October 2018 – The G-cat

What’s the (allele) frequency, Kenneth?

October 24, 2018October 24, 2018thegcatblog5 Comments

Allele frequency

A number of times before on The G-CAT, we’ve discussed the idea of using the frequency of different genetic variants (alleles) within a particular population or species to test a number of different questions about evolution, ecology and conservation. These are all based on the central notion that certain forces of nature will alter the distribution and frequency of alleles within and across populations, and that these patterns are somewhat predictable in how they change.

One particular distinction we need to make early here is the difference between allele frequency and allele identity. In these analyses, often we are working with the same alleles (i.e. particular variants) across our populations, it’s just that each of these populations may possess these particular alleles in different frequencies. For example, one population may have an allele (let’s call it Allele A) very rarely – maybe only 10% of individuals in that population possess it – but in another population it’s very common and perhaps 80% of individuals have it. This is a different level of differentiation than comparing how different alleles mutate (as in the coalescent) or how these mutations accumulate over time (like in many phylogenetic-based analyses).

Allele freq vs identity figure.jpg — An example of the difference between allele frequency and identity. In this example (and many of the figures that follow in this post), the circle denote different populations, within which there are individuals which possess either an A gene (blue) or a B gene. **Left:** If we compared Populations 1 and 2, we can see that they both have A and B alleles. However, these alleles vary in their frequency within each population, with an equal balance of A and B in Pop 1 and a much higher frequency of B in Pop 2. **Right:** However, when we compared Pop 3 and 4, we can see that not only do they vary in frequencies, they vary in the presence of alleles, with one allele in each population but not the other.

Non-adaptive (neutral) uses

Testing neutral structure

Arguably one of the most standard uses of allele frequency data is the determination of population structure, one which more avid The G-CAT readers will be familiar with. This is based on the idea that populations that are isolated from one another are less likely to share alleles (and thus have similar frequencies of those alleles) than populations that are connected. This is because gene flow across two populations helps to homogenise the frequency of alleles within those populations, by either diluting common alleles or spreading rarer ones (in general). There are a number of programs that use allele frequency data to assess population structure, but one of the most common ones is STRUCTURE.

Gene flow homogeneity figure — An example of how gene flow across populations homogenises allele frequencies. We start with two initial populations (1 and 2 from above), which have very different allele frequencies. Hybridising individuals across the two populations means some alleles move from Pop 1 and Pop 2 into the hybrid population: which alleles moves is random (the smaller circles). Because of this, the resultant hybrid population has an allele frequency somewhere in between the two source populations: think of like mixing red and blue cordial and getting a purple drink.

Simple YPP structure figure.jpg — An example of a Structure plot which long-term *The G-CAT* readers may be familiar with. This is taken from Brauer *et al.* (2013), where the authors studied the population structure of the Yarra pygmy perch. Each small column represents a single individual, with the colours representing how well the alleles of that individual fit a particular genetic population (each population has one colour). The numbers and broader columns refer to different ‘localities’ (different from populations) where individuals were sourced. This shows clear strong population structure across the 4 main groups, except for in Locality 6 where there is a mixture of Eastern and Merri/Curdies alleles.

Determining genetic bottlenecks and demographic change

Other neutral aspects of population identity and history can be studied using allele frequency data. One big component of understanding population history in particular is determining how the population size has changed over time, and relating this to bottleneck events or expansion periods. Although there are a number of different approaches to this, which span many types of analyses (e.g. also coalescent methods), allele frequency data is particularly suited to determining changes in the recent past (hundreds of generations, as opposed to thousands of generations ago). This is because we expect that, during a bottleneck event, it is statistically more likely for rare alleles (i.e. those with low frequency) in the population to be lost due to strong genetic drift: because of this, the population coming out of the bottleneck event should have an excess of more frequent alleles compared to a non-bottlenecked population. We can determine if this is the case with tests such as the heterozygosity excess, M-ratio or mode shift tests.

