# Validation of Automated Chromosome Recovery in the Reconstruction of Ancestral Gene Order
## 5 The Monocots  
The RACCROCHE method has been applied to six genomes drawn from a broad range of monocot orders:  
1. _Acorus calamus_ (sweet flag) from the order Acorales;
2. _Spirodela polyrhiza_ (duckweed) from the order Alismatales;
3. _Dioscora rotundata_ (yam) from the order Dioscorales;
4. _Asparagus officinalis_ (asparagus) from the order Aspargales;
5. _Elaes guiutensis_ (African oil palm) from the order Arecales;
6. _Ananas comosus_ (pineapple) from the order Poales.  
These species belong to lineages that diverged over 110 Mya. The phylogenetic relationship of the six extant species according to APG IV [14] is summarized in Figure 1, where ancestors (internal nodes) are labeled with numbers from 1 to 4 in chronological order, and the root is located between _Acorus_ and Ancestor 1.  
Almost all known flowering plant genomes have had at least one whole genome doubling or tripling event (WGD and WGT, respectively), with some often having several, in their lineages since the ancestral angiosperm. It is known that there were two WGDs between Ancestor 1 and _Spirodela_ and one WGD between each ancestor and its immediate  
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline
**Grayscale intensity** & 1 & 2 & 3 & 4 & 5 & 6 & 7 & & \\ \hline
**Proportion of pixels** & 50\% & 15\% & 10\% & 6\% & 4\% & 4\% & 4\% & 6.5\% & 0.5\% \\ \hline \end{tabular}
\end{table}
Table 1: The fixed proportion of pixels with its corresponding grayscale intensity in each data group shown in the heat maps.  
extant genome(s) in the tree, except _Spirodela_. The ancestral genomes reconstructed in [5], also confirmed the tetraploidization event \"tau\" [15] in the stem lineage between the alismatids (Acorales and Alismatales) and the lilioids (Dioscorales and Aspargales).  
The long period of evolutionary divergence and the complexity of the recurrent cycle of WGD and fractionation affecting these genomes represent a challenge for any ancestral genome reconstruction method. RACCROCHE calculates several measures of the quality of its reconstructions, including statistics on contig length and coherence between successive ancestors, and indicators of the numbers of different types of chromosomal rearrangement the reconstruction implies.  
The most telling evidence of the credibility of a reconstruction is the final chromosome structure it produces. The heat map visualization of RACCROCHE applied to the monocot data in Figure 2 shows a completely unambiguous clustering of the contigs into seven chromosomes of comparable size, for all four ancestors. Although this is gratifying, there are no genomes available from plants as old as these ancestors to verify the reconstruction.  
Figure 1: The phylogenetic relationship of the six extant monocot species according to APG IV is summarized in this phylogenetic tree, where ancestors (internal nodes) are labeled with numbers from 1 to 4. The root of this tree is between _Acorus_ and Ancestor 1.  
Figure 2: _Cont._  
Lacking ground truth about ancestral genomes dating from 100 Mya or more, one way we can assess the quality of reconstruction is through simulation, as discussed in Section 6. Thus, the main contribution from the current paper is a close simulation of monocot evolution, followed by an application of RACCROCHE to the simulated genomes, and a comparison of the results of the simulation input with the ancestor output by RACCROCHE.