An identity crisis: using genomics to determine species identities

This is the fourth (and final) part of the miniseries on the genetics and process of speciation. To start from Part One, click here.

In last week’s post, we looked at how we can use genetic tools to understand and study the process of speciation, and particularly the transition from populations to species along the speciation continuum. Following on from that, the question of “how many species do I have?” can be further examined using genetic data. Sometimes, it’s entirely necessary to look at this question using genetics (and genomics).

Cryptic species

A concept that I’ve mentioned briefly previously is that of ‘cryptic species’. These are species which are identifiable by their large genetic differences, but appear the same based on morphological, behavioural or ecological characteristics. Cryptic species often arise when a single species has become fragmented into several different populations which have been isolated for a long time from another. Although they may diverge genetically, this doesn’t necessarily always translate to changes in their morphology, ecology or behaviour, particularly if these are strongly selected for under similar environmental conditions. Thus, we need to use genetic methods to be able to detect and understand these species, as well as later classify and describe them.

Cryptic species fish
An example of cryptic species. All four fish in this figure are morphologically identical to one another, but they differ in their underlying genetic variation (indicated by the different colours of DNA). Thus, from looking at these fish alone we would not perceive any differences, but their genetic make-up might suggest that there are more than one species…
Cryptic species heatmap example
The level of genetic differentiation between the fish in the above example. The phylogenies on the left and top of the figure demonstrate the evolutionary relationships of these four fish. The matrix shows a heatmap of the level of differences between different pairwise comparisons of all four fish: red squares indicate zero genetic differences (such as when comparing a fish to itself; the middle diagonal) whilst yellow squares indicate increasingly higher levels of genetic differentiation (with bright yellow = all differences). By comparing the different fish together, we can see that Fish 1 and 2, and Fish 3 and 4, are relatively genetically similar to one another (red-deep orange). However, other comparisons show high level of genetic differences (e.g. 1 vs 3 and 1 vs 4). Based on this information, we might suggest that Fish 1 and 2 belong to one cryptic species (A) and Fish 3 and 4 belong to a second cryptic species (B).

Genetic tools to study species: the ‘Barcode of Life’

A classically employed method that uses DNA to detect and determine species is referred to as the ‘Barcode of Life’. This uses a very specific fragment of DNA from the mitochondria of the cell: the cytochrome c oxidase I gene, CO1. This gene is made of 648 base pairs and is found pretty well universally: this and the fact that CO1 evolves very slowly make it an ideal candidate for easily testing the identity of new species. Additionally, mitochondrial DNA tends to be a bit more resilient than its nuclear counterpart; thus, small or degraded tissue samples can still be sequenced for CO1, making it amenable to wildlife forensics cases. Generally, two sequences will be considered as belonging to different species if they are certain percentage different from one another.

Annotated mitogeome
The full (annotated) mitochondrial genome of humans, with the different genes within it labelled. The CO1 gene is labelled with the red arrow (sometimes also referred to as COX1) whilst blue arrows point to other genes often used in phylogenetic or taxonomic studies, depending on the group or species in question.

Despite the apparent benefits of CO1, there are of course a few drawbacks. Most of these revolve around the mitochondrial genome itself. Because mitochondria are passed on from mother to offspring (and not at all from the father), it reflects the genetic history of only one sex of the species. Secondly, the actual cut-off for species using CO1 barcoding is highly contentious and possibly not as universal as previously suggested. Levels of sequence divergence of CO1 between species that have been previously determined to be separate (through other means) have varied from anywhere between 2% to 12%. The actual translation of CO1 sequence divergence and species identity is not all that clear.

Gene tree – species tree incongruences

One particularly confounding aspect of defining species based on a single gene, and with using phylogenetic-based methods, is that the history of that gene might not actually be reflective of the history of the species. This can be a little confusing to think about but essentially leads to what we call “gene tree – species tree incongruence”. Different evolutionary events cause different effects on the underlying genetic diversity of a species (or group of species): while these may be predictable from the genetic sequence, different parts of the genome might not be as equally affected by the same exact process.

A classic example of this is hybridisation. If we have two initial species, which then hybridise with one another, we expect our resultant hybrids to be approximately made of 50% Species A DNA and 50% Species B DNA (if this is the first generation of hybrids formed; it gets a little more complicated further down the track). This means that, within the DNA sequence of the hybrid, 50% of it will reflect the history of Species A and the other 50% will reflect the history of Species B, which could differ dramatically. If we randomly sample a single gene in the hybrid, we will have no idea if that gene belongs to the genealogy of Species A or Species B, and thus we might make incorrect inferences about the history of the hybrid species.

