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Plastome phylogenetics
in Populus
Daisie Huang and Quentin Cronk
Relationships within Populus
•

P. trichocarpa and P.
balsamifera are sister
species: balsam
poplars (section
Tacamahaca)
...
Relationships within Populus
•
•
•

The sister relationship of
P. balsamifera and P.
trichocarpa is supported
by most mole...
POPCAN dataset
•

Genome BC’s POPCAN project is investigating the
genomics of Populus trichocarpa and P. balsamifera.

•

...
•

We can make a complete plastome phylogeny of the
North American cottonwoods and balsam poplars.

•
•
•

27 samples of P...
Plastome alignment
• We can align WGS
short reads to the
P. trichocarpa
reference
plastome.
Plastome alignment
• Very high coverage!
• 1000-8000x
coverage for most
positions.
Plastome alignment
• For most positions,
all reads agree on
the allele called.

• We used a

conservative
approach: infer
...
Plastome alignment
• What about

ascertainment bias?

• Aligning to a

reference of an
ingroup species
could cause
artifac...
• Alignment to different reference works as well!
• Slow-evolving plastome means that much of the
plastome is conserved ac...
• More missing data, but high coverage of genes.
• Populus alignment has 3% missing data.
• Manihot alignment has 44% miss...
Salix eriocephala
Salix interior
Populus tomentosa
P. angustifolia GHE1
P. angustifolia Coal1
P. angustifolia AZBR107
P. d...
•

P. trichocarpa and P.
balsamifera are in two
separate clades: clade B
and clade T.

•

Clade A contains the
cottonwoods...
•

Each clade is highly
supported.

•

Divergence between
clade B and clade T is
greater than divergence
within clade A, w...
•

Based on the Populus/Salix divergence date of 45 Mya:

•
•

Each of the three clades dates to 13-15 Mya.
The MRCA of th...
•

These dates of plastome divergence are much older than
the estimated nuclear divergence between P. trichocarpa
and P. b...
Hypotheses
•
•
•

Chloroplast capture
Stochastic processes
Divergent selection on cytoplasmic genes
Chloroplast capture
Salix eriocephala

•
•

•

Unlikely.
In this case, the
capturing species should
have one haplotype tha...
Incomplete lineage sorting
Salix eriocephala

•
•
•

Unlikely.
The two clades share
many nuclear alleles and
experience on...
CA

2
1_
rp
o

C1

rp

cD
ac

C2

o
rp

2
rps I
atp

ps
ppsb E
pssbbF
bJL

H
atp
F
atp

rpl2
rps 0
rpl318
psa 3
ttr J
pren...
CA

2
1_
rp
o

C1

rp

_1

oC

rp
o

B

pe
trn tN
C-G

trnT
nE -G
trnY -U GU
U
trnD -GUA
-GU C
C
psb
M

psbD

ps aB

rps14...
Conclusions
•

Plastome selection, while still not ruled out, is
not a complete explanation.

•
•
•

The Aigeros cottonwoo...
Conclusions
•

Plastome phylogeny tells a detailed story of a
single facet of the history of these two species:
this story...
Acknowledgements
• Cronk Lab collaborators:
• Charles Hefer, Armando Geraldes, Natalia
Kolosova

• POPCAN project PIs and ...
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Plastome phylogenomics in Populus

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Plastome phylogenomics in Populus

