I came across this article as I was starting a new project for my course in Evolution. A previous blog post has already dealt with the topic, but I wanted to expand on this and show how a news story doesn’t always cover everything about an article. The original article can be found at https://peerj.com/preprints/13/ 

This article caught my attention partially due to the news story, and partially due to the topic. Modern-day evolution is something that a lot of people don’t see evidence for, but it IS occurring. I live in an urban environment, but I grew up in a rural one. I spent a lot of time doing research with normal lab mice. To me, it makes sense that the mice in a city would be different than the mice in the country, and few studies compare the two environments!

 The article began by talking about urban ‘exploiters’ versus ‘avoiders’: organisms that either adapt and thrive in cities, or ones that don’t. Either way, the construction and growth in cities massively disrupts normal habitats (water and food sources, nesting sites, etc.) and produces huge changes in the environment (pavement, city-treated drinking water, pollution, pesticides and herbicides, etc.). Some critters can deal with that, and some can’t. The question is why—what enables some to adapt, while others can’t? What genes might be involved?

To study that, we’d have to find an organism that lives in both the city and the country. Mice. Mice are great for that. Not your standard lab mice (Mus musculus), but a different type of mouse: the white-footed Peromyscus leucopus mouse.
The authors note that inside New York City, these mice have a lot of variation in genes that don’t seem to affect their ability to survive or reproduce (neutral loci). However, they don’t seem to migrate very often and some genetic differences between populations have been observed. “High genetic diversity within and low to nonexistent migration between most NYC populations suggests that selection can operate efficiently within these geographically isolated populations, either on standing genetic variation or de novo mutations.” (Harris, 2013). The topics of genetic diversity, migration, and mutation are key concepts in Evolution, and ones that we explore in detail during the course. Basically, some of the previous studies imply that city mice and country mice might be different.

The researchers went out and caught 89 white-footed mice from several locations in NYC: the New York Botanical Gardens, Central Park, Ridgewood Reservoir, and Flushing Meadows-Willow Lake. They also caught 22 country mice from Harriman State Park. Next, they isolated mRNA from the liver, brain, and gonads of the mice and copied it backwards into cDNA (normally, information goes from DNA to RNA).

First, they compared  their sequencing results to those of normal lab mice and lab rats (because we know a lot more about what their genes are & what they do).  They found about 11,355 genes that they could identify, and about 43% of those (6,385 genes) that actually made proteins that we know of.  The other 57% of the genes?  They may not have matched up well to lab mouse/lab rat genes, and not all genes code for proteins; for some genes, we still have no idea what they make or do.  Figure 3 of their paper showed the distribution of what they found.  The blue bars represent the total number of genes identified for each tissue (liver, testis, brain, and ovary).  The red bars are the genes that the researchers could identify based on known sequences that came from lab mice/rats.  Notice how the blue bars are usually taller than the red bars?  That’s because they got cDNA that didn’t match up to stuff we know about or stuff that matched lab critters.  That could actually include genes that are only found in (specific to) the white-footed mouse.  The green bars represent genes that actually make proteins–and again, the green bars are lower than the red bars, because a lot of genes still have no known function and many don’t code for proteins.  there are a couple of interesting features.  Take a look at the brain.  ALL of the genes found in the brain corresponded to known genes (freaky), but not all made proteins.  Also, take a look at the ovary. This organ was interesting because it had the closest bar heights–that means that most of the genes were identifiable, and most made proteins.  That kind of makes me curious about what’s going on in those blue bars for the genes they couldn’t identify!

Figure 3.  Annotation of final reference transcriptome.  Number of assembled P. leucopus contigs from four different tissue types that had significant hits with known proteins on BLASTX, and GO term annotations from reference databases using Blast2Go; Blue= Total number of contigs, Red = BLASTX hits, Green=number of annotated contigs.