Gene tree incongruence figure
A diagram of gene tree – species tree incongruence. Each individual coloured line represents a single gene as we trace it back through time; these are mostly bound within the limits of species divergences (the black borders). For many genes (such as the blue ones), the genes resemble the pattern of species divergences very well, albeit with some minor differences in how long ago the splits happened (at the top of the branches). However, the red genes contrast with this pattern, with clear movement across species (from and into B): this represents genes that have been transferred by hybridisation. The green line represents a gene affected by what we call incomplete lineage sorting; that is, we cannot trace it back far enough to determine exactly how/when it initially diverged and so there are still two separate green lines at the very top of the figure. You can think of each line as a separate phylogenetic tree, with the overarching species tree as the average pattern of all of the genes.

There are a number of other processes that could similarly alter our interpretations of evolutionary history based on analysing the genetic make-up of the species. The best way to handle this is simply to sample more genes: this way, the effect of variation of evolutionary history in individual genes is likely to be overpowered by the average over the entire gene pool. We interpret this as a set of individual gene trees contained within a species tree: although one gene might vary from another, the overall picture is clearer when considering all genes together.

Species delimitation

In earlier posts on The G-CAT, I’ve discussed the biogeographical patterns unveiled by my Honours research. Another key component of that paper involved using statistical modelling to determine whether cryptic species were present within the pygmy perches. I didn’t exactly elaborate on that in that section (mostly for simplicity), but this type of analysis is referred to as ‘species delimitation’. To try and simplify complicated analyses, species delimitation methods evaluate possible numbers and combinations of species within a particular dataset and provides a statistical value for which configuration of species is most supported. One program that employs species delimitation is Bayesian Phylogenetics and Phylogeography (BPP): to do this, it uses a plethora of information from the genetics of the individuals within the dataset. These include how long ago the different populations/species separated; which populations/species are most related to one another; and a pre-set minimum number of species (BPP will try to combine these in estimations, but not split them due to computational restraints). This all sounds very complex (and to a degree it is), but this allows the program to give you a statistical value for what is a species and what isn’t based on the genetics and statistical modelling.

Vittata cryptic species
The cryptic species of pygmy perches identified within my research paper. This represents part of the main phylogenetic tree result, with the estimates of divergence times from other analyses included. The pictures indicate the physiology of the different ‘species’: Nannoperca pygmaea is morphologically different to the other species of Nannoperca vittata. Species delimitation analysis suggested all four of these were genetically independent species; at the very least, it is clear that there must be at least 2 species of Nannoperca vittata since is more related to N. pygmaea than to other N. vittata species. Photo credits: N. vittata = Chris Lamin; N. pygmaea = David Morgan.

The end result of a BPP run is usually reported as a species tree (e.g. a phylogenetic tree describing species relationships) and statistical support for the delimitation of species (0-1 for each species). Because of the way the statistical component of BPP works, it has been found to give extremely high support for species identities. This has been criticised as BPP can, at time, provide high statistical support for genetically isolated lineages (i.e. divergent populations) which are not actually species.

Improving species identities with integrative taxonomy

Due to this particular drawback, and the often complex nature of species identity, using solely genetic information such as species delimitation to define species is extremely rare. Instead, we use a combination of different analytical techniques which can include genetic-based evaluations to more robustly assign and describe species. In my own paper example, we suggested that up to three ‘species’ of N. vittata that were determined as cryptic species by BPP could potentially exist pending on further analyses. We did not describe or name any of the species, as this would require a deeper delve into the exact nature and identity of these species.

As genetic data and analytical techniques improve into the future, it seems likely that our ability to detect and determine species boundaries will also improve. However, the additional supported provided by alternative aspects such as ecology, behaviour and morphology will undoubtedly be useful in the progress of taxonomy.

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.

 

What is a species, anyway?

This is Part 1 of a four part miniseries on the process of speciation; how we get new species, how we can see this in action, and the end results of the process. This week, we’ll start with a seemingly obvious question: what is a species?

The definition of a ‘species’

‘Species’ are a human definition of the diversity of life. When we talk about the diversity of life, and the myriad of creatures and plants on Earth, we often talk about species diversity. This might seem glaringly obvious, but there’s one key issue: what is a species, anyway? While we might like to think of them as discrete and obvious groups (a dog is definitely not the same species as a cat, for example), the concept of a singular “species” is actually the result of human categorisation.

In reality, the diversity of life is spread across a huge spectrum of differentiation: from things which are closely related but still different to us (like chimps), to more different again (other mammals), to hardly relatable at all (bacteria and plants). So, what is the cut-off for calling something a species, and not a different genus, family, or kingdom? Or alternatively, at what point do we call a specific sub-group of a species as a sub-species, or another species entirely?