  1. 1. Plastome phylogenetics in Populus Daisie Huang and Quentin Cronk
  2. 2. Relationships within Populus • P. trichocarpa and P. balsamifera are sister species: balsam poplars (section Tacamahaca) • Closely related to the cottonwoods (section Aigeros). • Natural hybrids form at contact zones between species.
  3. 3. Relationships within Populus • • • The sister relationship of P. balsamifera and P. trichocarpa is supported by most molecular data. Levsen et al estimated a very recent date of divergence of ~75 Kya. Recent molecular phylogenies do not agree on the relative placement of the cottonwoods and the balsam poplars. P. balsamifera P. trichocarpa P. tremula P. angustifolia P. fremontii adapted from Levsen et al 2012
  4. 4. POPCAN dataset • Genome BC’s POPCAN project is investigating the genomics of Populus trichocarpa and P. balsamifera. • Main approach is to align short-read wholegenome shotgun sequences to reference genome of P. trichocarpa. • • • Resequenced >300 samples of P. trichocarpa. Resequenced >200 samples of P. balsamifera. Resequenced many samples of other species of Populus for phylogenetic context.
  5. 5. • We can make a complete plastome phylogeny of the North American cottonwoods and balsam poplars. • • • 27 samples of P. trichocarpa 21 samples of P. balsamifera Seven samples of other cottonwoods
  6. 6. Plastome alignment • We can align WGS short reads to the P. trichocarpa reference plastome.
  7. 7. Plastome alignment • Very high coverage! • 1000-8000x coverage for most positions.
  8. 8. Plastome alignment • For most positions, all reads agree on the allele called. • We used a conservative approach: infer ambiguities and low-coverage bases as missing data.
  9. 9. Plastome alignment • What about ascertainment bias? • Aligning to a reference of an ingroup species could cause artifacts.
  10. 10. • Alignment to different reference works as well! • Slow-evolving plastome means that much of the plastome is conserved across taxa.
  11. 11. • More missing data, but high coverage of genes. • Populus alignment has 3% missing data. • Manihot alignment has 44% missing data.
  12. 12. Salix eriocephala Salix interior Populus tomentosa P. angustifolia GHE1 P. angustifolia Coal1 P. angustifolia AZBR107 P. deltoides Missou7Raju P. deltoides Pdel181 Clade A P. fremontii Pfre186 P. balsamifera ROS4 P. deltoides SO539SL P. trichocarpa HALS30 Manihot P. trichocarpa JASP30 P. trichocarpa PITS29 P. trichocarpa NASD8 P. trichocarpa STKG4 P. trichocarpa LAFY30 P. trichocarpa JEFF30 P. trichocarpa NPLN30 P. trichocarpa NECA14 P. trichocarpa TOBA23 P. trichocarpa NBON29 Clade T P. trichocarpa CARS29 P. trichocarpa HAZH10 P. trichocarpa BULG11 P. trichocarpa ISKA6 P. trichocarpa SKNM10 P. trichocarpa KIMB16 Populus Salix spp. Populus tomentosa P. balsamifera MIN11 P. balsamifera RNA13 P. balsamifera LAR15 P. balsamifera DEN5 P. balsamifera DUN13 P. balsamifera GIL7 P. balsamifera STO13 P. balsamifera KUU3 P. balsamifera MAN9 P. balsamifera FRE14 P. balsamifera CAR14 P. trichocarpa WLOW15 P. balsamifera SOU5 P. balsamifera EDM5 P. trichocarpa SHEL15 P. balsamifera FBK10 P. balsamifera FTM8 P. trichocarpa MCGR15 P. balsamifera AP5454 P. balsamifera POR1 P. balsamifera NWL2 P. balsamifera LAR6 P. balsamifera GRA10 P. angustifolia GHE1 P. angustifolia Coal1 P. angustifolia AZBR107 P. deltoides Pdel181 P. deltoides Missou7Raju P. fremontii Pfre186 P. balsamifera ROS4 P. deltoides SO539SL P. trichocarpa HALS30 P. trichocarpa JASP30 P. trichocarpa PITS29 P. trichocarpa STKG4 P. trichocarpa NASD8 P. trichocarpa NPLN30 P. trichocarpa Nisqually-1 P. trichocarpa NBON29 P. trichocarpa TOBA23 P. trichocarpa NECA14 P. trichocarpa CARS29 P. trichocarpa LONG29 P. trichocarpa JEFF30 P. trichocarpa LAFY30 P. trichocarpa QFRS16 P. trichocarpa ISKA6 P. trichocarpa MCHA19 P. trichocarpa HAZH10 P. trichocarpa BULG11 P. trichocarpa SKNM10 P. trichocarpa CHKD19 P. trichocarpa BELC18 P. trichocarpa KIMB16 P. trichocarpa ALAA20 P. trichocarpa LONG29 P. trichocarpa QFRS16 P. trichocarpa MCHA19 P. trichocarpa ALAA20 P. trichocarpa CHKD19 Clade B P. trichocarpa