Plastome phylogenetics in Populus

Daisie Huang and Quentin Cronk

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

• 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 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?

• Chloroplast capture

• Stochastic processes

• Divergent selection on cytoplasmic genes

Hypotheses

• 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.

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