AMPK Methods and Protocols

11. Use then the Shimodaira–Hasegawa test [44] implemented
    into RAxML to test whether the ML tree explains the data
    significantly better than the alternative tree, in which the fungi
    are placed such that they agree with the species phylogeny. To
    do so runraxml –n outputFileName –s alignmentFilename –m
    modelOfChoice –f h –t mlTreeFilename –z alternativeTreeFile-
    name. The program will then tell you whether or not the ML
    tree is significantly better than the alternative tree. In the
    example of Fig.11, the sequence tree modified to reflect the
    evolutionary relationships of the species is only 3.9 log likeli-
    hood units worse than the ML tree. According to the SH test,
    this difference is not significant (p<0.01), and thus both trees
    explain the data equally well. It is therefore unnecessary to
    invoke complex evolutionary scenarios (seeFig. 10d) to explain
    the deviations of the ML tree from the species tree topology.

0.5

PRKAG1-3 Arabidopsis thaliana

KING1 Arabidopsis thaliana

PRKAG1-3 Sorangium cellulosum

SDS23 Candida parapsilosis

PRKAG1-3 Nematostella vectensis

KING1 Oryza sativa

SDS23 Saccharomyces cerevisiae

KING1 Selaginella moellendorffii

KING1 Capsaspora owczarzaki

PRKAG1 Homo sapiens

PRKAG1-3 Drosophila melanogaster

KING1 Dictyostelium discoideum

SDS23 Spizellomyces punctatus

PRKAG3 Homo sapiens

PRKAG1-3 Ectocarpus siliculosus

PRKAG1-3 Pyrobaculum neutrophilum

PRKAG1-3 Chlamydomonas reinhardtii

PRKAG2 Homo sapiens

SDS23 Kluyveromyces lactis

PRKAG1-3 Saccharomyces cerevisiae

PRKAG1-3 Selaginella moellendorffii

PRKAG1-3 Batrachochytrium dendrobatidis

KING1 Polysphondylium pallidum

PRKAG1-3 Phytophthora sojae

PRKAG1-3 Ustilago maydis

0.5

Species tree topology

Maximum likelihood tree topology

LogLik = -16,307.6 LogLik = -16,311.5

40

99

54

76

84

66

92

100

100

95

86

100

62

82

60

78

81

99

90

100

51

76

*

* *

Fig. 11The evolutionary histories of the human AMPKγsubunit and of its homologs in plants, AMPKβγand
KING1. The tree to the left represents the maximum likelihood (ML) topology computed with RAxML
(substitution model: LG [42]þIþG). Branch labels denote percent bootstrap support. Asterisks indicate
gene duplication events. The subtree harboring AMPKγof animals together with its fungal and plant orthologs
is shown in black. A gene duplication event in the last common ancestor of the eukaryotes gave rise to the
gray subtree, which harbors KING1 and its orthologs. This rejects the hypothesis that KING1 is the plant
counterpart to animal AMPKγ(see[12] for details). The positioning of the fungal sequences in both trees
(hatched clades) is at odds with the placement of fungi in the eukaryote species phylogeny (right tree). The log
likelihoods of the data given the ML tree and given a tree placing the fungal sequences at the expected
position are given below the respective trees.Seethe main text on tree topology testing for further details

Tracing AMPK Evolution 135