This might seem like a simple question: we look at two things, and they look different, so they must be different species, right? Well, of course, nature is never simple, and the line between “different” and “not different” is very blurry. Here’s an example: consider that you knew nothing about the history, behaviour or genetics of dogs. If you simply looked at all the different breeds of dogs on Earth, you might suggest that there are hundreds of species of domestic dogs. That seems a little excessive though, right? In fact, the domestic dog, Eurasian wolf, and the Australian dingo are all the same species (but different subspecies, along with about 38 others…but that’s another issue altogether).

Dogs
Morphology can be misleading for identifying species. In this example, we have A) a dog, B) also a dog, C) still a dog, D) yet another dog, and E) not a dog. For the record, A-D are all Canis lupus of some variety; and are domestic dogs (Canis lupus familiaris), C is a dingo (Canis lupus dingo) and is a grey wolf (Canis lupus lupus). E, however, is the Ethiopian wolf, Canis simensis.

How do we describe species?

This method of describing species based on how they look (their morphology) is the very traditional approach to taxonomy. And for a long time, it seemed to work…until we get to more complex scenarios like the domestic dog. Or scenarios where two species look fairly similar, but in reality have evolved entirely differently for a very, very long time. Or groups which look close to more than one other species. So how do we describe them instead?

Cats and foxes
A), a fox. B), a cat. C), a foxy cat? A catty fox? A cat-fox hybrid? Something unrelated to cat or a fox?

 

Believe it or not, there are dozens of ways of deciding what is a species and what isn’t. In Speciation (2004), Coyne & Orr count at least 25 different reported Species Concepts that had been suggested within science, based on different requirements such as evolutionary history, genetic identity, or ecological traits. These different concepts can often contradict one another about where to draw the line between species…so what do we use?

The Biological Species Concept (BSC)

The most commonly used species concept is called the Biological Species Concept (BSC), which denotes that “species are groups of interbreeding natural populations that are reproductively isolated from other such groups” (Mayr, 1942). In short, a population is considered a different species to another population if an individual from one cannot reliably breed to form fertile, viable offspring with an individual from the other. We often refer to this as “reproductive isolation.” It’s important to note that reproductive isolation doesn’t mean they can’t breed at all: just that the hybrid offspring will not live a healthy life and produce its own healthy offspring.

For example, a horse and zebra can breed to produce a zorse, however zorse are fundamentally infertile (due to the different number of chromosomes between a horse and a zebra) and thus a horse is a different species to a zebra. However, a German Shepherd and a chihuahua can breed and make a hybrid mutt, so they are the same species.

zorse
A zorse, which shows its hybrid nature through zebra stripes and horse colouring. These two are still separate species since zorses are infertile, and thus are not a singular stable entity.

You might naturally ask why reproductive isolation is apparently so important for deciding species. Most directly, this means that groups don’t share gene pools at all (since genetic information is introduced and maintained over time through breeding events), which causes them to be genetically independent of one another. Thus, changes in the genetic make-up of one species shouldn’t (theoretically) transfer into the gene pool of another species through hybrids. This is an important concept as the gene pool of a species is the basis upon which natural selection and evolution act: thus, reproductively isolated species may evolve in very different manners over time.

RI example
An example of how reproductive isolation maintains genetic and evolutionary independence of species. In A), our cat groups are robust species, reproductively isolated from one another (as shown by the black box). When each species undergoes natural selection and their genetic variation changes (colour changes on the cats and DNA), these changes are kept within each lineage. This contrasts to B), where genetic changes can be transferred between species. Without reproductive isolation, evolution in the orange lineage and the blue lineage can combine within hybrids, sharing the evolutionary pathways of both ancestral species.

Pitfalls of the BSC

Just because the BSC is the most used concept doesn’t make it infallible, however. Many species on Earth don’t easily demonstrate reproductive isolation from one another, nor does the concept even make sense for asexually reproducing species. If an individual reproduced solely asexually (like many bacteria, or even some lizards), then by the BSC definition every individual is an entirely different species…which seems a little excessive. Even in sexually reproducing organisms, it can be hard to establish reproductive isolation, possibly because the species never come into contact physically.

This raises the debate of whether two species could, let alone will, hybridise in nature, which can be difficult to determine. And if two species do produce hybrid offspring, assessing their fertility or viability can be difficult to detect without many generations of breeding and measurements of fitness (hybrids may not be sustainable in nature if they are not well adapted to their environment and thus the two species are maintained as separate identities).

