Incomplete lineage sorting through Pachinko – a visual analogy

Reconstructing evolutionary history

Unravelling the evolutionary history of organisms – one of the main goals of phylogenetic research – remains a challenging prospect due to a number of theoretical and analytical aspects. Particularly, trying to reconstruct evolutionary patterns based on current genetic data (the most common way phylogenetic trees are estimated) is prone to the erroneous influence of some secondary factors. One of these is referred to as ‘incomplete lineage sorting’, which can have a major effect on how phylogenetic relationships are estimated and the statistical confidence we may have around these patterns. Today, we’re going to take a look at incomplete lineage sorting (shortened to ILS for brevity herein) using a game-based analogy – a Pachinko machine. Or, if you’d rather, the same general analogy also works for those creepy clown carnival games, but I prefer the less frightening alternative.

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Rebuilding the genomic architecture of evolution

Beyond mutations in the genome

Although genetic variation is, in itself, often considered to be one of the fundamental underpinnings of adaptation by natural selection, it can appear through a number of different forms. Typically, we think of genetic variation in terms of individual mutations at a single site (referred to as ‘single nucleotide polymorphisms’, or SNPs), which may vary in frequency across a population or species in response to selective pressures. However, we’ve also discussed some other types of genetic-related variation within The G-CAT before, such as differential gene expression or epigenetic markers.

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Evolutionary clocks out of sync

Evolutionary time

It shouldn’t come as a surprise to anyone with a basic understanding of evolution that it is a temporal (and also spatial concept). Time is a fundamental aspect of the process of evolution by natural selection, and without it evolution wouldn’t exist. But time is also a fickle thing, and although it remains constant (let’s not delve into that issue here) not all things experience it in the same way.

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Genes in parallel

Adaptation from genetic variation

One of the central themes of this blog, and indeed of evolutionary biology as a whole, is the notion that adaptation is often underpinned by genes. Genetic variation acts as the basis for natural selection to favour or disfavour traits: while this is directly through phenotypic traits (e.g. fur colour, morphology, behaviour), these traits are typically determined by a genetic component. In the early stages of adaptation, evolution can often be observed by changes in the frequency of genetic variants (alleles) within a species or population over time as natural selection acts, gradually leading to the observable (and sometimes dramatic) change in species over time.

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Changing the (water)course of history

The structure of a river system

For anyone who has had to study geography at some point in their education, you’d likely be familiar with the idea of river courses drawn on a map. They’re so important, in fact, that they are often the delimiting factor in the edges of countries, states or other political units. Water is a fundamental requirement of all forms of life and the riverways that scatter the globe underpin the maintenance, structure and accumulation of a large swathe of biodiversity.

So, what is a river?

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

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 purpleSource: 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.


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).

Drosophila speciation experiment
A simplified summary of the speciation experiment in Drosophila, starting with a single species and resulting in two reproductively isolated species based on mating and food preference. Source: Ilmari Karonen, adapted from here.

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.

Darwin's finches drawings
Some of Darwin’s early drawings of the morphological differences in Galapagos finch beaks, which lead to the formulation of the theory of evolution by natural selection.

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.

Zebra finches.jpg
A pair of (very cute) model zebra finches. Source: Michael Lawton via



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).

Zebrafish embryo
The transparent nature of zebrafish embryos make them ideal for studying the development of organisms in early stages. Source:

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.

Origination of adaptation: the old and the new (genes)

Adaptation is arguably the most critical biological process in the evolution of species. The process of evolution by natural selection is the cornerstone of evolutionary biology (and indeed, all of contemporary biology!) and adaptation remains fundamental to the process. We know that adaptation is based on the idea that some genetic variants are ‘better’ adapted than others, and thus are unequally shared across a population. But where does this genetic variation come from?

The accumulation of new genetic variation

The classic way for new genetic variants to appear is often thought of as mutation: changes in a single base in the DNA are caused by various external processes such as chemical, physical or environmental influences (such as the sci-fi classics like UV rays or toxic chemicals). Although these forms of mutations happen very rarely and certainly don’t have the same effects comic books would leave you to believe, new mutations can occur relatively rapidly depending on the characteristics of the species. However, the most common way for new mutations to occur is actually part of the DNA replication process: copying DNA is not always perfect and even though the relevant proteins essentially run a spellcheck, sometimes the copy is not 100% perfect and new mutations occur.