BELC18 P. balsamifera MIN11 P. balsamifera RNA13 P. balsamifera LAR15 P. balsamifera GIL7 P. balsamifera DEN5 P. balsamifera DUN13 P. balsamifera STO13 Clade A P. balsamifera MAN9 P. balsamifera FRE14 P. balsamifera KUU3 P. balsamifera SOU5 P. trichocarpa MCGR15 Clade B P. balsamifera EDM5 P. balsamifera FBK10 Clade T P. trichocarpa SHEL15 P. trichocarpa WLOW15 P. balsamifera CAR14 P. balsamifera FTM8 P. balsamifera AP5454 P. balsamifera POR1 P. balsamifera LAR6 P. balsamifera NWL2 P. balsamifera GRA10 • Best ML trees from RAxML, bold lines indicate >95% bootstrap support. • • Rooted using Salix and an Old World Populus species. Both alignments resolve the same topology.
  13. 13. • P. trichocarpa and P. balsamifera are in two separate clades: clade B and clade T. • Clade A contains the cottonwoods: P. deltoides, P. fremontii, P. angustifolia. • Clades A, B, and T diverged at approximately the same time.
  14. 14. • Each clade is highly supported. • Divergence between clade B and clade T is greater than divergence within clade A, which has multiple cottonwood species.
  15. 15. • Based on the Populus/Salix divergence date of 45 Mya: • • Each of the three clades dates to 13-15 Mya. The MRCA of the balsam poplars and cottonwoods is ~25 Mya.
  16. 16. • These dates of plastome divergence are much older than the estimated nuclear divergence between P. trichocarpa and P. balsamifera. • How to explain this discrepancy?
  17. 17. Hypotheses • • • Chloroplast capture Stochastic processes Divergent selection on cytoplasmic genes
  18. 18. Chloroplast capture Salix eriocephala • • • Unlikely. In this case, the capturing species should have one haplotype that reflects the species it captured from. Both P. balsamifera and P. trichocarpa have significant sequence variation within each clade. Populus tomentosa P. balsamifera MIN11 P. balsamifera RNA13 P. balsamifera LAR15 P. balsamifera DEN5 P. balsamifera DUN13 P. balsamifera GIL7 P. balsamifera STO13 P. balsamifera KUU3 P. balsamifera MAN9 P. balsamifera FRE14 P. balsamifera CAR14 P. trichocarpa WLOW15 P. balsamifera SOU5 P. balsamifera EDM5 P. trichocarpa SHEL15 P. balsamifera FBK10 P. balsamifera FTM8 P. trichocarpa MCGR15 P. balsamifera AP5454 P. balsamifera POR1 P. balsamifera NWL2 P. balsamifera LAR6 P. balsamifera GRA10 P. angustifolia GHE1 P. angustifolia Coal1 P. angustifolia AZBR107 P. deltoides Pdel181 P. deltoides Missou7Raju P. fremontii Pfre186 P. balsamifera ROS4 P. deltoides SO539SL P. trichocarpa HALS30 P. trichocarpa JASP30 P. trichocarpa PITS29 P. trichocarpa STKG4 P. trichocarpa NASD8 P. trichocarpa NPLN30 P. trichocarpa Nisqually-1 P. trichocarpa NBON29 P. trichocarpa TOBA23 P. trichocarpa NECA14 P. trichocarpa CARS29 P. trichocarpa LONG29 P. trichocarpa JEFF30 P. trichocarpa LAFY30 P. trichocarpa QFRS16 P. trichocarpa ISKA6 P. trichocarpa MCHA19 P. trichocarpa HAZH10 P. trichocarpa BULG11 P. trichocarpa SKNM10 P. trichocarpa CHKD19 P. trichocarpa BELC18 P. trichocarpa KIMB16 P. trichocarpa ALAA20 Clade B Clade A Clade T
  19. 19. Incomplete lineage sorting Salix eriocephala • • • Unlikely. The two clades share many nuclear alleles and experience ongoing gene flow. Plastome data supports this: several sampled individuals show mismatched plastome haplotypes. Populus tomentosa P. balsamifera MIN11 P. balsamifera RNA13 P. balsamifera LAR15 P. balsamifera DEN5 P. balsamifera DUN13 P. balsamifera GIL7 P. balsamifera STO13 P. balsamifera KUU3 P. balsamifera MAN9 P. balsamifera FRE14 P. balsamifera CAR14 P. trichocarpa WLOW15 P. balsamifera SOU5 P. balsamifera EDM5 P. trichocarpa SHEL15 P. balsamifera FBK10 P. balsamifera FTM8 P. trichocarpa MCGR15 P. balsamifera AP5454 P. balsamifera POR1 P. balsamifera NWL2 P. balsamifera LAR6 P. balsamifera GRA10 P. angustifolia GHE1 P. angustifolia Coal1 P. angustifolia AZBR107 P. deltoides Pdel181 P. deltoides Missou7Raju P. fremontii Pfre186 P. balsamifera ROS4 P. deltoides SO539SL P. trichocarpa HALS30 P. trichocarpa JASP30 P. trichocarpa PITS29 P. trichocarpa STKG4 P. trichocarpa NASD8 P. trichocarpa NPLN30 P. trichocarpa Nisqually-1 P. trichocarpa NBON29 P. trichocarpa TOBA23 P. trichocarpa NECA14 P. trichocarpa CARS29 P. trichocarpa LONG29 P. trichocarpa JEFF30 P. trichocarpa LAFY30 P. trichocarpa QFRS16 P. trichocarpa ISKA6 P. trichocarpa MCHA19 P. trichocarpa HAZH10 P. trichocarpa BULG11 P. trichocarpa SKNM10 P. trichocarpa CHKD19 P. trichocarpa BELC18 P. trichocarpa KIMB16 P. trichocarpa ALAA20 Clade B Clade A Clade T
