plastome phylogenomics in populus
TRANSCRIPT
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.
Relationships within Populus
adapted from Levsen et al 2012
• 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
POPCAN dataset
• Genome BC’s POPCAN project is investigating the genomics of Populus trichocarpa and P. balsamifera.
• Main approach is to align short-read whole-genome 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.
• 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
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.
Plastome alignment
• What about ascertainment bias?
• Aligning to a reference of an ingroup species could cause artifacts.
• Alignment to different reference works as well!
• Slow-evolving plastome means that much of the plastome is conserved across taxa.
• More missing data, but high coverage of genes.
• Populus alignment has 3% missing data.
• Manihot alignment has 44% missing data.
• 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.
Populus
Manihot
Salix eriocephalaSalix interior
Populus tomentosaP. angustifolia GHE1
P. angustifolia Coal1 P. angustifolia AZBR107
P. deltoides Missou7RajuP. deltoides Pdel181
P. fremontii Pfre186P. balsamifera ROS4P. deltoides SO539SL
P. trichocarpa HALS30P. trichocarpa JASP30
P. trichocarpa PITS29P. trichocarpa NASD8 P. trichocarpa STKG4
P. trichocarpa LAFY30P. trichocarpa JEFF30P. trichocarpa NPLN30P. trichocarpa NECA14
P. trichocarpa TOBA23P. trichocarpa NBON29P. trichocarpa CARS29
P. trichocarpa HAZH10P. trichocarpa BULG11P. trichocarpa ISKA6 P. trichocarpa SKNM10
P. trichocarpa KIMB16P. trichocarpa LONG29
P. trichocarpa QFRS16P. trichocarpa MCHA19P. trichocarpa ALAA20P. trichocarpa CHKD19P. trichocarpa BELC18
P. balsamifera MIN11 P. balsamifera RNA13
P. balsamifera LAR15 P. balsamifera GIL7P. balsamifera DEN5P. balsamifera DUN13 P. balsamifera STO13
P. balsamifera MAN9P. balsamifera FRE14
P. balsamifera KUU3P. balsamifera SOU5
P. trichocarpa MCGR15P. balsamifera EDM5P. balsamifera FBK10
P. trichocarpa SHEL15P. trichocarpa WLOW15
P. balsamifera CAR14 P. balsamifera FTM8
P. balsamifera AP5454P. balsamifera POR1
P. balsamifera LAR6P. balsamifera NWL2
P. balsamifera GRA10
Clade B
Clade T
Clade A
Clade B
Clade T
Clade A
P. trichocarpa HAZH10P. trichocarpa BULG11P. trichocarpa SKNM10P. trichocarpa CHKD19P. trichocarpa BELC18P. trichocarpa KIMB16P. trichocarpa ALAA20
P. trichocarpa HALS30P. trichocarpa JASP30P. trichocarpa PITS29
P. trichocarpa STKG4 P. trichocarpa NASD8 P. trichocarpa Nisqually-1P. trichocarpa NBON29P. trichocarpa TOBA23P. trichocarpa NECA14P. trichocarpa CARS29P. trichocarpa LONG29P. trichocarpa JEFF30P. trichocarpa LAFY30P. trichocarpa QFRS16P. trichocarpa ISKA6 P. trichocarpa MCHA19
Populus tomentosaSalix spp.
P. balsamifera MIN11P. balsamifera RNA13
P. balsamifera LAR15P. balsamifera DEN5P. balsamifera DUN13P. balsamifera GIL7P. balsamifera STO13
P. balsamifera KUU3P. balsamifera MAN9P. balsamifera FRE14
P. balsamifera CAR14P. trichocarpa WLOW15P. balsamifera SOU5P. balsamifera EDM5P. trichocarpa SHEL15P. balsamifera FBK10P. balsamifera FTM8P. trichocarpa MCGR15
P. balsamifera AP5454P. balsamifera POR1P. balsamifera NWL2P. balsamifera LAR6
P. balsamifera GRA10P. angustifolia GHE1
P. angustifolia Coal1P. angustifolia AZBR107
P. deltoides Pdel181P. deltoides Missou7Raju
P. fremontii Pfre186P. balsamifera ROS4
P. deltoides SO539SL
P. trichocarpa NPLN30
• 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.
• Each clade is highly supported.
