype connection?
More homozygotes young stand than
mature
Is D connected to Ho?
Effect of removing inferior trees on genetic
diversity
Effect of
removing
inferior/superior
trees on genetic
diversity
NO MARKED
EFFECTS
Retrospective thinning: only rare
alleles that are affected
Modelling sample size for SSR
studies
Optimal
sample sizes
for SSR
studies
Sample size does not affect heterozygosity
Conclusions
• Natural spatial genetic structure is random,
approx. 3 groups
• Natural regeneration leads to solid groups
with high risk for inbreeding
• Diversity of artificial planting is similar to
naturally-born stands
• Thinning <30 % no significant effects, >30%
rare alleles lost
• Sample size = 20-25
Effects of warm winters
• 1st year is for free growth and no true
dormancy.
• 1st years seedlings are sensitive to
temperature fluctuations(milijonai).
• Question: amplitude of warm spels,
timing and pop effect ?
Effects of warm winters
Kaip tolimų populiacijų
palikuonys reaguoja ir
kodėl?
Koks poveikis medelių
ramybės būsenai?
Suomij
a
Material: 5 populations
Ispanij
a
Rusija
First part of winter 2013/2014. Pops after 3 weeks of
forcing treatments
3 weeks of
+20C was
enough to
force active
growth
T3 poveikyje
RU aktyviausi
Populiacijų sėjinukų ramybės būsenos palyginimas po pirmų trijų tyrimo savaičių 2013-12-09.
Vertikalios linijos rodo standartines paklaidas. Skaičiai virš stulpelių – sėjinukų skaičių
populiacijoje.
After new year the forcing effects were stronger
Kontinentinės populiacijos
stipriai pažeistos,
Tyrimo metodika: raudona- 2 sav. +15C, balta lauko sąlygose.
Antroje žiemos pusėje
poveikis stipresnis
Pirma žiemos pusė (T2) (poveikis silpnesnis, nes medeliai gilesnėje ramybės būsenoje
Ispanija
Lietuva
Čekija
Udmurtia
Suomija
Antra žiemos pusė (T1) (poveikis stipresnis, kadangi prarasta ramybės būsena
Udmurtia
Suomija
Lietuva
Čekija
Ispanija
T5 surprisingly well: warm cancels chilling, cold: regains hardiness
T1
T5
Weather conditions for high risk of winter frost
damage to 1 year old seedlings
0C
Šaltas ruduo = anksti
patenkintas vėsių temp.
poreikis = ramybės
būsena prarasta
ANKSTI
Šilta rudens pabaiga ir
pirma žiemos pusė =
patenkintas šilumos
poreikis aktyviam
metabolizmui = žemas
užsigrūdinimas
Šalta, su išreikštais
kirtimais iki -15 ir > C
temp. antroje žiemos
pusėje = pažeidimai
Day number under forcing to initiate
active growth after chilling treatments
250
Lietuva, Kazlų Rūda
Rusija, Udmurtija
210
200
192
175
150
131
127
105
100
50
41
0
0
2
4
6
Chilling treatment, weeks
2013.10.30
8
9S
10
Dienų skaičius +15C poveikyje iki augimo
pradžios
Number of days in forcing (+15C)
250
209
200
184
170
150
108
100
73
50
42
32
0
0
2
4
6
8
9S
10
Chilling treatment, weeks
70
2. Dienų skaičius DPB ir DAP
250
Suomija
206
200
Spain
192
155
150
101
100
50
0
2
4
6
Vėsių temperatūrų poveikio trukmė,
savaitėmis
2013.10.30
8
Dienų skaičius +15 C poveikyje iki
augimo pradžios
Dienų skaičius +15C poveikyje iki augimo
pradžios
250
196
200
179
155
150
130
100
50
0
2
4
6
8
Vėsių temperatūrų poveikio trukmė, savaitėmis
71
Apical dominance problems
Using NIR imaging for genetic
diversity studies
M&M
• HCam capturing detached
needles with pixel size
6.45×6.45 μm.
• = can detect change in cell
structure,
• 400 – 1000 nm spectral
interval, in step of 0.6 nm
• 1000 spectral bands=
1000 variables with
reflectance % for each
sample)
Provenances in the trial
Properties of green vegetation spectral
reflectance
VIS: prov
effect on
active
growth
VIS
chlorophyll
absorption
zone
NIR: prov
NIR
effect
after
bigger cell sizes growth
& spaces
cessation
among cells=
high NIR
reflection
Active growth= gradient
south-north
Least active
Most active
Growth cessation
Udm hardier
than Arch?
Shift in ranking
cessation
Hardening start
Hardening peak
• NIR informative during hardiness
development
• Continental prov is outstanding
To follow that after dormancy release
Hardy dormant
Conclusions
• There are significant provenance attributable and
interpretable differences in spectral reflectance
of Scots pine needles
• Providing a good opportunity for detecting this
spectral variation with the hyperspectral imaging
technique.
• Next is flying
Acknowledgements
Project TVARIPUSIS, Lithuanian Ministry of Education
Bavarian Forest Seed and Planting ASP help
Virgilijus Baliuckas
Darius Kavaliauskas
Vilma Kerpauskaite
Jurata Buchovska
ANOVAs
• Branch (tree)
effect= n.s.
• tree (prov)=
significant
• VIR significant at
active growth
• NIR significant at
hardening
Cessation
Hardening
Genetinė įvairovė individo lygmenyje
• Ar skiriasi viršutinėje ir apatinėje
lajos dalyse surinktų sėklų
genetinė įvairovė.
•Kokia įvairovė medyje?
O renkame iš
apačios...
•Kiek tėvų apvaisina vieną
kankorėžį
DNR tyrimai: tėvų skaičius
1-nas
tėvas
Kontinentinės populiacijos ramybės
būseną praranda greičiau
Udmurtia- kontinentine
Lietuva
Active growth
Shift in ranking
• Both VIS and NIR important