Genetic drift and allele freq figure — A diagram of how allele frequencies change in genetic bottlenecks due to genetic drift. **Left:** Large circles again denote a population (although across different sequential times), with smaller circle denoting which alleles survive into the next generation (indicated by the coloured arrows). We start with an initial ‘large’ population of 8, which is reduced down to 4 and 2 in respective future times. Each time the population contracts, only a select number of alleles (or individuals) ‘survive’: assuming no natural selection is in process, this is totally random from the available gene pool. **Right:** We can see that over time, the frequencies of alleles A and B shift dramatically, leading to the ‘extinction’ of Allele B due to genetic drift. This is because it is the less frequent allele of the two, and in the smaller population size has much less chance of randomly ‘surviving’ the purge of the genetic bottleneck.

Adaptive (selective) uses

Testing different types of selection

We’ve also discussed previously about how different types of natural selection can alter the distribution of allele frequency within a population. There are a number of different predictions we can make based on the selective force and the overall population. For understanding particular alleles that are under strong selective pressure (i.e. are either strongly adaptive or maladaptive), we often test for alleles which have a frequency that strongly deviates from the ‘neutral’ background pattern of the population. These are called ‘outlier loci’, and the fact that their frequency is much more different from the average across the genome is attributed to natural selection placing strong pressure on either maintaining or removing that allele.

Other selective tests are based on the idea of correlating the frequency of alleles with a particular selective environmental pressure, such as temperature or precipitation. In this case, we expect that alleles under selection will vary in relation to the environmental variable. For example, if a particular allele confers a selective benefit under hotter temperatures, we would expect that allele to be more common in populations that occur in hotter climates and rarer in populations that occur in colder climates. This is referred to as a ‘genotype-environment association test’ and is a good way to detect polymorphic selection (i.e. when multiple alleles contribute to a change in a single phenotypic trait).

Genotype by environment figure.jpg — An example of how the frequency of alleles might vary under natural selection in correlation to the environment. In this example, the blue allele A is adaptive and under positive selection in the more intense environment, and thus increases in frequency at higher values. Contrastingly, the red allele B is maladaptive in these environments and decreases in frequency. For comparison, the black allele shows how the frequency of a neutral (non-adaptive or maladaptive) allele doesn’t vary with the environment, as it plays no role in natural selection.

Taxonomic (species identity) uses

At one end of the spectrum of allele frequencies, we can also test for what we call ‘fixed differences’ between populations. An allele is considered ‘fixed’ it is the only allele for that locus in the population (i.e. has a frequency of 1), whilst the alternative allele (which may exist in other populations) has a frequency of 0. Expanding on this, ‘fixed differences’ occur when one population has Allele A fixed and another population has Allele B fixed: thus, the two populations have as different allele frequencies (for that one locus, anyway) as possible.

Fixed differences are sometimes used as a type of diagnostic trait for species. This means that each ‘species’ has genetic variants that are not shared at all with its closest relative species, and that these variants are so strongly under selection that there is no diversity at those loci. Often, fixed differences are considered a level above populations that differ by allelic frequency only as these alleles are considered ‘diagnostic’ for each species.

Fixed differences figure.jpg — An example of the difference between fixed differences and allelic frequency differences. In this example, we have 5 cats from 3 different species, sequencing a particular target gene. Within this gene, there are three possible alleles: **T, A** or G respectively. You’ll quickly notice that the T allele is both unique to Species A and is present in all cats of that species (i.e. is fixed). This is a fixed difference between Species A and the other two. Alleles A and G, however, are present in both Species B and C, and thus are not fixed differences even if they have different frequencies.

Intrapopulation (relatedness) uses

Allele frequency-based methods are even used in determining relatedness between individuals. While it might seem intuitive to just check whether individuals share the same alleles (and are thus related), it can be hard to distinguish between whether they are genetically similar due to direct inheritance or whether the entire population is just ‘naturally’ similar, especially at a particular locus. This is the distinction between ‘identical-by-descent’, where alleles that are similar across individuals have recently been inherited from a similar ancestor (e.g. a parent or grandparent) or ‘identical-by-state’, where alleles are similar just by chance. The latter doesn’t contribute or determine relatedness as all individuals (whether they are directly related or not) within a population may be similar.

To distinguish between the two, we often use the overall frequency of alleles in a population as a basis for determining how likely two individuals share an allele by random chance. If alleles which are relatively rare in the overall population are shared by two individuals, we expect that this similarity is due to family structure rather than population history. By factoring this into our relatedness estimates we can get a more accurate overview of how likely two individuals are to be related using genetic information.

