Gene Trees and Species Trees:Lessons from morning glories

Lauren A. Eserman & Richard E. MillerDepartment of Biological SciencesSoutheastern Louisiana University

Introduction

• DNA sequences are an important source of data for phylogenetic reconstruction

• Single-gene trees were considered exciting and sufficient at one time

• Chase and 41 other authors, 1993• Phylogeny of

angiosperms using rbcL

Introduction

• Next sequenced additional gene regions– Philosophical argument for “total evidence” –more

data will strengthen the ability to determine species relationships

– Used concatenated datasets to implement this idea – Still dominates the way species trees are estimated

Introduction

• Population genetics and coalescent theory emphasize that genes have unique histories– Gene trees do not always reflect the true species

history

Introduction

– Gene duplication events

– Horizontal gene transfer

– Incomplete lineage sorting (deep coalescence)

– Branch length heterogeneity

• Gene tree heterogeneity can come about by:

Edwards, 2009

• This provides evidence against concatenation

Introduction• Paradigm shift in systematics? (Edwards, 2009)– Moving away from notion that gene trees show true

species relationships– Promotes synergism of phylogenetic systematics

with population genetics and coalescent theory

Introduction• Application of the paradigm shift: – Use collective information from multiple gene trees

to estimate a species tree – Consider conflicting results, valid alternative

hypotheses for species relationships

Research Objectives

1. Explore how gene trees with different phylogenetic signal influence the estimated species tree– Using 28 gene trees– Effects of concatenation on estimated species

tree

2. Alternative objective is to obtain an understanding of species relationships for the organisms of interest (not discussed here)

• Morning glories are generally species of the genus Ipomoea (not monophyletic)

• Focus on tribe Ipomoeeae− Ipomoea + 9 other genera− c. 900 species− Distributed throughout the

subtropics and tropics of the world

Study Organisms

Ipomoea nil

Methods1. Bayesian phylogenetic analysis

of 28 gene trees

• Obtained 28 gene regions for species of Ipomoeeae based on our research and additional genes from GenBank

• Number of taxa ranged from 6 to 129• Alignments using MAFFT and manually adjusted• Models of nucleotide substitution chosen using jModelTest• Gene trees constructed using MrBayes v3.1.2

– 4 runs, 4 chains sampling every 100-200 generations– Runs were continued until stationary distribution was estimated– Burn-in determined as asymptote in plots of total tree length by

generations– Convergence criteria:

• Same topology among 4 runs• PP of clade support ±3% among 4 runs

– Majority rule consensus tree constructed from a combination of post-burnin trees from all 4 runs

• ITS tree used as working hypothesis– Densest taxon sampling (129 species)– Good intrageneric resolution– NOTE: Not assuming this is the species tree –