  20. 20. CA 2 1_ rp o C1 rp cD ac C2 o rp 2 rps I atp ps ppsb E pssbbF bJL H atp F atp rpl2 rps 0 rpl318 psa 3 ttr J prenP penW-UG ttG -CCG L A A atp U -UC trnR -UCC trnG U S-GC trnbI ps K psb -UUG trnQ clpP petB psbH psbN psbT psbB trnK-UUU matK SNPs on Populus trichocarpa reference plastome Polymorphism within clade T psbA trnH-GUG rps19 rpl2 rpl23 trnI-CAU petD Polymorphism within clade B rpl14 rps8 rpl36 rps11 rpoA Fixed polymorphism between T and B rpl16 ycf1 5 trnL -CA A nd hB ycf 2 rps 7 trn rrn V-G AC 16 trn I-G AU trn A- s7 rp 23 C UG rrn 4.5 CG rrnn5 -A UU rrrnR -G t N trn 1 ycf ndhA 5 rps1 ndhH ndhD ndhI ndhG ndhE psaC ccsA trnL-UAG ndhF ycf1 3 trnN -G trnR UU rrrn4 -ACG r n5.5 rrn2 t rnA -UG trn C I-G AU C r rn1 trn 6 VGA hB nd AA CA Lt trn f15 yc SNP comparisons between clades B and T show that some genes are more variable than others. ycf2 AU nI-C trpl23 r rpl2 9 rps12 rpl2 rps3 • Selection acting on a gene or genes in the plastome in one lineage could lead to plastome divergence. pe t ce A m A yc ps f4 aI • _1 oC rp o B pe trn tN C-G trnT nE -G trnY -U GU U trnD -GUA -GU C C psb M psbD ps aB rps14 trnfM-CAU trnG-UCC psbZ trnS-UG A psbC psaA ycf3 C ndhhK nd J ndh A -GA trnF -UAA trnL U 0UG trnT4 rps GA trnS-G cL rb B atp E AU C atp M--UAC trnnV tr Differential selection
  21. 21. CA 2 1_ rp o C1 rp _1 oC rp o B pe trn tN C-G trnT nE -G trnY -U GU U trnD -GUA -GU C C psb M psbD ps aB rps14 trnfM-CAU trnG-UCC psbZ trnS-UG A psbC psaA ycf3 C ndhhK nd J ndh A -GA trnF -UAA trnL U 0UG trnT4 rps GA trnS-G cL rb cD ac The most variable genes between clades are psbA, rbcL, ycf1, and rpoC. pe t ce A m A yc ps f4 aI C2 o rp 2 rps I atp ps ppsb E pssbbF bJL H atp F atp rpl2 rps 0 rpl318 psa 3 ttr J prenP penW-UG ttG -CCG L A • B atp E AU C atp M--UAC trnnV tr Differential selection A atp U -UC trnR -UCC trnG U S-GC trnbI ps K psb -UUG trnQ clpP Polymorphism within clade T petD Fixed polymorphism between T and B rpl16 ycf2 AU nI-C trpl23 r rpl2 9 rps12 rpl2 rps3 ycf1 5 trnL -CA A nd hB ycf 2 rps 7 trn rrn V-G AC 16 trn I-G AU trn A- s7 rp 23 C UG rrn 4.5 CG rrnn5 -A UU rrrnR -G t N trn 1 ycf ndhA 5 rps1 ndhH ndhD ndhI ndhG ndhE psaC ccsA trnL-UAG ndhF ycf1 3 trnN -G trnR UU rrrn4 -ACG r n5.5 rrn2 t rnA -UG trn C I-G AU C r rn1 trn 6 VGA hB nd AA CA Lt trn f15 yc Using branch-site tests of selection, found significant differential selection on ycf1 in clade A, the Aigeros cottonwoods. psbA trnH-GUG rps19 rpl2 rpl23 trnI-CAU Polymorphism within clade B rpl14 rps8 rpl36 rps11 rpoA • petB psbH psbN psbT psbB • No significant evidence of selection between clades T and B. trnK-UUU matK SNPs on Populus trichocarpa reference plastome
  22. 22. Conclusions • Plastome selection, while still not ruled out, is not a complete explanation. • • • The Aigeros cottonwoods, as a group, may be undergoing differential plastome selection. If differential selection is acting on clades B and T, evidence is not strong. Still possible that selection may be acting on the tightly linked mitochondrial genome.
  23. 23. Conclusions • Plastome phylogeny tells a detailed story of a single facet of the history of these two species: this story is obscured by more recent events in the nuclear loci. • Population genetic studies confirm the sister relationship of P. trichocarpa and P. balsamifera. • However, the cytonuclear discordance must be capturing a real historical event that predates the Pleistocene bottleneck.
  24. 24. Acknowledgements • Cronk Lab collaborators: • Charles Hefer, Armando Geraldes, Natalia Kolosova • POPCAN project PIs and funders: • Carl Douglas, Shawn Mansfield • University of British Columbia • Genome BC

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