Hybrid birds
An example of unfit hybrids causing effective reproductive isolation. In this example, we have two different bird species adapted to very different habitats; a smaller, long-tailed bird (left) adapted to moving through dense forest, and a large, longer-legged bird (right) adapted to traversing arid deserts. When (or if) these two species hybridised, the resultant offspring would be middle of the road, possessing too few traits to be adaptive in either the forest or the desert and no fitting intermediate environment available. Measuring exactly how unfit this hybrid would be is a difficult task in establishing species boundaries.

 

Integrative taxonomy

To try and account for the issues with the BSC, taxonomists try to push for the usage of “integrative taxonomy”. This means that species should be defined by multiple different agreeing concepts, such as reproductive isolation, genetic differentiation, behavioural differences, and/or ecological traits. The more traits that can separate the two, the greater support there is for the species to be separated: if they disagree, then more information is needed to determine exactly whether or not that should be called different species. Debates about taxonomy are ongoing and are likely going to be relevant for years to come, but form critical components of understanding biodiversity, patterns of evolution, and creating effective conservation legislation to protect endangered or threatened species (for whichever groups we decide are species).

 

How did pygmy perch swim across the desert?

“Pygmy perch swam across the desert”

As regular readers of The G-CAT are likely aware, my first ever scientific paper was published this week. The paper is largely the results of my Honours research (with some extra analysis tacked on) on the phylogenomics (the same as phylogenetics, but with genomic data) and biogeographic history of a group of small, endemic freshwater fishes known as the pygmy perch. There are a number of different messages in the paper related to biogeography, taxonomy and conservation, and I am really quite proud of the work.

Southern_pygmy_perch 1 MHammer
A male southern pygmy perch, which usually measures 6-8 cm long.

To my honest surprise, the paper has received a decent amount of media attention following its release. Nearly all of these have focused on the biogeographic results and interpretations of the paper, which is arguably the largest component of the paper. In these media releases, the articles are often opened with “…despite the odds, new research has shown how a tiny fish managed to find its way across the arid Australian continent – more than once.” So how did they manage it? These are tiny fish, and there’s a very large desert area right in the middle of Australia, so how did they make it all the way across? And more than once?!

 The Great (southern) Southern Land

To understand the results, we first have to take a look at the context for the research question. There are seven officially named species of pygmy perches (‘named’ is an important characteristic here…but we’ll go into the details of that in another post), which are found in the temperate parts of Australia. Of these, three are found with southwest Western Australia, in Australia’s only globally recognised biodiversity hotspot, and the remaining four are found throughout eastern Australia (ranging from eastern South Australia to Tasmania and up to lower Queensland). These two regions are separated by arid desert regions, including the large expanse of the Nullarbor Plain.

Pygmyperch_distributionmap
The distributions of pygmy perch species across Australia. The dots and labels refer to different sampling sites used in the study. A: the distribution of western pygmy perches, and essentially the extent of the southwest WA biodiversity hotspot region. B: the distribution of eastern pygmy perches, excluding N. oxleyana which occurs in upper NSW/lower QLD (indicated in C). C: the distributions relative to the map of Australia. The black region in the middle indicates the Nullarbor Plain. 

 

The Nullarbor Plain is a remarkable place. It’s dead flat, has no trees, and most importantly for pygmy perches, it also has no standing water or rivers. The plain was formed from a large limestone block that was pushed up from beneath the Earth approximately 15 million years ago; with the progressive aridification of the continent, this region rapidly lost any standing water drainages that would have connected the east to the west. The remains of water systems from before (dubbed ‘paleodrainages’) can be seen below the surface.

Nullarbor Plain photo
See? Nothing here. Photo taken near Watson, South Australia. Credit: Benjamin Rimmer.

Biogeography of southern Australia

As one might expect, the formation of the Nullarbor Plain was a huge barrier for many species, especially those that depend on regular accessible water for survival. In many species of both plants and animals, we see in their phylogenetic history a clear separation of eastern and western groups around this time; once widely distributed species become fragmented by the plain and diverged from one another. We would most certainly expect this to be true of pygmy perch.

But our questions focus on what happened before the Nullarbor Plain arrived in the picture. More than 15 million years ago, southern Australia was a massively different place. The climate was much colder and wetter, even in central Australia, and we even have records of tropical rainforest habitats spreading all the way down to Victoria. Water-dependent animals would have been able to cross the southern part of the continent relatively freely.

Biogeography of the enigmatic pygmy perches

This is where the real difference between everything else and pygmy perch happens. For most species, we see only one east and west split in their phylogenetic tree, associated with the Nullarbor Plain; before that, their ancestors were likely distributed across the entire southern continent and were one continuous unit.

