Experimental Evolution OF Evolution.

Evolution is something the vast majority of the 21st century agrees on.
TV shows like the big bang theory have created a cosy little bandwagon where after an 8 hour shift, Joe blow can switch off to the terminology and just go with the flow of the episode.

Knowledge of the basics and by extension their appreciation, is a luxury afforded to only those who sought it out in the first place. Without this, the ‘flow of the episode’ takes us where it wants us to go.

Going against the grain, and fighting against the flow of the episode is something that sets people apart, and this is not a concept lost on evolution itself.

We both know Joe blow isn’t going to be making any power moves in his life time, but what about his children?
If they are anything like their father, probably not. But if for some reason, their genes are expressed differently, they might just have the opportunity to ‘blow’ up the family name.

The world they grow up in will of course be different to their dad, but what if it wasn’t?
Would they still have the ability to change?
By how much would they change?

The study I chose to write this blog about asks that same question but in the context of Drosophila melanogaster fruit flies.

How does the expression of genes change when the environment is kept constant?
How does the expression of genes change when the environment itself changes regularly?

The Evolution of evolution guys yes.

Inception etc.

As a side note like any other dream, some parts are more memorable in the morning when you wake up than others, and my rendition of this paper works out a bit like that. Certain things have been left out for the greater good of the take home message. The take home message here is that he was stuck to the floor . . .  never mind the holes he knows are at the bottom of everyone’s feet. It’s deeper than that. Moooooving along . . .  .

To recreate the idea of our hypothetical Joe Blow, researchers used a field collected Drosophila Melanogaster raised on a standard corneal food to establish two other large populations. One was given the time to adapt to a salt-enriched diet and the other was given the time to adapt to a cadmium-rich diet.
A cross was made between both of these to create 20 smaller populations.

These were split into four different environments,

A Cadmium-rich diet and a Salt-rich diet where the flies were given either of the two every generation as a food source.
A temporally variable environment where flies were reared in alternating generations of Salt-rich followed by Cadmium-rich food.
And a Spatially variable environment where in each generation, half of the flies were fed on one diet and the other half on the other diet, separated from one another up until the point of mating.

Before a gene can become what it needs to be, it must first be written down like a post it note. At any given point in time researchers are able to take a snapshot of all the post it notes that are around in a cell before whoever needs to do what’s on them finally gets them done.

After 130 generations researchers took all the post it notes of the drosophila populations and compared them to see if they prioritized having certain things done more often than others.

This is what they called ‘RNA-seq data.’

This would be an example of what the post it notes would like in an inflamed leg muscle that is under exercise

In the same way the culture of the world would change between Joe Blow and his kids, the times and the place affected these genes within the drosophila. Different post it notes become more and more prevalent amongst the evolved populations.

When they compared the populations that were given Cadmium-rich OR Salt-rich food every generation, they found 546 genes that showed what they called a selection history effect.
This is essentially an evolved difference in the amount of a specific post it note relative to the other diets. 
In a previous study by the same researchers they measured how often certain alleles showed up between an ancestral salt-rich and cadmium-rich fed populations. These alleles are simply different versions of the same gene (before they are written down in a post it note). A bit like how you would get skim milk, soy milk, rice milk, oat milk, goat milk, and finally cow milk. It’s all drinkable but a little bit different. 

Combing that data set with their results from the current evolution experiment, they were able separate genes based on whether the particular variation of that allele was located in coding regions, noncoding regions, or located in the DNA sections between genes (intergenic).
They found that the genes that were expressed more (or had more post it notes) also were the same genes they noticed previously had higher numbers of variations in their intergenic regions. Why would a mutation that appears between genes affect them?
Cis acting factors. Like little poltergeists, you might not see them initially, but after months and months of confusing results and occurrences you’re left thinking somethings toying with you .

These are regions that are near-by certain genes which can impose a regulatory function, giving them an idea on just how many post it notes are left in the booklet, and how many they can afford to take out to write on.
The reoccurrence of mutations in these intergenic regions, together with the overlap of an increase in gene expression, suggests that cis acting factors contribute significantly to the evolved difference in the amount of post it notes between populations.
They next chose to examine whether the genes that have more post it notes in cadmium exposure relative to salt exposure in the Ancestral Populations, were also upregulated in the 5 constant Cad populations.

