In recent years, DNA sequences have become one of the most widely used sources for constructing phylogenetic trees. How can we test the accuracy of these construction efforts, considering that evolutionary events occurred in the past, mostly without human witnesses? Phylogenetic trees represent hypotheses of evolutionary relationships that can be explicitly tested using data. The example described in this animation does just that.
A group of investigators set up an experiment to track the evolution of a bacterial virus, called bacteriophage T7, in the laboratory. With the history of the T7 lineages known, the investigators could determine if a phylogenetic construction (based on the DNA sequences of the viruses at the endpoints of the lineages) matched the known history of the lineages.
If phylogenetic trees represent reconstructions of past events without human witnesses, how can we test the accuracy of phylogenetic methods? Biologists have conducted experiments both in living organisms and with computer simulations that have demonstrated the accuracy of phylogenetic methods.
One such experiment employed a bacterial virus called bacteriophage T7. A single culture of the virus was used as a starting point, and lineages were allowed to evolve from this ancestral virus in the laboratory. On the culture dish, each round plaque represents a clone of genetically identical viruses.
The initial culture was split into two separate lineages, one of which became the ingroup for analysis, and the other became the outgroup for rooting the tree. The test tubes for growing the viruses contained bacteria in a culture broth that was laced with a DNA mutagen. The mutagen increased the frequency of DNA sequence changes from one generation of viruses to the next.
A T7 bacteriophage injects its DNA into a host cell of E. coli, resulting in the production of numerous progeny viruses and the lysis of the cell. In this experiment, approximately two generations were allowed to reproduce in a test tube.
Let's look at the ingroup. A small sample of viruses was used to inoculate successive fresh cultures of E. coli. After five such cultures, the last generation of viruses was incubated on Petri plates with E. coli. The resulting plaques are clearings in the lawn of bacteria, with each consisting of a pool of identical viruses.
A single plaque was picked to start a new culture. This procedure was repeated about 40 times, meaning that the viruses reproduced through roughly 400 generations, all while being exposed to a DNA mutagen. A single plaque from the final generation was then used to inoculate two new series of tubes.
We can simplify this flow chart and represent it with a phylogenetic tree. Each node signifies a point at which the lineages were split in two. The outgroup was not split into separate lineages.
The lineages of the ingroup were split after every 400 generations, and samples of the virus were saved for analysis at each branching point. The lineages were allowed to evolve until there were eight lineages in the ingroup and one in the outgroup.
The investigators then sequenced DNA from viruses at the endpoints of the eight lineages, as well as from the ancestors at the branching points. They gave the sequences from the endpoints to other investigators to analyze, without revealing the known history of the lineages or the sequences of the ancestral viruses.
Using the sequence information from the endpoints, the second group of investigators reconstructed the branching order of the lineages exactly as it had occurred. Additionally, the investigators reconstructed with 98% accuracy the nucleotide sequences of the ancestral viruses. This investigation clearly illustrates the validity of phylogenetic reconstruction methods.
In an experiment to test the accuracy of phylogenetic reconstruction methods, investigators followed the evolution of a bacterial virus as it reproduced over more than 1000 generations. The growth medium during these generations included a mutagen, which caused more nucleotide changes per replication cycle than normal, essentially speeding up evolutionary change. From this experiment, the investigators produced a known phylogenetic tree of 9 lineages. DNA sequences from the viruses at the endpoints of the lineages were given to another set of investigators, who used the sequence to reproduce an accurate phylogenetic tree of the viral lineages. Thus, this experiment provided a known phylogenetic tree that allowed the investigators to declare that their phylogenetic reconstruction was indeed accurate.