The importance of horizontal gene transfer (HGT) is likely understated in science. It was discussed only rarely in the past and, despite its current popularity and resurgence in relevance today, it continues to retain further secrets within the natural world that we have yet to uncover. We now openly know and have showcased over the past several decades just how relevant HGT is to bacteria, since it is one of the primary drivers of overall evolution and trait change across that entire kingdom of life, including in others like archaea. 

This has contributed to the formation of the idea of pan-genomes, the collection of all the genes found within a clade of life and often are generally the critical genes needed for life in general to function, with some additions as well. Because of just how widespread gene sharing is in bacteria, it is difficult in many ways to even classify them into related groups, as massive parts of their genomes may have been obtained from unrelated species. Comparatively, this sort of idea within eukaryotes and multi-cellular organisms has been largely dismissed, since it is commonly believed that horizontal gene transfer only has had a minimal impact on the development of such more “complex” organisms. 

But is that true?

Finding The Truth of HGT

Even today, the debates range on, with examples of that ongoing conflict being seen between Husnik and McCutcheon (2018) and Martin (2018). And now there is yet another entry into the fight, with evidence of HGT playing a rather major role in at least some forms of eukaryotic life. In this case, unicellular red algae species. The Cyanidiales are one of the oldest eukaryotic groups and in the habitats that they continue to thrive in, they make up almost all of the available biomass, showing the success of their evolutionary development. HGT events are expected to have played a fairly noticeable role in their evolution of extremophile living, giving them capabilities including low pH tolerance, high temperature tolerance, and resistance to both salinity and heavy metal poisoning. 

All of these things are why the collaborative research team at Heinrich Heine University in Germany and both Rutgers University and Arizona State University in the United States chose Cyanidiales as their model organism group for testing HGT impact on the evolutionary development of eukaryotic species. Though they took pains to note that there are limits on what they can discover. HGT differences themselves are naturally complicated to pick out from a genome as something not normally formed within it and there is always the possibility of contamination or misreading of produced data. The math and required settings for analysis are not trivial to set up either. 

Despite all that, the researchers did their best to account for any possible erroneous variables in their methods and worked to expand the data sets they had available to run their programs on. They ended up with 13 different Cyanidiales red algae lineages spread across 9 unrelated habitats. To truly prove things one way or the other, they had to not only find HGT-inserted genes and show that they are not random data artefacts, but also showcase that these genes had a cumulative evolutionary effect on genome development and weren’t just one off incidents. The primary goal in such a case would be to show that the HGTs weren’t just removed through differential loss processes, which would mean they were never actually integrated into the genome in the first place.

Absence Is Not Nothing

The problem is that there is plenty of past evidence, especially on the part of Martin et al, that there are no cumulative effects on eukaryotic genomes and no pan-genomes that can be constructed through physically shared genes. The absence of both of these would insinuate the lack of HGT as a relevant mechanism in eukaryotes. So to showcase HGTs as having a major effect on these genomes, both of these must be explained properly. Additionally, explaining a mechanism for eukaryotes to uptake these genes would also be required, but this has luckily already been done in red algae thanks to Lee et al (2016) and others.

The research team first used their data to find an explanation for the lack of a eukaryotic pan-genome in the algae. They found that HGT is indeed fairly rare in red algae species and accumulates at a rate of a dozen or more million years per a single incorporation in the population. Even so, it still occurs at a decent enough rate. But it is prone to gene erosion over time depending on if the trait is properly utilized or not. Because natural selection processes do not matter in regards to where a gene comes from, only the improvement of fitness applies, the keeping of an HGT depends on if it gives such an improvement. If not, then it may be eroded away or only achieve partial fixation within a population.

They grouped the orthologs, mostly matching genes in two species that came from a common ancestor, into those that were likely influenced by an HGT event, ending up with 96 such orthogroups. For those, only 8 of the HGTs were found to be included in all 13 of the observed species, with the rest only found in a sub amount of the 13. Also, several dozen of them were shown to have eroded over time, with all but 1 of the truly ancient HGT events being lost.

However, when bringing in the context of the individual species habitats and the breadth of how the species are spread out within them, 84 of the 96 orthogroups were seen to confer ecologically relevant and useful traits. Because they are strongly related to stress condition resistance, this increases the likelihood of these traits being picked up via HGT plasmids in the environment from bacterial species undergoing the same stresses in their habitats. This also explains why such events are so rare, they only become necessary to obtain in times of high stress and only one real trait is needed for each type of stress, keeping the pool of taken up genes low overall. 

Discovering True Understanding

This is why the eukaryotic algae pan-genome does not expand over time, because the species do not accumulate more and more HGTs over that time. They have no need to do so. And the ones they do pick up are strictly related to their personal habitats, meaning only a few are found to have been taken up by all of the species.

And to show cumulative effects, one only has to see how HGT derived genes diverge from their original sequence over time, diversifying and obtaining their own sequence identity separate from the gene’s source. Interestingly, the data set the researchers were working with did not show such a cumulative effect across the species. What instead they found was a separation between HGTs with a closer sequence to the species originally versus those HGTs that were more foreign in origin overall.

The closer ones did show cumulative effects toward the host genome, but the more divergent ones did not, no matter how ancient they were. This shows that there is a general separation between the sources of the HGTs picked up by Cyanidiales. This strange result led the research team to a main conclusion about HGTs in red algae. 

The general hypothesis is that two forces may be working separately on HGT to cause a change in what happens to picked up genes. Selective pressures for adapting against stresses with new traits and also the integration specifically of genes that have a compatibility with the eukaryotic replication systems so that they can be properly included in genomes. The problem is that the second requirement can’t alter genes that have critical components that make the genes functional, thus making it so that they can’t be significantly altered in sequence over time. However, this means that they can be eroded more easily and lost due to poor conservation.

An Expansive Incorporation

Overall, HGTs made up around 1% of the total genome of red algae species, showing that they play an important role in those genomes, contributing to their extremophile capabilities. However, in order to keep these traits intact, the incorporated genes cannot be altered much at all. This leaves them open to being lost over time and limits the cumulative effects that can be observed across the evolutionary history of these species and thus making a eukaryotic pan-genomes impossible to chart accurately. 

The researchers hope to continue their testing by moving from genome data to lab experiments where they will see if they can cause these bacterial HGTs to activate under stress conditions, proving their benefits. With a new three years of funding obtained from NASA, it’s likely the Rutgers-based part of the team will be able to find out some of those answers in the coming future.

Press Article

Study Article

Photo CCs: Serrated wrack, red hornweed and other algae from Wikimedia Commons

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