Adaptation of mutation figure
An example of how adaptation can occur from a new mutation. In this example, we have one gene (TTXTT), with initial only one allele (variant), TTATT. In the second generation (row), a mutation occurs in one individual which creates a new, second allele: TTGTT. This allele is favoured over the TTATT allele, and in the next generation it’s frequency increases as the alternative allele frequency decreases (the pattern is shown in the frequency values on the right side).

It is important to remember that only mutations that are present in the reproductive cells (sperm and eggs) can be inherited and passed on, and thus be a source for adaptation. Mutations in other tissues of the body, such as within the skin, are not spread across the entire body of the subject and thus aren’t passed on to offspring.

Standing genetic variation

Alternatively, genetic variation might already be present within a species or population. This is more likely if population sizes are large and populations are well connected and interbreeding. We refer to this diverse initial gene pool as ‘standing genetic variation’: that is, the amount of genetic variation within the population or species before the selective pressure requiring adaptation. Standing genetic variation can be thought of as the ‘diversity of choices’ for natural selection to act upon: the variants are readily available, and if a good choice exists it will be favoured by natural selection and become more widespread within the population or species (i.e. evolve).

Adaptation of standing variation figure.jpg
A slightly more complex example of how adaptation can occur from standing variation, this time with two different genes. One codes for fur colour, with two different alleles: GCATA codes for orange fur, and GCGTA codes for grey fur. The other gene codes for ear tufts, with TTCCT coding for tufts and TCCCT coding for no tufts. Natural selection favours both orange fur and tufted ears, and cats with these traits reproduce more frequently than those without (see graph below). These cats probably look familiar.

Graph of standing variation.jpg
The frequency of all four alleles (i.e. either allele for both genes) over the generations in the above figure. Clearly, we can see how adaptation rapidly favours orange fur and tufted ears over grey fur and non-tufted ears with the shifts in frequencies over the different alleles.

We’ve discussed standing genetic variation before on The G-CAT, but often in a different light (and phrasing). For example, when we’ve talked about founder effect: that is, when a population is formed from only a few different individuals which causes it to be very genetically depauperate. In populations under strong founder effect, there is very little standing genetic variation for natural selection to act upon. This has long been an enigma for many pest species: how have they managed to proliferate so widely when they often originate from so few individuals and lack genetic diversity?

Adaptive variation

Adaptation may not require new genetic variants to be generated from mutation. If there are a large number of alleles within the gene pool to start with, then natural selection may favour one of those variants over others and allow adaptation to start immediately. Compared to the rate at which new mutations occur, are potentially corrected for in DNA repair, are potentially erased by genetic drift, and then put under selective pressure, adaptation from standing genetic variation can occur very quickly.

Rate of adaptation figure.jpg
A rough example of the speed of adaptation depending on how the adaptive allele originated: whether it was already present (in the form of standing variation), or whether it was created by a new mutation. As one would expect, there is a significant lag delay in adaptation in the mutation scenario, based on the time it takes for said adaptive mutation to be created through relatively random processes. Thus, a positively selected allele from standing variation can allow a species to adapt much faster than waiting for a positive mutation to occur.

Conserving genetic variation

Given the adaptive potential provided by maintaining a good amount of standing genetic variation, it is imperative to conserve genetic diversity within populations in conservation efforts. This is why we often equate genetic diversity with ‘adaptive potential’ of species, although the exact amount of genetic diversity required for adaptive potential depends on a large number of other factors. Clearly, in some instances species show the ability to adapt to new pressures or novel environments even without a large amount of standing genetic variation.

It is important to remember that standing genetic variation consists of two types: neutral genetic diversity, which is not necessarily under selection at the time, and adaptive genetic diversity, which is directly under selection (although this can be either for or against the given variant). However, currently neutral genetic variants may become adaptive variants in the future if selective pressures change: although those different variants aren’t necessarily beneficial or detrimental at the moment, that may change in the future. Thus, conserving both types of genetic diversity is important for the survivability and longevity of populations under conservation programs.