Using the Grand Ancestor population as a point of reference (because it was naïve to BOTH diets), researchers identified 905 genes that showed a significant change in expression when the flies are reared on cadmium-rich food compared to salt.

They then again checked for overlap with the 546 genes that showed that selection history effect from before.
108 genes overlapped between the two gene sets, and further computer analysis showed that they had a reoccurring theme of being involved with the cell membrane.
In 91% of these genes, their response in terms of how many post it notes they had was opposite and contradictory to what appeared in the ancestral populations.

That is to say for example, A gene with more post it notes in the Ancestral populations will have evolved to have less post it notes in the populations that have been given the time to adapt. 
It is an example of what they called counter gradient evolution.
Where the genetic influences on a trait, oppose the environmental influences, creating far less of a change than what you would expect from the environment.

We expect that in a new environment, there will be far much more to do i.e. – more post it notes to deal with the changes, but what researchers found was the opposite of that.
There were less post it notes for the same genes even though the environments were different.
There are two common reasons behind the emergence of a counter gradient pattern.
If natural selection favours the same amount of post it notes across all the environments, but one environment induces a change, then opposing genetic changes are expected to evolve from what was normal.

The other reason is related to the stress that is experienced by a population exposed to a new environment. We expect that this stress would cause different post it notes to appear in higher amounts to cope with the changes. The result would be an abnormal display as above.

Abnormal displays like this build character, and after enough of them you might fight yourself changed for the better.
Someone who had already adapted however, would not warrant the same stress response as someone who wasn’t ‘built for it’.
The likely scenario is that after the 5 Cadmium populations had been given the time to adapt to the diet, they no longer needed to exhibit this stress specific response that was seen in the Naïve Ancestor.
This is the reason behind there being less post it notes for the same genes between the populations that are stressed and those who are not, who have already adapted.

The level of change that could appear in the amount of post it notes for a particular gene set is what these researchers termed its expression plasticity. We would expect a higher expression plasticity for the diets that vary spatially and temporally, compared to the two that were fed consistently every generation.

The researchers could not test this however just using the complete set of post it notes from a given population. They needed to first, identify specific genes where they expected either an increase or decrease before they were written down.

To do this, the screened for genes that could meet two criteria.
First, they required a significant difference in the amount of post it notes from the optimal that is seen in the Ancestral Salt/Cadmium populations.
Second, they needed to exclude genes that naturally have high levels of change in their number of post it notes between populations.

109 genes passed this screening test and for each gene in each population they calculated the change across diets in such a way that a positive value indicates the post it notes changed adaptively.

For each population they averaged the values across all 109 genes to obtain a single measure of adaptive plasticity that they could then compare between diets.
The mean score for both the spatially and temporally varying diets was significantly greater than 0.
This same mean score for the consistent diets was practically zero.

Their hypothesis that adaptive changes arise more readily and to a greater extent in these heterogeneous environments was confirmed once they assessed these results.
The change in expression that has been measures thus far is not the same as measuring how adaptive expression is in either diet.

To do this, the researchers figured out a mathematical formula that allowed them to represent how far the expression levels of genes are from their optimum.
As before the formula equates to a number that is a unique distance from zero, where zero represents optimal gene expression.

The Metric Φ in the Temporally and spatially variable diets was close to 0 for both, indicating them being close to optimal expression – i.e. the perfect amount of post it notes for what needs doing in that environment.
The same metric in a consistently fed population that has been transferred to either of the variable diets, was found to be significantly higher and much further away from optimal expression.

Obviously because they haven’t had the time to adapt.

The patterns of counter gradient variation that they see represent evolutionary responses that attempt to restore just the right amount of post it notes to handle the situation.

If the Goldilocks was built to handle the different types of porridge, then we wouldn’t have had our beloved nursery rhyme. It’s fair to say that when she sampled each of them, a bit of counter gradient variation occurred. She stalled too long making the post it notes that she needed to digest the meal, fell asleep, and the rest is history.