The wild world of allele frequency

Despite appearances, this is just a brief foray into the many applications of allele frequency data in evolution, ecology and conservation studies. There are a plethora of different programs and methods that can utilise this information to address a variety of scientific questions and refine our investigations.

Short essay: Real life or (‘just’) fantasy?

October 18, 2018thegcatblogLeave a comment

The fantastical

Like many people, from a young age I was obsessed and interested in works of fantasy and science fiction. To feel transported to magical worlds of various imaginative creatures and diverse places. The luxury of being able to separate from the mundanity of reality is one many children (or nostalgic adults) will be able to relate to upon reflection. Worlds that appear far more creative and engaging than our own are intrinsically enticing to the human psyche and the escapism it allows is no doubt an integral part of growing up for many people (especially those who have also dealt or avoided dealing with mental health issues).

The biological

The intricate connection to the (super)natural world drove me to fall in love with the natural world. Although there might seem to be an intrinsic contrast between the two – the absence or presence of reality – the truth is that the world is a wondrous place if you observe it through an appropriate lens. Dragons are real, forms of life are astronomically varied and imaginative, and there we are surrounded by the unknown and potentially mythical. To see the awe and mystification on a child’s face when they see a strange or unique animal for the very first time bears remarkable parallels to the expression when we stare into the fantasy of Avatar or The Lord of the Rings.

Combined dragon images — Two (very different) types of real life dragons. On the left, a terrifying dragon fish brought up from the abyssal depths by the CSIRO *RV Investigator* expedition. On the right, the minuscule but beautiful blue dragon (*Glaucus atlanticus*), which is actually a slug.

It might seem common for ‘nerds’ (at least under the traditional definition of being obsessed with particular aspects of pop culture) to later become scientists of some form or another. And I think this is a true reflection: particularly, I think the innate personality traits that cause one to look at the world of fantasy with wonder and amazement also commonly elicits a similar response in terms of the natural world. It is hard to see an example where the CGI’d majesty of contemporary fantasy and sci-fi could outcompete the intrigue generated by real, wondrous plants and animals.

Seeing the divine in the mundane

Although we often require a more tangible, objective justification for research, the connection of people to the diversity of life (whether said diversity is fictitious or not) should be a significant driving factor in the perceived importance of conservation management. However, we are often degraded to somewhat trivial discussions: why should we care about (x) species? What do they do for us? Why are they important?

Combined baobab images — Sometimes the ‘mundane’ (real) can inspire the ‘fantasy’… On the left, a real baobab tree (genus *Adansonia*: this one is *Adansonia grandidieri*) from Madagascar. On the right, the destructive baobab trees threaten to tear apart the prince’s planet in ‘*The Little Prince’* by Antoine de Saint-Exupéry.

If we approach the real world and the organisms that inhabit it with truly the same wonder as we approach the fantastical, would we be more successful in preserving biodiversity? Could we reverse our horrific trend of letting species go extinct? Every species on Earth represents something unique: a new perspective, an evolutionary innovation, a lens through which to see the world and its history. Even the most ‘mundane’ of species represent something critical to functionality of ecosystems, and their lack of emphasis undermines their importance.

Dementor wasp.png — …and sometimes, the fantasy inspires the reality. This is the dementor wasp (*Ampulex dementor*), named after the frightening creatures from the ‘*Harry Potter*‘ series. The name was chosen by the public based on the behaviour of the wasp to inject a toxin into its cockroach prey, which effectively turns them into mindless zombies and makes them unable to resist being pulled helplessly into the wasp’s nest. Absolutely terrifying.

The biota of Earth are no different to the magical fabled beasts of science fiction and fantasy, and we’re watching it all burn away right in front of our eyes.

You’re perfect, you’re beautiful, you look like a model (species)

October 11, 2018thegcatblog1 Comment

What is a ‘model’?

There are quite literally millions of species on Earth, ranging from the smallest of microbes to the largest of mammals. In fact, there are so many that we don’t actually have a good count on the sheer number of species and can only estimate it based on the species we actually know about. Unsurprisingly, then, the number of species vastly outweighs the number of people that research them, especially considering the sheer volumes of different aspects of species, evolution, conservation and their changes we could possibly study.