Next, they tried to figure out whether the genes were unique to the tissue or whether they were just generic. For example, when they were looking at gonad tissue, were most of the genes involved in reproduction, or were they more like genes involved in glycolysis (that goes on in all our cells)?   Figure 4 showed a series of overlapping circles that did NOT help me understand anything.  However, these are supposed to represent genes that were really common in the tissue (like the brain) that were also really common in gonad, liver, or both.  Basically, what A tells us is that they found 69 genes that were really common in all 3 tissues, 62 that were common to brain and gonad, and 19 that were common to brain and liver.  I would expect these to be non-specific genes; ones that are probably involved in normal cell processes.  For the brain (A), the most common genes included genes for regulating behavior, actin binding, ion channel activity (which is involved in neurotransmission), motor activity (surprising, since the brain doesn’t move), and calcium ion binding.  For the liver (B), the most common genes dealt with metabolic processes like binding ATP and GTP, NADH dehydrogenase (converts NADH->NAD) and electron carrier activity.  This kind of implies that the liver is really metabolically active, and that matches up with what we know about anatomy and physiology.  The gonads (testis and/or ovary) had more genes related to reproduction, including cell division and cell cycle (to make lots-o-sperm), cilia (to move sperm, though you’d expect flagella here), and transcriptional and epigenetic regulation (gene expression stuff).

Figure 4.  Over-represented GO terms from pairwise comparisons (FDR ≤ 0.05). (a) Comparison of brain transcriptome to liver and gonad. (b) Comparison liver to brain and gonad. (c) Comparison of gonad to liver and brain.

Okay, so, the liver of white-footed mice seems to do metabolism, the brains of the mice seem to be weirdly motor-function-oriented (maybe because wild mice run a lot?) and the gonads make gametes.  Those aren’t exactly surprises, but we didn’t know much about the genetics of THESE mice to start off.  So what evidence do we have that these genes evolved?  For a gene to be selected FOR (a beneficial gene), we can get an idea of this based on the kinds of mutations we see. Some mutations are silent–they don’t change the amino acid or impact the protein. These are also called synonymous mutations (we cover this in my course!), or Ps.  Then there are other mutations that DO produce changes in the amino acids–and possibly in the proteins.  Those are non-synonymous mutations, or Pn.  If Pn/Ps > 1, then a gene is considered to be beneficial–under positive selection (natural selection is trying to keep that gene).  If Pn/Ps < 1, then the mutation is bad (deleterious), and natural selection is attempting to remove it from the population.

So all we have to do is count the number and types of mutations!  They found that some types of genes were definitely different between the city and country mice and had Pn/Ps > 1 (positive selection).  Some of these genes were involved in drug/pollution metabolism, and it makes sense for these to be preferred traits in an urban environment.  There were some differences in sperm production, too, for reasons that are less clear.  The proportions of mice they caught were about the same in urban and rural environments: about 53-54% male.  In class, we learn that more males tends to stimulate sperm production in chickens… but that wouldn’t account for a difference between city (53%) and country (54%) male mice.  They also found that at least one gene involved in epigenetic regulation was different: this gene helped remove methyl groups, which typically turn off or silence genes, and this seemed to be important for city mice.  They also found some genes that showed signs of being under negative selection (Pn/Ps < 1): these genes were involved in immune system function (there are different immune stressors in rural/urban environments), reproduction (different genes, but same system), and epigenetics (again, different genes, same system).  It was interesting to me that the immune system genes were for innate immunity, which is the non-specific immunity: things like allergies (think histamine) and inflammation.  Along with the drug & pollution metabolism differences, it makes sense that the immune system responses would also change.

Even within the city, though, the mice from different areas were different.  They found immune system and metabolic differences between the different populations (so, for example, Central Park mice were different from New York Botanical Garden mice).  That may make some sense, too; even within a city, there may be different food sources in different neighborhoods, different pollution levels and types, etc.

So, looking at all of that was an interesting experience for me.  I don’t know much about full-genome sequencing and I had to look up a lot of terms.  I had to try to interpret what I read and what their data showed, and it was tougher than I expected!  This review took me about a full day to do (which is longer than some), but the gist of what I get out of it is that the city versus country mice have differences in metabolism, reproduction (especially sperm production), and epigenetics.  I can’t tell if it’s enough to separate a species, but the reproductive changes and the epigenetic changes could eventually separate the two groups enough that they can’t interbreed–so I guess it’s possible.  More importantly, though, it shows that natural selection (the main force of evolution) is actively working on these mice–trying to keep some genes and trying to remove others, even in the same system!  Neat!