Other types of adaptation

Although genetic diversity is clearly critically important for adaptive potential, alternative mechanisms for adaptation also exist. One of these relies less on the actual genetic variants being different, but rather how individual genes are used. This can happen in a few different ways, but mostly commonly this is through alternative splicing: when a gene is being ‘read’ and a protein is produced, different parts of the gene can be used (and in different order) to make a completely different protein.

Alternate splicing figure.jpg
An extreme example of alternate splicing of one gene. We start with a single gene, composed of 5 (AE) main gene elements (exons). Different environmental pressures (like fire risk, flooding, cold weather or predators, for example) cause the organism to use different combinations of these exons to make different proteins (right side; AD). Actual alternate splicing is not usually this straight-forward (one gene doesn’t conveniently split into four forms depending on the threat), but the process is generally the same.

Believe it or not, we’ve sort of discussed the effects of alternative splicing before. Phenotypic plasticity occurs when a single organism can have very different physiological traits depending on the environment: even though the genes are the same, they are utilised in different ways to make a different body shape. This is how some species can look incredibly different when they live in different places even if they’re genetically very similar. That said, for the vast majority of species maintaining good levels of genetic diversity is critical for the survivability of said species.

It takes (at least) two: coevoultion and species interactions

The environmental context of adaptation

We’ve talked many times before about how species evolve in response to some kind of environmental pressure, which favours (or disfavours) certain traits within that species. Over time, this drives changes in the frequencies of species traits and alters the overall average phenotype of that species (sometimes slowly, sometimes rapidly).

While we usually talk about the environment in terms of abiotic conditions such as temperature or climate, biotic factors are equally important: that is, the parts of the environment which are themselves also alive. Because of this, changes in one species can have profound repercussions on other species linked within the ecosystem. Thus, the evolution of one species is intrinsically linked to the evolution of other relevant species within the ecosystem: often, these connected evolutionary pathways battle with one another as each one changes. Let’s take a look at a few different examples of how evolution of one species may impact the evolution of another.

Predator-prey coevolution

One of the most obvious ways the evolution of two different species can interact is in predator and prey relationships. Naturally, prey species evolve to be able to defend themselves from predators in various ways, such as crypsis (e.g. camouflage), toxicity or behavioural changes (such as nocturnalism or group herding). Contrastingly, predators will evolve new and improved methods for detecting and hunting prey, such as enhanced senses, venom and stealth (through soft-padded feet, for example).

There are millions of possible examples of predator-prey coevolution that could be used as examples here, based on the continual drive for one species to get the upper hand over the other. But one that comes to mind is of a creature that I learnt about while on holiday in Scandinavia: the pine marten, and how it affects squirrels.

This photo is one that I took whilst on a lunch break at a bakery in the Norwegian mountains, of a small critter running among the rocks by the lakeside. Not sure exactly what species it was, I asked the tour director who excitedly told me that it was a pine marten. After doing a bit of research on them (and trying to figure out what the difference between a pine marten, a stoat, and a weasel is), I’ve discovered that it’s actually more likely to be a stoat than a pine marten, based on size and colour. But pine martens are still an intriguing species in their own right (and also found in Norway, so the confusion is understandable).

The pine marten is a species in the mustelid family, along with otters, weasels, stoats, and wolverines. Like many mustelids, they are carnivorous mammals which feed on a variety of different prey items like rodents, small birds and insects. One of the more abundant species that they prey upon are squirrels: both red squirrels and grey squirrels are potential food for the cute yet savage pine marten.

However, within the distribution of pine martens (across much of Europe), red squirrels are the native species and grey squirrels are invasive, originating from North America. Because of the long-lasting relationship between red squirrels and pine martens, they’ve co-evolved: most notably, by red squirrels changing to a mostly arboreal lifestyle and avoiding the ground as much as possible. Grey squirrels, however, have not had the evolutionary history to learn this lesson and are easy food for a smart pine marten. Thus, in regions where pine martens have been conserved or reintroduced, they are actively controlling the invasive grey squirrel population, which in turn boosts the native red squirrel population by reduction of competition. The coevolutionary link between red squirrels and pine martens is critical for combating the invasive species.


