# Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns
## 3 Results  
### Different Tree Space Occupied by the 11 Segments  
Multi-dimensional scaling of the random, post-burn-in sampling of BEAST trees for each of the 11 segments revealed that segments 4 and 9 occupy distinct tree spaces from each other and the rest of the genome (Figure 2). Segments 10 and 11 occupied indistinguishable tree space indicating close geodesic distances and high levels of evolutionary linkage between them. Segments 3 (VP3), 5 (NSP1), and 6 (VP6) also shared highly overlapping tree space with one another. Segment 2's (VP2) evolutionary history was most like segment 6 (VP6). None of the segments overlapped with segment 1 (VP1) except for segment 7 (NSP3). Segment 7 also had the most phylogenetic uncertainty of all 11 segments as shown by the larger spread around the plot of the post-burn-in trees in Figure 1. The best-supported trees are depicted for segments 1-3 (Figure 3), segments 4-6, 9 (Figure 4), and segments 7, 8, 10, and 11 (Figure 5), with the host species coded on the tree and the branch lengths color-coded by relative evolutionary rate (Figure 3). While segment 4 had a more independent evolutionary history from other segments, its tree and AI/PS statistics suggested this segment has stricter host boundaries than segment 9 (Figure 4, Table 1), indicating either less opportunity for divergence due to selection responses to host species change or that segment 4 has a stronger role in host determination. The AI and PS statistics calculated from the post-burn-in random trees (Table 1, Figures 2-5) comparing human to non-human host spillover events, indicated segment 4 was least likely to switch hosts (AI = 1.23, PS = 11.71) while segment 9 was the most prone to switching hosts (AI = 3.83, PS = 25.80). The AI and PS statistic for separately tested non-human host phylogenies (SI files S24 and S25) using each specific host species as a state, was 3.20 for segment 4 and 4.18 for segment 9. A lower AI statistic indicates a stronger correlation between trait and phylogeny, so this result also indicates VP7 has less strict host boundaries relative to the other segments. We note that as the 11 trees used for the whole-genome comparison were under-sampled for non-human hosts, a larger analysis should be done with less-biased host sampling before confident conclusions can be formed regarding a comparison of relative host associations between all of the 11 segments. \begin{table}
\begin{tabular}{c c c c c} \hline
**Segment** & **Association Index (AI)** & _p-Value_ & **Parsimony Score (PS)** & _p-Value_ \\ \hline Segment 1 & 1.79 & 0.00 & 16.78 & 0.00 \\ Segment 2 & 1.97 & 0.00 & 18.63 & 0.00 \\ Segment 3 & 2.88 & 0.00 & 22.86 & 0.00 \\ Segment 4 & 1.23 & 0.00 & 11.71 & 0.00 \\ Segment 5 & 2.11 & 0.00 & 17.87 & 0.00 \\ Segment 6 & 2.68 & 0.00 & 19.19 & 0.00 \\ Segment 7 & 1.60 & 0.00 & 18.03 & 0.00 \\ Segment 8 & 1.89 & 0.00 & 19.66 & 0.00 \\ Segment 9 & 3.83 & 0.00 & 25.79 & 0.00 \\ Segment 10 & 2.44 & 0.00 & 17.26 & 0.00 \\ Segment 11 & 2.11 & 0.00 & 18.99 & 0.00 \\ Segment 4 * & 3.11 & 0.00 & 31.88 & 0.00 \\ Segment 9 * & 4.18 & 0.00 & 34.15 & 0.00 \\ \hline \end{tabular}
\end{table} 
Table 1: Association Index and Parsimony score statistics calculated in BaTS. The statistics for segments 1-11 was calculated based on 300 randomly sampled-post burn-in trees (Figures 3–5) in which states for each isolate were marked as either human or non-human host. The * next to segments 4 and 9 rows at the bottom of the table is indicating the statistics are for the separately made trees for segments 4 and 9, using all available complete sequences of both VP4 and VP7 available for non-human host isolates, with the AI and PS statistics being calculated using the specific host as the state for each isolate. Figure 2: Multi-dimensional scaling plot using 350 post-burn-in BEAST trees from each of the 11 RVA segments. Randomly sampled post-burn-in trees were taken from each segment to account for phylogenetic uncertainty. Points sharing the same color are from the same segment as shown in the legend.