• Divergence between clade B and clade T is greater than divergence within clade A, which has multiple cottonwood species.
• 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.
• These dates of plastome divergence are much older than the estimated nuclear divergence between P. trichocarpa and P. balsamifera.
• How to explain this discrepancy?
• 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.
Chloroplast capture
P. trichocarpa HAZH10P. trichocarpa BULG11P. trichocarpa SKNM10P. trichocarpa CHKD19P. trichocarpa BELC18P. trichocarpa KIMB16P. trichocarpa ALAA20
P. trichocarpa HALS30P. trichocarpa JASP30
P. trichocarpa PITS29P. trichocarpa STKG4
P. trichocarpa NASD8
P. trichocarpa Nisqually-1P. trichocarpa NBON29P. trichocarpa TOBA23P. trichocarpa NECA14P. trichocarpa CARS29
P. trichocarpa LONG29P. trichocarpa JEFF30P. trichocarpa LAFY30P. trichocarpa QFRS16
P. trichocarpa ISKA6 P. trichocarpa MCHA19
Salix eriocephalaSalix interior
Populus tomentosaP. balsamifera MIN11
P. balsamifera RNA13P. balsamifera LAR15P. balsamifera DEN5P. balsamifera DUN13P. balsamifera GIL7P. balsamifera STO13
P. balsamifera KUU3P. balsamifera MAN9
P. balsamifera FRE14P. balsamifera CAR14
P. trichocarpa WLOW15P. balsamifera SOU5P. balsamifera EDM5P. trichocarpa SHEL15P. balsamifera FBK10
P. balsamifera FTM8P. trichocarpa MCGR15
P. balsamifera AP5454P. balsamifera POR1P. balsamifera NWL2P. balsamifera LAR6
P. balsamifera GRA10P. angustifolia GHE1
P. angustifolia Coal1P. angustifolia AZBR107
P. deltoides Pdel181P. deltoides Missou7Raju
P. fremontii Pfre186P. balsamifera ROS4
P. deltoides SO539SL
P. trichocarpa NPLN30
Clade B
Clade T
Clade A
• Unlikely.
• The two clades share many nuclear alleles and experience ongoing gene flow.
• Plastome data supports this: several sampled individuals show mismatched plastome haplotypes.
Incomplete lineage sorting
P. trichocarpa HAZH10P. trichocarpa BULG11P. trichocarpa SKNM10P. trichocarpa CHKD19P. trichocarpa BELC18P. trichocarpa KIMB16P. trichocarpa ALAA20
P. trichocarpa HALS30P. trichocarpa JASP30
P. trichocarpa PITS29P. trichocarpa STKG4
P. trichocarpa NASD8
P. trichocarpa Nisqually-1P. trichocarpa NBON29P. trichocarpa TOBA23P. trichocarpa NECA14P. trichocarpa CARS29
P. trichocarpa LONG29P. trichocarpa JEFF30P. trichocarpa LAFY30P. trichocarpa QFRS16
P. trichocarpa ISKA6 P. trichocarpa MCHA19
Salix eriocephalaSalix interior
Populus tomentosaP. balsamifera MIN11
P. balsamifera RNA13P. balsamifera LAR15P. balsamifera DEN5P. balsamifera DUN13P. balsamifera GIL7P. balsamifera STO13
P. balsamifera KUU3P. balsamifera MAN9
P. balsamifera FRE14P. balsamifera CAR14
P. trichocarpa WLOW15P. balsamifera SOU5P. balsamifera EDM5P. trichocarpa SHEL15P. balsamifera FBK10
P. balsamifera FTM8P. trichocarpa MCGR15
P. balsamifera AP5454P. balsamifera POR1P. balsamifera NWL2P. balsamifera LAR6
P. balsamifera GRA10P. angustifolia GHE1
P. angustifolia Coal1P. angustifolia AZBR107
P. deltoides Pdel181P. deltoides Missou7Raju
P. fremontii Pfre186P. balsamifera ROS4
P. deltoides SO539SL
P. trichocarpa NPLN30
Clade B
Clade T
Clade A
• Selection acting on a gene or genes in the plastome in one lineage could lead to plastome divergence.
