Ancestral and More Recently Acquired Syntenic Relationships of MADS-box Genes Uncovered by the
Physcomitrella patens Pseudochromosomal Genome Assembly
Plant Cell Reports
E. I. Barker N. W. Ashton
Department of Biology, University of Regina, Regina, SK, S4S 0A2, Canada
Author for correspondence:
Neil W. Ashton
e-mail: physcomitrella@gmail.com
OR3 New phylogenetic analysis of Physcomitrella patens MADS-box genes and pseudogenes
Supplementary Introduction
We previously constructed separate trees of type I, MIKCC and MIKC* MADS-box genes in
Physcomitrella and a poorly resolved composite tree of MADS-box genes from several plant taxa (Barker and
Ashton 2013). For our current analysis, we included the pseudogenes, PPTIM6 and PPMA5. Here, we present
the major results that are referred to in our current paper.
Supplementary Results
Weighted Maximum Parsimony (WMP) cladograms of Physcomitrella Type I, Type II (MIKCC) and
Type II (MIKC*) MADS-box genes are illustrated below.
The WMP tree of type I genes contained a clade of Mβ genes that was sister to a clade comprising two
subclades, one containing Mα genes and another containing three type I genes that cannot be classified as
belonging to any of the three major clades of angiosperm type I genes, Mα, Mβ and Mγ (Pařenicová et al.
2003). The pseudogene, PPTIM6, grouped with the Mβ genes. In the Bayesian and Maximum Likelihood (ML)
trees, the branch leading to the clades of Mα genes and unclassified genes was collapsed into a polytomy.
The WMP and Bayesian trees of type II MIKCC genes both contained two major clades of MIKCC
genes, one comprising PPM2 and its sister clade containing PPM1 and PpMADS1. The second clade contained
PpMADS-S as sister to a clade containing PPMC5 and PPMC6. In the ML tree, only the PPM2 clade was
resolved.
The WMP and Bayesian trees of type II MIKC* genes contained two major clades, which we designate
as the PpMADS2 clade and the PPMA9 clade. PPMA5, the pseudogene, grouped with PPM7. In the ML tree,
the branch leading to the PpMADS2-PPMA12 and PpMADS3-PPMA8 subclades was collapsed into a
polytomy.
WMP cladograms of Physcomitrella Type I, Type II (MIKCC) and Type II (MIKC*) MADS-box genes. The
main clades within each of these major groups are shaded differentially. Pseudogenes are designated by Ψ.
Bootstrap support values (or Bayesian posterior probabilities) are shown above (WMP) or below
(Bayesian/ML) branches.
Supplementary Material and Methods
For the phylogenetic tree of type I genes, we used 50 amino acid residues from the most conserved
middle portion of the encoded MADS domain of the Physcomitrella type I genes (except PPTIM6 which
possesses a truncated MADS-box) and the single MADS domain protein from each of two algal species,
Ostreococcus lucimarinus and O. tauri, that were used to root the tree. For the MIKCC tree rooted with the
MIKC* genes, PpMADS2 and PPM6, the complete protein sequences encoded by the MIKCC genes, along
with the MADS domain, a portion of the I domain and the extended K domain (Krogan and Ashton 2000)
encoded by the MIKC* genes were aligned in Clustal Omega (Sievers et al. 2011) and adjusted manually by
eye in Mesquite (Maddison and Maddison 2015). For the tree of MIKC* genes rooted with the MIKCC genes,
PPM1 and PPMC5, the complete protein sequences encoded by the MIKC* genes (except for the
polyglutamine tract of the C-terminal domain) and the N-terminal domain, the MADS domain, a portion of
the I domain and the extended K domain encoded by the MIKCC genes were aligned using the same procedure.
(All alignments are available upon request.) The DNA sequences were then aligned according to the protein
alignments. Model testing for the Bayesian and ML trees was performed using jModeltest 2.1.7 (Guindon and
Gascuel 2003; Darriba et al. 2012) and the default settings except Base Tree Search=Best. The Corrected
Akaike Information Criterion (Akaike 1974) was used to choose the best model (TPM2+I for the type I genes,
TIM3+G for the MIKCC genes and TrN+I+G for the MIKC* genes). WMP and ML trees were constructed in
PAUP 4.0 beta (Swofford 2002) using the default settings except that the initial trees were obtained by random
stepwise addition. WMP and ML trees were bootstrapped with 1000 replicates, with the exception that the ML
tree of MIKC* genes was bootstrapped with 100 replicates. Bayesian trees were constructed using MrBayes
(Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). For the type I, MIKCC and MIKC* trees,
50,000 generations, 300,000 generations and 1,000,000 generations respectively were run. Trees were
visualised in TreeGraph2 (Stöver et al. 2010).
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