Martens and squirrels figure.jpg
The relationship between pine marten abundance and the abundance of both red (native) and grey (invasive) squirrels. On the left, without pine martens the invasive species runs rampant, outcompeting the native species. However, as pine martens increase in the ecosystem, the grey squirrels are predated on much more than the red squirrels due to their naivety, leading to the ‘natural’ balance on the right.

Martens and squirrels stats.jpg
A diagram of how the abundance of squirrels changes relative to the number of pine martens. The invasive grey squirrels are significantly depleted by pine marten presence, which in turn allows the native red squirrels to increase in population size after being freed from competition.

Host-parasite coevolution

In a similar vein to predator and prey coevolution, pathogenic species and their unfortunate hosts also undergo a sort of ‘arms race’. Parasites must keep evolving new ways to infect and transmit to hosts as the hosts evolve new methods of resisting and avoiding the infecting species. This spiralling battle of evolutionary forces is dubbed as the ‘Red Queen hypothesis’, formulated in 1973 by Leigh Van Valen and used to describe many other forms of coevolution. The name comes from Lewis Carroll’s Through the Looking Glass, and one quote in particular:

‘Now, here, you see, it takes all the running you can do, to keep in the same place’.

The quote references how species must continually adapt and respond to the evolution of other species just keep existing and prevent extinction. Species that remain static and stop evolving will inevitably go extinct as the world around them changes.


Plenty of other strange and unique mechanisms of coevolution exist within nature. One of them is mimicry, the process by which one species attempts to look like another to protect itself. The most iconic group known for this is butterflies: many species, although they may be evolutionarily very different, share similar colouration patterns and body shapes as mimics. Depending on the nature of the copy, mimicry can be classified into two broad categories. In either case, the initial ‘reference’ species is toxic or unpalatable to predators and uses a type of colour signal to communicate this: think of the bright yellow colours of bees and wasps or the red of ladybirds. Where the two categories change is in the nature of the ‘mimic’ species.

Müllerian mimicry

If the mimic is also toxic or unpalatable, we call this Müllerian mimicry (after Johann Friedrich Theodor Müller). By sharing the same colouration patterns and both being toxic, the two mimicking species boost the potential for the signal to be learnt by predators. If a predator eats either species, it will associate that colour pattern with toxicity and neither species are as likely to be preyed upon in the future. In this sense, it is a cooperative coevolutionary relationship between the two physically similar species.

Mullerian mimicry figure
A (somewhat familiar) example of Müllerian mimicry with two species of butterflies, the monarch and the viceroy. Although this has traditionally been thought of as a textbook case of Batesian mimicry (see below), the toxicity of both species likely makes it a scenario of Müllerian mimicry instead. Since both butterflies share the same pattern and both are toxic, it sends a strong signal to predators such as wasps to avoid them both.

Batesian mimicry

In contrast, the mimic might not actually be toxic or unpalatable, and simply copying a toxic species. This is referred to as Batesian mimicry (after Henry Walter Bates), and involves a mimic species relying on the association of colour and toxicity to have been learnt by predators through the ‘reference’ species. Although the mimic is not toxic, it is essentially piggy-backing on the hard evolutionary work that has already been done by the actually toxic species. In this case, the coevolutionary relationship is more parasitic as the mimic benefits from the ‘reference’ but the favour is not returned.

Batesian mimicry figure
An example of Batesian mimicry, with hoverflies and wasps. Hoverflies are not at all toxic, and are generally harmless; however, by mimicking the clear bright yellow warning systems of more dangerous species like wasps and bees, they avoid being eaten by predators such as birds.

Coevolution of species and the importance of species interactions

There are countless of other species interactions which could drive coevolutionary relationships in nature. These can include various forms of symbiosis, or the response of different species to ecosystem engineers: that is, species that can change and shape the environment around them (such as corals in reef systems). Understanding how a species evolves within its environment thus needs to consider how many other local species are also evolving and responding in their own ways.