Species on Earth estimate figure — Some estimations on the number of eukaryotic species (i.e. not including things like bacteria), with the number of known species in blue and the predicted number of total species on Earth in purple. Source: Census of Marine Life.

This is partly where the concept of a ‘model’ comes into it: it’s much easier to pick a particular species to study as a target, and use the information from it to apply to other scenarios. Most people would be familiar with the concept based on medical research: the ‘lab rat’ (or mouse). The common house mouse (Mus musculus) and the brown rat (Rattus norvegicus) are some of the most widely used models for understanding the impact of particular biochemical compounds on physiology and are often used as the testing phase of medical developments before human trials.

So, why are mice used as a ‘model’? What actually constitutes a ‘model’, rather than just a ‘relatively-well-research-species’? Well, there are a number of traits that might make certain species ideal subjects for understanding key concepts in evolution, biology, medicine and ecology. For example, mice are often used in medical research given their (relative) similar genetic, physiological and behavioural characteristics to humans. They’re also relatively short-lived and readily breed, making them ideal to observe the more long-term effects of medical drugs or intergenerational impacts. Other species used as models primarily in medicine include nematodes (Caenorhabditis elegans), pigs (Sus scrofa domesticus), and guinea pigs (Cavia porcellus).

The diversity of models

There are a wide variety and number of different model species, based on the type of research most relevant to them (and how well it can be applied to other species). Even with evolution and conservation-based research, which can often focus on more obscure or cryptic species, there are several key species that have widely been applied as models for our understanding of the evolutionary process. Let’s take a look at a few examples for evolution and conservation.

Drosophila

It would be remiss of me to not mention one of the most significant contributors to our understanding of the genetic underpinning of adaptation and speciation, the humble fruit fly (Drosophila melanogaster, among other species). The ability to rapidly produce new generations (with large numbers of offspring with very short generation time), small fully-sequenced genome, and physiological variation means that observing both phenotypic and genotypic changes over generations due to ‘natural’ (or ‘experimental’) selection are possible. In fact, Drosphilia spp. were key in demonstrating the formation of a new species under laboratory conditions, providing empirical evidence for the process of natural selection leading to speciation (despite some creationist claims that this has never happened).

Darwin’s finches

The original model of evolution could be argued to be Darwin’s finches, as the formed part of the empirical basis of Charles Darwin’s work on the theory of evolution by natural selection. This is because the different species demonstrate very distinct and obvious changes in morphology related to a particular diet (e.g. the physiological consequences of natural selection), spread across an archipelago in a clear demonstration of a natural experiment. Thus, they remain the original example of adaptive radiation and are fundamental components of the theory of evolution by natural selection. However, surprisingly, Darwin’s finches are somewhat overshadowed in modern research by other species in terms of the amount of available data.

Zebra finches

Even as far as birds go, one species clearly outshines the rest in terms of research. The zebra finch is one of the most highly researched vertebrate species, particularly as a model of song learning and behaviour in birds but also as a genetic model. The full genome of the zebra finch was the second bird to ever be sequenced (the first being a chicken), and remains one of the more detailed and annotated genomes in birds. Because of this, the zebra finch genome is often used as a reference for other studies on the genetics of bird species, especially when trying to understand the function of genetic changes or genes under selection.

Fishes

Fish are (perhaps surprisingly) also relatively well research in terms of evolutionary studies, largely due to their ancient origins and highly diverse nature, with many different species across the globe. They also often demonstrate very rapid and strong bouts of divergence, such as the cichlid fish species of African lakes which demonstrate how new species can rapidly form when introduced to new and variable environments. The cichlids have become the poster child of adaptive radiation in fishes much in the same way that Darwin’s finches highlighted this trend in birds. Another group of fish species used as a model for similar aspects of speciation, adaptive divergence and rapid evolutionary change are the three-spine and nine-spine stickleback species, which inhabit a variety of marine, estuarine and freshwater environments. Thus, studies on the genetic changes across these different morphotypes is a key in understanding how adaptation to new environments occur in nature (particularly the relatively common transition into different water types in fishes).

cichlid diversity figure — The sheer diversity of species and form makes African cichlids an ideal model for testing hypotheses and theories about the process of evolution and adaptive radiation. Figure sourced from Brawand *et al.* (2014) in Nature.