Not for pygmy perch, though. Our phylogenetic patterns show that there were multiple splits between eastern and western ancestral pygmy perch. We can see this visually within the phylogenetic tree; some western species of pygmy perches are more closely related, from an evolutionary perspective, to eastern species of pygmy perches than they are to other western species. This could imply a couple different things; either some species came about by migration from east to west (or vice versa), and that this happened at least twice, or that two different ancestral pygmy perches were distributed across all of southern Australia and each split east-west at some point in time. These two hypotheses are called “multiple invasion” and “geographic paralogy”, respectively.

MCC_geographylabelled
The phylogeny of pygmy perches produced by this study, containing 45 different individuals across all species of pygmy perch. Species are labelled in the tree in brackets, and their geographic location (east or west) is denoted by the colour on the right. This tree clearly shows more than one E/W separation, as not all eastern species are within the same clade. For example, despite being an eastern species, N. variegata is more closely related to Nth. balstoni or N. vittata than to the other eastern species (N. australisN. obscuraN. oxleyana and N. ‘flindersi’.

So, which is it? We delved deeper into this using a type of analysis called ‘ancestral clade reconstruction’. This tries to guess the likely distributions of species ancestors using different models and statistical analysis. Our results found that the earliest east-west split was due to the fragmentation of a widespread ancestor ~20 million years ago, and a migration event facilitated by changing waterways from the Nullarbor Plain pushing some eastern pygmy perches to the west to form the second group of western species. We argue for more than one migration across Australia since the initial ancestor of pygmy perches must have expanded from some point (either east or west) to encompass the entirety of southern Australia.

BGB_figure
The ancestral area reconstruction of pygmy perches, estimated using the R package BioGeoBEARS. The different pie charts denote the relative probability of the possible distributions for the species or ancestor at that particular time; colours denote exactly where the distribution is (following the legend). As you can see, the oldest E/W split at 21 million years ago likely resulted from a single widespread ancestor, with it’s range split into an east and west group. The second E/W event, at 15 million years ago, most likely reflects a migration from east to west, resulting in the formation of the N. vittata species group. This coincides with the Nullarbor Plain, so it’s likely that changes in waterway patterns allowed some eastern pygmy perch to move westward as the area became more arid.

So why do we see this for pygmy perch and no other species? Well, that’s the real mystery; out of all of the aquatic species found in southeast and southwest Australia, pygmy perch are one of the worst at migrating. They’re very picky about habitat, small, and don’t often migrate far unless pushed (by, say, a flood). It is possible that unrecorded extinct species of pygmy perch might help to clarify this a little, but the chances of finding a preserved fish fossil (let alone for a fish less than 8cm in size!) is extremely unlikely. We can really only theorise about how they managed to migrate.

Pygmy perch biogeo history
A diagram of the distribution of pygmy perch species over time, as suggested by the ancestral area reconstruction. A: the initial ancestor of pygmy perches was likely found throughout southern Australia. B: an unknown event splits the ancestor into an eastern and western group; the sole extant species of the W group is Nth. balstoniC: the ancestor of the eastern pygmy perches spreads towards the west, entering part of the pre-Nullarbor region. D: due to changes in the hydrology of the area, some eastern pygmy perches (the maroon colour in C) are pushed towards the west; these form N. vittata species and N. pygmaea. The Nullarbor Plain forms and effectively cuts off the two groups from one another, isolating them.

What does this mean for pygmy perches?

Nearly all species of pygmy perch are threatened or worse in the conservation legislation; there have been many conservation efforts to try and save the worst-off species from extinction. Pygmy perches provide a unique insight to the history of the Australian climate and may be a key in unlocking some of the mysteries of what our land was like so long ago. Every species is important for conservation and even those small, hard-to-notice creatures that we might forget about play a role in our environmental history.

The direction of evolution: divergence vs. convergence

Direction of evolution

We’ve talked previously on The G-CAT about how the genetic underpinning of certain evolutionary traits can change in different directions depending on the selective pressure it is under. Particularly, we can see how the frequency of different alleles might change in one direction or another, or stabilise somewhere in the middle, depending on its encoded trait. But thinking bigger picture than just the genetics of one trait, we can actually see that evolution as an entire process works rather similarly.

Divergent evolution

The classic view of the direction of evolution is based on divergent evolution. This is simply the idea that a particular species possess some ancestral trait. The species (or population) then splits into two (for one reason or another), and each one of these resultant species and populations evolves in a different way to the other. Over time, this means that their traits are changing in different directions, but ultimately originate from the same ancestral source.

