AMPK Methods and Protocols

duplicated gene (Fig.10d). If gene loss appears prevalent in

one subtree, it is worthwhile considering a tree reconstruction

artifact rather than a gene duplication (Fig.11;seeNote 18).

9. When a sequence tree does not follow your expectation
   (Fig.10), you can test whether it explains the data significantly
   better than the tree you were expecting [43]. For example, the
   ML tree in Fig.11 places the fungal sequences at positions that
   disagree with the commonly accepted eukaryote phylogeny.


10. To test whether this discrepancy is indeed significantly sup-
    ported by the data, modify a copy of the maximum likelihood
    tree such that it reflects the expected tree (seeNote 19).

(A)

Y 1 At 1 H 1? Ec 1

(B)

Yeast Human A. thaliana E. coli

Y 1 H 1 At 1 Ec 1

time time

(C) (D)

YY 11 aa HH 11 aa AAtt 11 aaYY 11 bbb HH 11 b AAtt 11 bb EEcc 11

Gene

duplication

S1

S3

S2

time

Gene

duplication

S1

S3

S2

time

YY 11 aa AAtt 11 aa HH 11 bb EEcc 11

S3

S2

S1

Fig. 10The evolution of species and of their genes. (a) The outer tree in black represents the evolutionary
relationships of the species, and we refer to it as thespecies tree. S1–S3 denote speciation events. The
sequence tree(syn.Gene tree) connecting the four proteins Y1, H1, At1, and Ec1 is shown in gray. As the
evolutionary lineages of the four proteins were separated by a speciation event rather than by a gene
duplication, we call themorthologs.The sequence tree connecting the four proteins is, thus, congruent to
the species tree. (b) Quite often, the reconstructed sequence tree deviates from the species tree. This can
indicate tree reconstruction artifacts resulting in sequence tree not accurately reflecting the phylogenetic
signal in the data. Alternatively, the sequences placed at unexpected positions might not be orthologous
(denoted here by the “?” that is appended to H1). (c) An idealized sequence tree of proteins which evolutionary
histories include a gene duplication (paralogs). Note that the sequence subtrees originating at the gene
duplication event each reflect the evolutionary relationships of the species that diversified after the gene
duplication event (black subtrees). (d) The minimal evolutionary scenario required to explain the gene tree in
(b). It invokes one gene duplication and three independent gene losses

134 Arpit Jain et al.