Notes from the Field: Octoroks

Scientific name

Octorokus infletus

Meaning: Octorokus from [octorok] in Hylian; infletus from [inflate] in Latin.

Translation: inflating octorok; all varieties use an inflatable air sac derived from the swim bladder to float and scan the horizon.


Octorokus infletus hydros [aquatic morphotype]

Octorokus infletus petram [mountain morphotype]

Octorokus infletus silva [forest morphotype]

Octorokus infletus arctus [snow morphotype]

Octorokus infletus imitor [deceptive morphotype]

All octoroks.jpg
The various morphotypes of inflating octoroksA: The water octorok, considered the morphotype closest to the ancestral physiology of the species. B: The forest octorok, with grass camouflage. C: The deceptive octorok, which has replaced its tufted vegetation with a glittering chest as bait. D: The mountainous octorok, with rock camouflage. E: The snow octorok, with tundra grass camouflage.

Common name

Variable octorok

Taxonomic status

Kingdom Animalia; Phylum Mollusca; Class Cephalapoda; Order Octopoda; Family Octopididae; Genus Octorokus; Species infletus

Conservation status

Least Concern


The species is found throughout all major habitat regions of Hyrule, with localised morphotypes found within specific habitats. The only major region where the variable octorok is not found is within the Gerudo Desert, suggesting some remnant dependency of standing water.

Octorok distribution.jpg
The region of Hyrule, with the distribution of octoroks in blue. The only major region where they are not found is the Gerudo Desert in the bottom left.


Habitat choice depends on the physiology of the morphotype; so long as the environment allows the octorok to blend in, it is highly likely there are many around (i.e. unseen).

Behaviour and ecology

The variable octorok is arguably one of the most diverse species within modern Hyrule, exhibiting a large number of different morphotypic forms and occurring in almost all major habitat zones. Historical data suggests that the water octorok (Octorokus infletus hydros) is the most ancestral morphotype, with ancient literature frequently referring to them as sea-bearing or river-traversing organisms. Estimates from the literature suggests that their adaptation to land-based living is a recent evolutionary step which facilitated rapid morphological radiation of the lineage.

Several physiological characteristics unite the variable morphological forms of the octorok into a single identifiable species. Other than the typical body structure of an octopod (eight legs, largely soft body with an elongated mantle region), the primary diagnostic trait of the octorok is the presence of a large ‘balloon’ with the top of the mantle. This appears to be derived from the swim bladder of the ancestral octorok, which has shifted to the cranial region. The octorok can inflate this balloon using air pumped through the gills, filling it and lifting the octorok into the air. All morphotypes use this to scan the surrounding region to identify prey items, including attacking people if aggravated.

inflated octorok
A water morphotype octorok with balloon inflated.

Diets of the octorok vary depending on the morphotype and based on the ecological habitat; adaptations to different ecological niches is facilitated by a diverse and generalist diet.


Although limited information is available on the amount of gene flow and population connectivity between different morphotypes, by sheer numbers alone it would appear the variable octorok is highly abundant. Some records of interactions between morphotypes (such as at the water’s edge within forested areas) implies that the different types are not reproductively isolated and can form hybrids: how this impacts resultant hybrid morphotypes and development is unknown. However, given the propensity of morphotypes to be largely limited to their adaptive habitats, it would seem reasonable to assume that some level of population structure is present across types.

Adaptive traits

The variable octorok appears remarkably diverse in physiology, although the recent nature of their divergence and the observed interactions between morphological types suggests that they are not reproductively isolated. Whether these are the result of phenotypic plasticity, and environmental pressures are responsible for associated physiological changes to different environments, or genetically coded at early stages of development is unknown due to the cryptic nature of octorok spawning.

All octoroks employ strong behavioural and physiological traits for camouflage and ambush predation. Vegetation is usually placed on the top of the cranium of all morphotypes, with the exact species of plant used dependent on the environment (e.g. forest morphotypes will use grasses or ferns, whilst mountain morphotypes will use rocky boulders). The octorok will then dig beneath the surface until just the vegetation is showing, effectively blending in with the environment and only occasionally choosing to surface by using the balloon. Whether this behaviour is passed down genetically or taught from parents is unclear.