rather, a working hypothesis to compare to other gene trees

• Topology and clade support of 27 other gene trees compared to ITS gene tree

Methods1. Synthesis of 28 gene trees

I alba MDR6

I hederacea MDR6

I nil MDR6

I lindheimeri MDR6

I purpurea MDR6

I tricolor MDR6

I coccinea MDR6

I hederifolia MDR6

I neei MDR6

I quamoclit MDR6

I lacunosa MDR6

I triloba MDR6

I trifida MDR6

100

98

100

52

100

100

100

100

100

Majority rule

myb1

PHAR

MINA

DFRB-2I batatas FG

I batatas FY

I horsfalliae MDR

I nil SI1

I nil SI2

I purpurea ST

I quamoclit MDR

I tenuiloba MDR

I coccinea MDR

T oblongata MDR

M dissecta MDR

97

99

100

85

100

100

100

100

Majority rule

PHAR

MINA

Results1. Same relationships between ITS and other genes

Results2. Individual species with unique positions not shown in

any other gene treeI nil 554 A1

I nil 225 C2

I nil 830 O3

I nil 420 T

I nil 163 AD4

I nil 422 AJ

I nil 679 AK

I nil 447 AL

I nil 449 BG8

I nil 418 BA6

I nil 845 BC7

I nil 767 BJ

I nil 195 BS11

I nil 414 BW12

I nil 164 BY

I nil 771 BZ

I indica 481 A

I indica 166 C

I indica 253 D

I indica 168 E

I indica 602 F

I indica 130 B

I indica 424 G

I neurocephala 222 A1

I alba 129

I nil 547 BM

I nil 427 BQ10

I nil SI1 AM5

I nil 766 AY

I nil 776 BI

I nil 371 BK

I nil 706 BL

I nil 779 BN9

I hederifolia 506

99

78

76

82

81

100

80

100

100

62

100

8298

100100

76

79

Majority rule

bHLH3

PHAR

I batatas FG

I batatas FY

I horsfalliae MDR

I nil SI1

I nil SI2

I purpurea ST

I quamoclit MDR

I tenuiloba MDR

I coccinea MDR

T oblongata MDR

M dissecta MDR

97

99

100

85

100

100

100

100

Majority rule

DFRB-2

PHAR

MINA

Results3. Major alternative topology in CHSE

CHSEI alba MTC4

I purpurea SI4 A1

I nil MTC4 A

I nil SI5 B

I lobata MTC4

I quamoclit MTC4

I cordatotriloba MTC4

I amnicola MTC4

I argillicola MTC4

I wrightii MTC4

I platensis MTC4

I saintronanensis MTC4

I aquatica MTC4

I diamantinensis MTC4

I eriocarpa MTC4

I plebia MTC4

I ochracea MTC4

I pes tigridis MTC4

P hybrida MTC4

100

82

98

100

69

100

56

100

95

100

100

99

100

70

100

Majority rule

PHAR

MINA

Results4. Identify new unnamed clades

I alba MDR4

I hederacea MDR4

I nil TKS InbHLH2

I purpurea

I tricolor SI3

I coccinea MDR4

I quamoclit MDR4

I horsfalliae MDR4

I lacunosa MDR4

I trifida MDR4

I hochstetteri MDR4

I violacea MDR4

O pteripes MDR4

54

100

100

99

100

100

100

87

100

100

Majority rulebHLH2

PHAR

MINA

BATA

‘VIOL’

A nervosa 20T holubiiI pes tigris 12S beraviensisS tiliifolia 62I obscura 26I ochracea 22I arachnospermaI pedicellaris 97L owariensisI eriocarpaI lonchophylla 96I plebeia 18I albivenia 125I cairica 91I sepiaria 98I diamantinensis 45I aquatica 7I coccinea 13I sesscosiana 143I lutea 141I neei 140I funis 123I lobata 39I quamoclit 14I orizabensis 142I dumetorum 147I lindheimeri 25I indica 130I hederacea 10I nil 11 AI nil 127 BI purpurea 9 AI purpurea 131 BI pubescens 76I mairetii 137I barbatisepala 90I marginisepala 148I tricolor 128 BI tricolor 16 AI parasitica 5 AI stans 136I expansa 135I seducta 146I ampullacea 124I ternifolia 47I neurocephala 145I muricata 144I parasitica 15 BI alba 129I santillanii 138I conzattii 44I graminea 46I platensis 49I carnea 21I costata 55I polpha 56I wolcottiana 38I saintronanensis 50I leptophylla 4I pandurata 48I muelleri 36I gracilis 24I argillicola 34I asarifolia 8 A1I amnicola 3I imperati 53I sumatrana W22I batatas 1 A1I lacunosa 40I umbraticola 29I setosa 6I wrightii 33I sagittata 132I polymorphaM tuberosa 19O brownii 63

100

98

62

90 70

100

95 58

100

94

70

60

95

61

79

7598

100

99

100

90100

97

97

76

82

58

81

6499

59

54 99

56 100

100100

100

5272 100

100100

70

67 72

53

6298 96

98 71

Majority rule waxy 1‘OBSC’

MINA

PHAR

TRIC

CALO

‘AMNI’

BATA

Methods2. Concatenated dataset

• To address the issue of concatenation, constructed concatenated dataset using 10 genes