Zebra fish

More similar to the medical context of lab rats is the zebrafish (ironically, zebra themselves are not considered a model species). Zebrafish are often used as models for understanding embryology and the development of the body in early formation given the rapid speed at which embryonic development occurs and the transparent body of embryos (which makes it easier to detect morphological changes during embryogenesis).

Using information from model species for non-models

While the relevance of information collected from model species to other non-model species depends on the similarity in traits of the two species, our understanding of broad concepts such as evolutionary process, biochemical pathways and physiological developments have significantly improved due to model species. Applying theories and concepts from better understood organisms to less researched ones allows us to produce better research much faster by cutting out some of the initial investigative work on the underlying processes. Thus, model species remain fundamental to medical advancement and evolutionary theory.

That said, in an ideal world all species would have the same level of research and resources as our model species. In this sense, we must continue to strive to understand and research the diversity of life on Earth, to better understand the world in which we live. Full genomes are progressively being sequenced for more and more species, and there are a number of excellent projects that are aiming to sequence at least one genome for all species of different taxonomic groups (e.g. birds, bats, fish). As the data improves for our non-model species, our understanding of evolution, conservation management and medical research will similarly improve.

Lost in a forest of (gene) trees

October 4, 2018thegcatblog3 Comments

Using genetics to understand species history

The idea of using the genetic sequences of living organisms to understand the evolutionary history of species is a concept much repeated on The G-CAT. And it’s a fundamental one in phylogenetics, taxonomy and evolutionary biology. Often, we try to analyse the genetic differences between individuals, populations and species in a tree-like manner, with close tips being similar and more distantly separated branches being more divergent. However, this runs on one very key assumption; that the patterns we observe in our study genes matches the overall patterns of species evolution. But this isn’t always true, and before we can delve into that we have to understand the difference between a ‘gene tree’ and a ‘species tree’.

A gene tree or a species tree?

Our typical view of a phylogenetic tree is actually one of a ‘gene tree’, where we analyse how a particular gene (or set of genes) have changed over time between different individuals (within and across populations or species) based on our understanding of mutation and common ancestry.

However, a phylogenetic tree based on a single gene only demonstrates the history of that gene. What we assume in most cases is that the history of that gene matches the history of the species: that branches in the genetic tree mirror when different splits in species occurred throughout history.

The easiest way to conceptualise gene trees and species trees is to think of individual gene trees that are nested within an overarching species tree. In this sense, individual gene trees can vary from one another (substantially, even) but by looking at the overall trends of many genes we can see how the genome of the species have changed over time.

Gene tree incongruence

Different genes may have different patterns for a number of reasons. Changes in the genetic sequences of organisms over time don’t happen equally across the entire genome, and very specific parts of the genome can evolve in entirely different directions, or at entirely different rates, than the rest of the genome. Let’s take a look at a few ways we could have conflicting gene trees in our studies.

Incomplete lineage sorting

One of the most prolific, but more complicated, ways gene trees can vary from their overarching species tree is due to what we call ‘incomplete lineage sorting’. This is based on the idea that species and the genes that define them are constantly evolving over time, and that because of this different genes are at different stages of divergence between population and species. If we imagine a set of three related populations which have all descended from a single ancestral population, we can start to see how incomplete lineage sorting could occur. Our ancestral population likely has some genetic diversity, containing multiple alleles of the same locus. In a true phylogenetic tree, we would expect these different alleles to ‘sort’ into the different descendent populations, such that one population might have one of the alleles, a second the other, and so on, without them sharing the different alleles between them.

If this separation into new populations has been recent, or if gene flow has occurred between the populations since this event, then we might find that each descendent population has a mixture of the different alleles, and that not enough time has passed to clearly separate the populations. For this to occur, sufficient time for new mutations to occur and genetic drift to push different populations to differently frequent alleles needs to happen: if this is too recent, then it can be hard to accurately distinguish between populations. This can be difficult to interpret (see below figure for a visualisation of this), but there’s a great description of incomplete lineage sorting here.