Management actions

Few management actions are recommended for this highly abundant species. However, further research is needed to better understand the highly variable nature and the process of evolution underpinning their diverse morphology. Whether morphotypes are genetically hardwired by inheritance of determinant genes, or whether alterations in gene expression caused by the environmental context of octoroks (i.e. phenotypic plasticity) provides an intriguing avenue of insight into the evolution of Hylian fauna.

Nevertheless, the transition from the marine environment onto the terrestrial landscape appears to be a significant stepping stone in the radiation of morphological structures within the species. How this has been facilitated by the genetic architecture of the octorok is a mystery.


From mutation to speciation: the genetics of species formation

The genetics of speciation

Given the strong influence of genetic identity on the process and outcomes of the speciation process, it seems a natural connection to use genetic information to study speciation and species identities. There is a plethora of genetics-based tools we can use to investigate how speciation occurs (both the evolutionary processes and the external influences that drive it). One clear way to test whether two populations of a particular species are actually two different species is to investigate genes related to reproductive isolation: if the genetic differences demonstrate reproductive incompatibilities across the two populations, then there is strong evidence that they are separate species (at least under the Biological Species Concept; see Part One for why!). But this type of analysis requires several tools: 1) knowledge of the specific genes related to reproduction (e.g. formation of sperm and eggs, genital morphology, etc.), 2) the complete and annotated genome of the species (to be able to find and analyse the right genes properly) and 3) a good amount of data for the populations in question. As you can imagine, for people working on non-model species (i.e. ones that haven’t had the same history and detail of research as, say, humans and mice), this can be problematic. So, instead, we can use other genetic information to investigate and suggest patterns and processes related to the formation of new species.

Is reproductive isolation naturally selected for or just a consequence?

A fundamental aspect of studies of speciation is a “chicken or the egg”-type paradigm: does natural selection directly select for rapid reproductive isolation, preventing interbreeding; or as a secondary consequence of general adaptive differences, over a long history of evolution? This might be a confusing distinction, so we’ll dive into it a little more.

Of the two proposed models of speciation, the by-product of natural selection (the second model) has been the more favoured. Simply put, this expands on Darwin’s theory of evolution that describes two populations of a single species evolving independently of one another. As these become more and more different, both in physical (‘phenotype’) and genetic (‘genotype’) characteristics, there comes a turning point where they are so different that an individual from one population could not reasonably breed with an individual from the other to form a fertile offspring. This could be due to genetic incompatibilities (such as different chromosome numbers), physiological differences (such as changes in genital morphology), or behavioural conflicts (such as solitary vs. group living).

Certainly, this process makes sense, although it is debatable how fast reproductive isolation would occur in a given species (or whether it is predictable just based on the level of differentiation between two populations). Another model suggests that reproductive isolation actually might arise very quickly if natural selection favours maintaining particular combinations of traits together. This can happen if hybrids between two populations are not particularly well adapted (fit), causing natural selection to favour populations to breed within each group rather than across groups (leading to reproductive isolation). Typically, this is referred to as ‘reinforcement’ and predominantly involves isolating mechanisms that prevent individuals across populations from breeding in the first place (since this would be wasted energy and resources producing unfit offspring). The main difference between these two models is the sequence of events: do populations ecologically diverge, and because of that then become reproductively isolated, or do populations selectively breed (enforcing reproductive isolation) and thus then evolve independently?

Reinforcement figure.jpg
An example of reinforcement leading to speciation. A) We start with two populations of a single species (a red fish population and a green fish population), which can interbreed (the arrows). B) Because these two groups can breed, hybrids of the two populations can be formed. However, due to the poor combination of red and green fish genes within a hybrid, they are not overly fit (the red cross). C) Since natural selection doesn’t favour forming hybrids, populations then adapt to selectively breed only with similar fish, reducing the amount of interbreeding that occurs. D) With the two populations effectively isolated from one another, different adaptations specific to each population (spines in red fish, purple stripes in green fish) can evolve, causing them to further differentiate. E) At some point in the differentiation process, hybrids move from being just selectively unfit (as in B)) to entirely impossible, thus making the two populations formal species. In this example, evolution has directly selected against hybrids first, thus then allowing ecological differences to occur (as opposed to the other way around).