Evidence for divergent evolution is rife throughout nature, and is a fundamental component of all of our understanding of evolution. Divergent evolution means that, by comparing similar traits in two species (called homologous traits), we can trace back species histories to common ancestors. Some impressive examples of this exist in nature, such as the number of bones in most mammalian species. Humans have the same number of neck bones as giraffes; thus, we can suggest that the ancestor of both species (and all mammals) probably had a similar number of neck bones. It’s just that the giraffe lineage evolved longer bones whereas other lineages did not.

Homology figure
A diagrammatic example of homologous structures in ‘hand’ bones. The coloured bones demonstrate how the same original bone structures have diverged into different forms. Source: BiologyWise.

Convergent evolution

But of course, evolution never works as simply as you want it to, and sometimes we can get the direct opposite pattern. This is called convergent evolution, and occurs when two completely different species independently evolve very similar (sometimes practically identical) traits. This is often caused by a limitation of the environment; some extreme demand of the environment requires a particular physiological solution, and thus all species must develop that trait in order to survive. An example of this would be the physiology of carnivorous marsupials like Tasmanian devils or thylacines: despite being in another Class, their body shapes closely resemble something more canid. Likely, the carnivorous diet places some constraints on physiology, particularly jaw structure and strength.

Convergent evol intelligence
A surprising example of convergent evolution is cognitive ability in apes and some bird groups (e.g. corvids). There’s plenty of other animal groups more related to each of these that don’t demonstrate the same level of cognitive reasoning (based on the traits listed in the centre): thus, we can conclude that cognition has evolved twice in very, very different lineages. Source: Emery & Clayton, 2004.

A more dramatic (and potentially obvious) example of convergent evolution would be wings and the power of flight. Despite the fact that butterflies, bees, birds and bats all have wings and can fly, most of them are pretty unrelated to one another. It seems much more likely that flight evolved independently multiple times, rather than the other 99% of species that shared the same ancestor lost the capacity of flight.

Parallel evolution

Sometimes convergent evolution can work between two species that are pretty closely related, but still evolved independently of one another. This is distinguished from other categories of evolution as parallel evolution: the main difference is that while both species may have shared the same start and end point, evolution has acted on each one independent of the other. This can make it very difficult to diagnose from convergent evolution, and is usually determined by the exact history of the trait in question.

Parallel evolution is an interesting field of research for a few reasons. Firstly, it provides a scenario in which we can more rigorously test expectations and outcomes of evolution in a particular environment. For example, if we find traits that are parallel in a whole bunch of fish species in a particular region, we can start to look at how that particular environment drives evolution across all fish species, as opposed to one species case studies.

Marsupial handedness.jpg
Here’s another weird example; different populations of marsupials (particularly kangaroos and wallabies) show preferential handedness depending on where the population is. That is, different populations of different species of marsupials shows parallel evolution of handedness, since they’re related to one another but have evolved it independently of the other species. Source: Giljov et al. (2015).

Following from that logic, it is then important to question the mechanisms of parallelism. From a genetic point of view, do these various species use the same genes (and genetic variants) to produce the same identical trait? Or are there many solutions to the selective question in nature? While these questions are rather complicated, and there has been plenty of evidence both for and against parallel genetic underpinning of parallel traits, it seems surprisingly often that many different genetic combinations can be used to get the same result. This gives interesting insight into how complex genetic coding of traits can be, and how creative and diverse evolution can be in the real world.

Where is evolution going?

Cat phylogeny
An example of all three types of evolutionary trajectory in a single phylogeny of cats (you know how we do it here at The G-CAT). This phylogeny consists of two distinct genera; one with one species (P. aliquam) and another of three species (the red box indicates their distance). Our species have three main physical traits: coat colour, ear tufts and tail shape. At the ancestral nodes of the tree, we can see what the ancestor of these species looked like for these three traits. Each of these traits has undergone a different type of evolution. The tufts on the ears are the result of divergent evolution, since F. tuftus evolved the trait differently to its nearest relative, F. griseo. Contrastingly, the orange coat colour of F. tuftus and P. aliquam are the result of convergent evolution: neither of these species are very closely related (remembering the red box) and evolved orange coats independently of one another (since their ancestors are grey). And finally, the fluffy tails of F. hispida and F. griseo can be considered parallel evolution, since they’re similar evolutionarily (same genus) but still each evolved tail fluff independently (not in the ancestor). This example is a little convoluted, but if you trace the history of each trait in the phylogeny you can more easily see these different patterns.