• SNP comparisons between clades B and T show that some genes are more variable than others.
trnH-GUGpsbA
trnK-UUUmatK
trnQ-UUGpsbKpsbItrnS-GCUtrnG-UCCtrnR-UCUatpA
atpFatpH
atpIrps2
rpoC2
rpoC1_
1rpoC1_
2
rpoB
trnC-
GCA
petNps
bMtrnD-
GUC
trnY-
GUA
trnE-
UUC
trnT-
GG
Utrn
T-G
GU
psbDpsbC
trnS-
UGA
trnS-
UGA
psbZ
trnG
-UC
Ctrn
fM-C
AUrp
s14Baspps
aA
ycf3
trnS-
GG
Arp
s4trn
T0UG
Utrn
L-UA
Atrn
F-G
AAnd
hJnd
hKnd
hCtrn
V-UA
Ctrn
M-CAU
atpE
atpB
rbcL
rbcL
accD
psaI
ycf4
cemA
petA
psbJpsbLpsbFpsbE
petLpetGtrnW-CCA
trnP-UGG
psaJrpl33rps18rpl20
clpP
psbB
psbT psbN psbHpetB
petD
rpoArps11 rpl36rps8rpl14rpl16
rps3 rpl22 rps19rpl2
rpl23 trnI-CAU
ycf2
ycf15 trnL-CAA
trnL-CAA ndhB rps7
trnV-GAC
rrn16 trnI-GAU
trnA-UGC
rrn23
rrn4.5rrn5trnR-ACG
trnN-GUU
ycf1 ndhF
trnL-UAG
ccsA Dhdn psaC
ndhEndhGndhI
ndhA
ndhHrps15
ycf1
trnN-GUUtrnR-ACG
rrn5rrn4.5
rrn23trnA-UGC
trnI-GAU
rrn16
trnV-GAC
rps7
ndhB
trnL-CAA
ycf15
ycf2
trnI-CAUrpl23rpl2rps19
Polymorphism within clade T
SNPs on Populus trichocarpa reference plastome
Polymorphism within clade BFixed polymorphism between T and B
Differential selection
• The most variable genes between clades are psbA, rbcL, ycf1, and rpoC.
• No significant evidence of selection between clades T and B.
• Using branch-site tests of selection, found significant differential selection on ycf1 in clade A, the Aigeros cottonwoods.
trnH-GUGpsbA
trnK-UUUmatK
trnQ-UUGpsbKpsbItrnS-GCUtrnG-UCCtrnR-UCUatpA
atpFatpH
atpIrps2
rpoC2
rpoC1_
1rpoC1_
2
rpoB
trnC-
GCA
petNps
bMtrnD-
GUC
trnY-
GUA
trnE-
UUC
trnT-
GG
Utrn
T-G
GU
psbDpsbC
trnS-
UGA
trnS-
UGA
psbZ
trnG
-UC
Ctrn
fM-C
AUrp
s14Baspps
aA
ycf3
trnS-
GG
Arp
s4trn
T0UG
Utrn
L-UA
Atrn
F-G
AAnd
hJnd
hKnd
hCtrn
V-UA
Ctrn
M-CAU
atpE
atpB
rbcL
rbcL
accD
psaI
ycf4
cemA
petA
psbJpsbLpsbFpsbE
petLpetGtrnW-CCA
trnP-UGG
psaJrpl33rps18rpl20
clpP
psbB
psbT psbN psbHpetB
petD
rpoArps11 rpl36rps8rpl14rpl16
rps3 rpl22 rps19rpl2
rpl23 trnI-CAU
ycf2
ycf15 trnL-CAA
trnL-CAA ndhB rps7
trnV-GAC
rrn16 trnI-GAU
trnA-UGC
rrn23
rrn4.5rrn5trnR-ACG
trnN-GUU
ycf1 ndhF
trnL-UAG
ccsA Dhdn psaC
ndhEndhGndhI
ndhA
ndhHrps15
ycf1
trnN-GUUtrnR-ACG
rrn5rrn4.5
rrn23trnA-UGC
trnI-GAU
rrn16
trnV-GAC
rps7
ndhB
trnL-CAA
ycf15
ycf2
trnI-CAUrpl23rpl2rps19
Polymorphism within clade T
SNPs on Populus trichocarpa reference plastome
Polymorphism within clade BFixed polymorphism between T and B
Differential selection
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.
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.