Reproductive isolation through DMIs

The reproductive incompatibility of two populations (thus making them species) is often intrinsically linked to the genetic make-up of those two species. Some conflicts in the genetics of Population 1 and Population 2 may mean that a hybrid having half Population 1 genes and half Population 2 genes will have serious fitness problems (such as sterility or developmental problems). Dramatic genetic differences, particularly a difference in the number of chromosomes between the two sources, is a significant component of reproductive isolation and is usually to blame for sterile hybrids such as ligers, zorse and mules.

However, subtler genetic differences can also have a strong effect: for example, the unique combination of Population 1 and Population 2 genes within a hybrid might interact with one another negatively and cause serious detrimental effects. These are referred to as “Dobzhansky-Müller Incompatibilities” (DMIs) and are expected to accumulate as the two populations become more genetically differentiated from one another. This can be a little complicated to imagine (and is based upon mathematical models), but the basis of the concept is that some combinations of gene variants have never, over evolutionary history, been tested together as the two populations diverge. Hybridisation of these two populations suddenly makes brand new combinations of genes, some of which may be have profound physiological impacts (including on reproduction).

DMI figure
An example of how Dobzhansky-Müller Incompatibilities arise, adapted from Coyne & Orr (2004). We start with an initial population (center top), which splits into two separate populations. In this example, we’ll look at how 5 genes (each letter = one gene) change over time in the separate populations, with the original allele of the gene (lowercase) occasionally mutating into a new allele (upper case). These mutations happen at random times and in random genes in each population (the red letters), such that the two become very different over time. After a while, these two populations might form hybrids; however, given the number of changes in each population, this hybrid might have some combinations of alleles that are ‘untested’ in their evolutionary history (see below). These untested combinations may cause the hybrid to be infertile or unviable, making the two populations isolated species.

DMI table
The list of ‘untested’ genetic combinations from the above example. This table shows the different combinations of each gene that could be made in a hybrid if these two populations interbred. The red cells indicate combinations that have never been ‘tested’ together; that is, at no point in the evolutionary history of these two populations were those two particular alleles together in the same individual. Green cells indicate ones that were together at some point, and thus are expected to be viable combinations (since the resultant populations are obviously alive and breeding).

How can we look at speciation in action?

We can study the process of speciation in the natural world without focussing on the ‘reproductive isolation’ element of species identity as well. For many species, we are unlikely to have the detail (such as an annotated genome and known functions of genes related to reproduction) required to study speciation at this level in any case. Instead, we might choose to focus on the different factors that are currently influencing the process of speciation, such as how the environmental, demographic or adaptive contexts of populations plays a role in the formation of new species. Many of these questions fall within the domain of phylogeography; particularly, how the historical environment has shaped the diversity of populations and species today.

Phylogeo of speciation
An example of the interplay between speciation and phylogeography, taken from Reyes-Velasco et al. (2018). They investigated the phylogeographic history of several different groups of species within the frog genus Ptychadena; in this figure, we can see how the different species (indicated by the colours and tree on the left) relate to the geography of their habitat (right).

A variety of different analytical techniques can be used to build a picture of the speciation process for closely related or incipient species. A good starting point for any speciation study is to look at how the different study populations are adapting; is there evidence that natural selection is pushing these populations towards different genotypes or ecological niches? If so, then this might be a precursor for speciation, and we can build on this inference with other complementary analyses.

For example, estimating divergence times between populations can help us suggest whether there has been sufficient time for speciation to occur (although this isn’t always clear cut). Additionally, we could estimate the levels of genetic hybridisation (‘introgression’) between two populations to suggest whether they are reasonably isolated and divergent enough to be considered functional species.

The future of speciation genomics

Although these can help answer some questions related to speciation, new tools are constantly needed to provide a clearer picture of the process. Understanding how and why new species are formed is a critical aspect of understanding the world’s biodiversity. How can we predict if a population will speciate at some point? What environmental factors are most important for driving the formation of new species? How stable are species identities, really? These questions (and many more) remain elusive for a wide variety of life on Earth.