One of the most challenging evolutionary problems is locating the root of the eukaryote tree. We know that the root lies somewhere within the kingdom Protozoa, but the widespread view that early eukaryotes were amitochondrial has recently been dramatically overturned. Systematic biases in sequence evolution prevent the reliable inference of the eukaryote root from single‐gene trees. Concatenated sequence trees should be more reliable, but leave many possibilities open. In principle deletions/insertions or gene fusions should be superior for this purpose whenever ancestral and derived states are clearly distinguishable. Using a derived gene fusion between dihydrofolate reductase (DHFR) and thymidylate synthase (TS), genes we were able to greatly narrow down the position of the root. This gene fusion clearly divides eukaryotes into two clades: Amoebozoa plus opisthokonts (unikonts, which are ancestrally uniciliate) and bikonts, which are ancestrally biciliate. Another gene fusion between the first three enzymes of the six enzyme pyrimidine synthesis pathway supports this division. This derived three gene fusion is seen in Amoebozoa and opisthokonts (unikonts) but not in bikonts. A th
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