• All gene trees showed similar topologies I alba MTC

I nil MTC

I purpurea MTC

I lobata MTC

I quamlocit MTC

I amnicola MTC

I argillicola MTC

I wrightii MTC

I cordatotriloba MTC

I platensis MTC

I saintranensis MTC

I umbraticola MTC

I aquatica MTC1

I aquatica MTC2

I diamantinensis MTC

I pes tigridis MTC

I eriocarpa MTC

I plebia MTC

I ochracea MTC

I obscura MTC

100

75

100

76

100

97

100

100

86

100

60

100

100

Majority rule

DFRB-1I alba MTC A

I alba MDR B

I lobata MTC

I quamoclit MTC

I nil MTC A

I nil MDR B

I hederacea MDR

I purpurea MTC A

I purpurea MDR B

I cordatotriloba MTC

I batatas HN

I trifida MDR

I obscura MTC

I umbraticola MTC

I wrightii MTC

I amnicola MTC

I saintronanensis MTC

I argillicola MTC

I platensis MTC

I aquatica MTC

I diamantinensis MTC

I eriocarpa MTC

I plebia MTC

I pes tigridis MTC

I ochracea MTC

100

100

96

96

100

100

80

100

100

100

100

100

100

67

100

100

64

50

66

100

83

100

Majority rule

UF3GT CHII alba MTC4

I lobata MTC4

I quamoclit MTC4

I nil MTC4

I ampullacea REM

I neurocephala REM

I purpurea MTC4

I amnicola MTC4

I argillicola MTC4

I wrightii MTC4

I aquatica MTC4

I diamantinensis MTC4

I cordatotriloba MTC4

I umbraticola MTC4

I platensis MTC4

I saintronanensis MTC4

I eriocarpa MTC4

I pes tigridis MTC4

I plebia MTC4

I ochracea MTC4

I obscura MTC4

100

90

100

79

86

55

96

50

100

100

100

78

100

100

99

99

Majority rule

Results 10-gene

concatenated dataset

I alba

I nil

I purpurea

I lobata

I quamoclit

I cordatotriloba

I umbraticola

I platensis

I saintronanensis

I amnicola

I argillicola

I wrightii

I aquatica

I diamantinensis

I eriocarpa

I plebia

I pes tigridis

I ochracea

I obscura

100

100

100

99

100

100

100

100

100

100

100

100

100

100

100

100

Majority rule

• Maintains topologies of individual gene trees

Concatenated dataset

• What happens when one more gene is added?

• Add CHSE to 10-gene concatenated dataset– Alternative topology– All coding region– No indels

Results 11-gene concatenated

dataset

• Exhibits topology of CHSE – new gene overwhelms this analysis

I alba

I nil

I purpurea

I lobata

I quamoclit

I cordatotriloba

I amnicola

I argillicola

I wrightii

I platensis

I saintronanensis

I aquatica

I diamantinensis

I eriocarpa

I plebia

I pes tigridis

I ochracea

100

100

100

100

100

100

100

90

100

100

100

100

100

100

Majority rule

10-gene concatenated dataset 11-gene concatenated datasetI alba

I nil

I purpurea

I lobata

I quamoclit

I cordatotriloba

I amnicola

I argillicola

I wrightii

I platensis

I saintronanensis

I aquatica

I diamantinensis

I eriocarpa

I plebia

I pes tigridis

I ochracea

100

100

100

100

100

100

100

90

100

100

100

100

100

100

Majority rule

I alba

I nil

I purpurea

I lobata

I quamoclit

I cordatotriloba

I umbraticola

I platensis

I saintronanensis

I amnicola

I argillicola

I wrightii

I aquatica

I diamantinensis

I eriocarpa

I plebia

I pes tigridis

I ochracea

I obscura

100

100

100

99

100

100

100

100

100

100

100

100

100

100

100

100

Majority rule

BEST AnalysisBayesian Estimation of Species Trees

(Liu, 2008)

• Incorporates a multispecies coalescent model to estimate species tree from many gene trees

• Methods:– 11-gene concatenated dataset– 2 runs, 4 chains– 8 million generations (did not reach convergence

on topology)

BEST Analysis

• Results:– Clade present in

CHSE appears in BEST tree

– Overall topology differs

– Species pairs supported throughout

I alba

I amnicola

I argillicola

I wrightii

I platensis

I saintronanensis

I aquatica

I diamantinensis

I cordatotriloba

I lobata

I quamoclit

I nil

I purpurea

I eriocarpa

I plebia

I pes tigridis

I ochracea

100

99

75

73

78

100

98

100

100

84

100

100

100

100

Majority rule

Discussion

• Analysis of 28 gene trees – Provides an estimate of species tree– Alternative hypotheses for species relationships

have emerged– Overall congruence among gene trees

Discussion – Concatenated datasets

• Total evidence philosophically justified but misleads results because of gene tree heterogeneity– Shown clearly in 11-gene concatenated dataset

• Left with idea that we have two alternative hypotheses of species relationships– Two estimates of the species tree

Discussion – Concatenated datasets

• Can now appreciate how a single gene can overwhelm results of a concatenated dataset– Topology of CHSE dominated

I alba

I nil

I purpurea

I lobata

I quamoclit

I cordatotriloba

I amnicola

I argillicola

I wrightii

I platensis

I saintronanensis

I aquatica

I diamantinensis

I eriocarpa

I plebia

I pes tigridis

I ochracea

100

100

100

100

100

100

100

90

100

100

100

100

100

100

Majority rule

I alba

I nil

I purpurea

I lobata

I quamoclit

I cordatotriloba

I umbraticola

I platensis

I saintronanensis

I amnicola

I argillicola

I wrightii

I aquatica

I diamantinensis

I eriocarpa

I plebia

I pes tigridis

I ochracea

I obscura

100

100

100

99

100

100

100

100

100

100

100

100

100

100

100

100

Majority rule

Ipomoea purpurea

Seed Donations:M. Clegg, M. Rausher,J. A. McDonald, J. MillerP. Tiffin, B. Zufall, S.M. Chang

Research Assistants:A. McDaniel, K. Robichaux,W. Terry, S. Major, H. Echlin,F. St. Cyr

Acknowledgements