ILS_adaptedfigure — A demonstration of incomplete lineage sorting, generously adapted from a talk by fellow MELFU postdocs Dr Yuma (Jonathon) Sandoval-Castillo and Dr Catherine Attard. On the left is a depiction of a single gene coalescent tree over time: circles represent a single individual at a particular point in time (row) with the colours representing different alleles of that same gene. The tree shows how new mutations occur (colour changes along the branches) and spread throughout the descendent populations. In this example, we have three recently separated species, with a good number of different alleles. However, when we study these alleles in tree form (the phylogeny on the right), we see that the branches themselves don’t correlate well with the boundaries of the species. For example, the teal allele found within Species C is actually more similar to Species B alleles (purple and blue) than any other Species B alleles, based on the order and patterns of these mutations.

Hybridisation and horizontal transfer

Another way individual genes may become incongruent with other genes is through another phenomenon we’ve discussed before: hybridisation (or more specifically, introgression). When two individuals from different species breed together to form a ‘hybrid’, they join together what was once two separate gene pools. Thus, the hybrid offspring has (if it’s a first generation hybrid, anyway) 50% of genes from Species A and 50% of genes from Species B. In terms of our phylogenetic analysis, if we picked one gene randomly from the hybrid, we have 50% of picking a gene that reflects the evolutionary history of Species A, and 50% chance of picking a gene that reflects the evolutionary history of Species B. This would change how our outputs look significantly: if we pick a Species A gene, our ‘hybrid’ will look (genetically) very, very similar to Species A. If we pick a Species B gene, our ‘hybrid’ will look like a Species B individual instead. Naturally, this can really stuff up our interpretations of species boundaries, distributions and identities.

Hybridisation_figure — An example of hybridisation leading to gene tree incongruence with our favourite colourful fish. A) We have a hybridisation event between a red fish (Species A) and a green fish (Species B), resulting in a hybrid species (‘Species’ H). The red fish genome is indicated by the yellow DNA, the green fish genomes by the blue DNA, and the hybrid orange fish has a mixture of these two. B) If we sampled one set of genes in the hybrid, we might select a gene that originated from the red fish, showing that the hybrid is identical (or very similar) the Species A. D) Conversely, if we sampled a gene originating from the green fish, the resultant phylogeny might show that the hybrid is the same as Species B. C) If we consider these two patterns in combination, which see the true pattern of species formation, which is not a clear dichotomous tree and rather a mixture of the two sets of trees.

Paralogous genes

More confusingly, we can even have events where a single gene duplicates within a genome. This is relatively rare, although it can have huge effects: for example, salmon have massive genomes as the entire thing was duplicated! Each version of the gene can take on very different forms, functions, and evolve in entirely different ways. We call these duplicated variants paralogous genes: genes that look the same (in terms of sequence), but are totally different genes.

This can have a profound impact as paralogous genes are difficult to detect: if there has been a gene duplication early in the evolutionary history of our phylogenetic tree, then many (or all) of our study samples will have two copies of said gene. Since they look similar in sequence, there’s all possibility that we pick Variant 1 in some species and Variant 2 in other species. Being unable to tell them apart, we can have some very weird and abstract results within our tree. Most importantly, different samples with the same duplicated variant will seem similar to one another (e.g. have evolved from a common ancestor more recently) than it will to any sample of the other variant (even if they came from the exact same species)!

An example of how paralogous genes can confound species tree. We start with a single (purple) gene: at a particular point in time, this gene duplicates into a red and a blue form. Each of these genes then evolve and spread into four separate descendent species (A, B, C and D) but not in entirely the same way. However, since both the red and blue genetic sequences are similar, if we took a single gene from each species we might (somewhat randomly) sequence either the red or the blue copy. The different phylogenetic trees on the right demonstrate how different combinations of red and blue genes give very different patterns, since all blue copies will be more related to other blue genes than to the red gene of the same species. E.g. a blue A and a blue C are more similar than a blue A and a red A.

Overcoming incongruence with genomics

Although a tricky conundrum in phylogenetics and evolutionary genetics broadly, gene tree incongruence can largely be overcome with using more loci. As the random changes of any one locus has a smaller effect of the larger total set of loci, the general and broad patterns of evolutionary history can become clearer. Indeed, understanding how many loci are affected by what kind of process can itself become informative: large numbers of introgressed loci can indicate whether hybridisation was recent, strong, or biased towards one species over another, for example. As with many things, the genomic era appears poised to address the many analytical issues and complexities of working with genetic data.

The G-cat

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Month: October 2018