So, where is evolution going for nature? Well, the answer is probably all over the place, but steered by the current environmental circumstances. Predicting the evolutionary impacts of particular environmental change (e.g. climate change) is exceedingly difficult but a critical component of understanding the process of evolution and the future of species. Evolution continually surprises us with creative solution to complex problems and I have no doubt new mysteries will continue to be thrown at us as we delve deeper.

Not that kind of native-ity: endemism and invasion of Australia

The endemics of Australia

Australia is world-renowned for the abundant and bizarre species that inhabit this wonderful island continent. We have one of the highest numbers of unique species in the entire world (in the top few!): this is measured by what we call ‘endemism’. A species is considered endemic to a particular place or region if that it is the only place it occurs: it’s completely unique to that environment. In Australia, a whopping 87% of our mammals, 45% of our birds, 93% of our reptiles, 94% of our amphibians 24% of our fishes and 86% of our plants are endemic, making us a real biodiversity paradise! Some lists even label us as a ‘megadiverse country’, which sounds pretty awesome on paper. And although we traditionally haven’t been very good at looking after it, our array of species is a matter of some pride to Aussies.

Endemism map
A map representing the relative proportion of endemic species in Australia, generated through the Atlas of Living Australia. The colours range from no (white; 0% endemics) or little (blue) to high levels of endemism (red; 100% of species are endemic). As you can see, some biogeographic hotspots are clearly indicated (southwest WA, the east coast, the Kimberley ranges).

But the real question is: why are there so many endemics in Australia? What is so special about our country that lends to our unique flora and fauna? Although we naturally associate tropical regions with lush, vibrant and diverse life, most of Australia is complete desert. That said, most of our species are concentrated in the tropical regions of the country, particularly in the upper east coast and far north (the ‘Top End’).

There are a number of different factors which contribute to the high species diversity of Australia. Most notably is how isolated we are as a continent: Australia has been separated from most of the rest of the world for millions of years. In this time, the climate has varied dramatically as the island shifted northward, creating a variety of changing environments and unique ecological niches for species to specialise into. We refer to these species groups as ‘Gondwana relicts’, since their last ancestor with the rest of the world would have been distributed across the supercontinent Gondwana over 100 million years ago. These include marsupials, many birds groups (including ratites and megapodes), many fish groups and a plethora of others. A Gondwanan origin explains why they are only found within Australia, southern Africa and South America (the closest landmass that was also historically connected to Gondwana).

Early arrivals and naturalisation to the Australian ecosystem 

But not all of Australia’s species are so ancient and ingrained in the landscape. As Australia drifted northward and eventually collided with the Sunda plate (forming the mountain ranges across southeast Asia), many new species and groups managed to disperse into Australia. This includes the first indigenous people to colonise Australia, widely regarded as one of the oldest human civilisations and estimated to have arrived down under over 65 thousand years ago.

Eventually, this connection also brought with them one of our most iconic species; the dingo. Estimates of their arrival dates the migration at around 6 thousand years ago. As Australia’s only ‘native’ dog, there has been much debate about its status as an Australian icon. To call the dingo ‘native’ implies it’s always been there: but 6 thousand years is more than enough time to become ingrained within the ecosystem in a stable fashion. So, to balance the debate (and prevent the dingo from being labelled as an ‘invasive pest’ unfairly), we often refer to them as ‘naturalised’. This term helps us to disentangle modern-day pests, many of which our immensely destructive to the natural environment, from other species that have naturally migrated and integrated many years ago.

Patriotic dingo
Although it may not be a “true native”, the dingo will forever be a badge of our native species pride.

Invaders of the Australian continent

Of course, we can never ignore the direct impacts of humans on the ecosystem. Particularly with European settlement, another plethora of animals were introduced for the first time into Australia; these were predominantly livestock animals or hunting-related species (both as predators and prey). This includes the cane toad, widely regarded as one of the biggest errors in pest control on the planet.

When European settlers in the 1930s attempted to grow sugar cane in the far eastern part of the country, they found their crops decimated by a local beetle. In an effort to eradicate them, they brought over a species of cane toad, with the idea that they would control the beetle population and all would be well. Only, cane toads are particularly lazy and instead of targeting the cane beetles, they just thrived on all the other native invertebrates around. They’re also very resilient and adaptable (and highly toxic), so their numbers exploded and they’ve since spread across a large swathe of the country. Their toxic skin makes them fatal food objects for many native predators and they strongly compete against other similar native animals (such as our own amphibians). The cane toad introduction of 1935 is the poster child of how bad failed pest control can be.

DSC_0867_small
This guy here, he’s a bastard. Spotted in my parent’s backyard in Ipswich, QLD. Source: me, with spite.

But is native always better?