Ok so enough about Wolves, Joe blows, T-rexes and Leonardo DiCaprio, the bottom line really is as expected. Environments that have spatial or temporal variation elicit adaptive responses in the individuals who are naive. If an organism is already adapted to an environment then its response in terms of post it notes will not be adaptive in nature. It will know exactly what to do, how much to do, and at what times. No problem.

They mentioned some of the limitations they noticed in retrospect after having conducted the study.
They chose to count post it notes only from very young drosophila larvae, and this offers with it a problem when you attempt to extrapolate across other developmental stages.
The collections of genes they used were all reasonably highly expressed and that they could have been more liberal with their thresholds. This is a reoccurring problem in expression studies, where accepting a few more false positives would have given a higher resolution picture and idea of the post it notes involved in the specific response.

They also only took a single snapshot of the post it notes at a single time point in the populations and had they taken multiple, they would have noticed the inconsistencies that arise – as the tasks on the post it notes are being performed.

Moving forward, follow up studied perhaps using the same drosophila populations should have a focus on understanding (1) Why plasticity occurs

(2) What is the main cascade of events and who are the star players that underlie adaptation – i.e. where is the quarterback??

(3) How these events relate back to the change in post it notes that eventually cause different behaviour patterns and hopefully speciation away from the Joe Blow Lineage.

Thanks for reading !


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8 Responses to Experimental Evolution OF Evolution.

  1. jcturnbullnz says:

    Holy metaphor, Batman!

    Thanks Jesse, this was an interesting way to explain concepts & describe critical thinking.

  2. msbdavies says:

    Hi Jesse

    I love the post-it-notes image you edited. How exactly did the mathematically calculations the optimal expression? That sounds really interesting!


    • jessealbany says:

      They did not so much calculate the optimal expression as much as they did the distance from it. It made no sense to me. I’m going to try an explain it the same way they did. And I am betting I wont do it any justice.


      This was the formula for it.
      Φ represents the relative distance to the optimum for expression.
      in the diet “d” of gene “i” in population “j”

      Φ,d,i, is the expression for the sample that represents the “optimal state” for that diet.
      N d,i, is the expression for the sample that represents the “Non-adapted” state, for that diet.

      They don’t mention the meaning of the ” E ” however.
      I think they contrasted the two states in relation to 0, and in relation to one another.

      Just because its probably worth more than my interpretation.

      This is the original paper, If you control + F
      “To further examine how selective history alters expression on genes of interest”
      you’ll find the section where they explain it.

  3. aearnshaw9 says:

    Hi Jesse,

    Really appreciate the metaphor as it made what you were explaining much clearer. Are you able to clarify whether if they looked at genes from older drosophila, would this show the same expression as younger larvae? And would lower expressed genes show different patterns?


    • jessealbany says:

      Hey Alyssa,

      The genes are qualitatively the same between ages but quantitatively different over the lifespan. The researchers may have just chosen larvae over mature flies because of the high metabolic rate.
      This article on PubMed is a liiiittle old but goes a bit more into that.

      Lower expressed genes just reiterate the same patterns showing the Heterogenous populations are more adapted to both diets compared to the naïve alternative.

  4. annabehlingnz says:

    Hi Jesse,
    As others have already commented, I too really enjoyed the post-it note analogy.
    I haven’t viewed your topic paper, but am wondering if you could shed some light on the reason that cadmium and salt diets were chosen as the two conditions, and whether you think the same effect would be observed with a range of experimental conditions (diet-based and beyond)? Thanks 🙂

  5. jessealbany says:

    Hey anna,

    I have been wondering why they chose these two diets, especially seeing as Cadmium is such a target of Bioremediation, – and is actually toxic to the flies.

    I think just like how the the beaks of darwins finches are extrapolated into the bigger picture of evolution, this scenario of drosophila serves in the same purpose.
    It isn’t so clear cut though, and I’d imagine once future experiments move outside of simply diet, they may end up find results that would disagree with the patterns in this Paper.

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