History tells a very stark tale about the poor native animals and the ravenous, rampaging pest species. Because of this, it is a widely adopted philosophical viewpoint that ‘native is always best’. And while I don’t disagree with the sentiment (of course we need to preserve our native wildlife, and not the massively overabundant pests), there are rare examples where nature is a little more complicated. In Australia, this is exemplified in the noisy miner.

The noisy miner is a small bird which, much like its name implies, is incredibly noisy and aggressive. It’s highly abundant, found predominantly throughout urban and suburban areas, and seems to dominate the habitat. It does this by bullying out other bird species from nesting grounds, creating a monopoly on the resource to the exclusion of many other species (even larger ones such as crows and magpies). Despite being native, it seems to have thrived on human alteration of the landscape and is a serious threat to the survival and longevity of many other species. If we thought of it solely under the ‘nature is best’ paradigm, we would dismiss the noisy miner as ‘doing what it should be.’ The truth is really more of a philosophical debate: is it natural to let the noisy miner outcompete many other natives, possibly resulting in their extinction? Or is it only because of human interference (and thus is our responsibility to fix) that the noisy miner is doing so well in the first place? It’s not a simple question to answer, although the latter seems to be incredibly important.

Noisy miner harassing currawong
An example of the aggressive behaviour of the noisy miner (top), swooping down on a pied currawong (bottom). Despite the size differences, noisy miners will frequently attempt to harass and scare off other larger birds. Image source: Bird Ecology Study Group website.

The amazing biodiversity of Australia is a badge of honour we should wear with patriotic pride. Conservation efforts of our endemic fauna are severely limited by a lack of funding and resources, and despite a general acceptance of the importance of diverse ecosystems we remain relatively ineffective at preserving it. Understanding and connecting with our native wildlife, whilst finding methods to control invasive species, is key to conserving our wonderful ecosystems.

“How do you conserve genes?”: clarifying conservation genetics

Sometimes when I talk about the concept of conservation genetics to friends and family outside of the field, there can be some confusion about what this actually means. Usually, it’s assumed that means the conservation of genetics: that is, instead of trying to conserve individual animals or plants, we try to conserve specific genes. While in some cases this is partially true (there might be genes of particular interest that we want to maintain in a wild population), often what we actually mean is using genetic information to inform conservation management and to give us the best chance of long-term rescue for endangered species.

DNA Zoo comic
Don’t worry, it’s an open range zoo: the genes have plenty of room to roam.

See, the DNA of individuals contains much more information than just the genes that make up an organism. By looking at the number, frequency or distribution of changes and differences in DNA across individuals, populations or species, we can see a variety of different patterns. Typically, genetics-based conservation analysis is based on a single unifying concept: that different forces create different patterns in the genetic make-up of species and populations, and that these can be statistically evaluated using genetic data. The exact type or scale of effect depends on how the data is collected and what analysis we use to evaluate that data, although we could do multiple types of analysis using the same dataset.

Oftentimes, we want to know about the current or historical state of a species or population to best understand how to move forward: by understanding where a species has come from, what it has been affected by, and how it has responded to different pressures, we can start to suggest and best manage these species into the future.

However, there are lots of possible avenues for exploration: here are just a few…

Evolutionary significant units (ESUs) and management units (MUs)

One commonly used application of genetic information for conservation is the designation of what we call ‘Evolutionary Significant Units’ (ESUs). Using genetics, we can determine the boundaries of particular populations which correspond to their own unique evolutionary groups. These are often the results of historical processes which have separated and driven the independent evolution of each ESU, usually with low or no gene flow across these units. Generally, managing and conserving each of these can lead to overall more robust management of the species as a whole by making sure certain groups that have unique and potentially critical adaptations are maintained in the wild. Although ESUs can sometimes be arguable (particularly when there is some, but not much, gene flow across units), it forms an important aspect of conservation designations.

In cases of shorter term separations across these populations, where there are noticeable differences in the genetics of the populations but not necessarily massively different evolutionary histories, conservationists will sometimes refer to ‘Management Units’ (MUs). These have much weaker evolutionary pressure behind them but might be indicative of very recent impacts, such as human-driven fragmentation of habitat or contemporary climate change. MUs often reflect very sudden and recent changes in populations and might have profound implications for the future of these groups: thus, they are an important way of assessing the current state of the species. The next couple of figures demonstrate this from one of my colleagues’ research papers.

YPP_map
The geographic distributions of Yarra pygmy perch populations, generously taken from Brauer et al. (2013). Each dot and number on the map represents a single population of pygmy perch used in the analysis. The colour of the population represents which MU it belongs to, whilst the shape of the marker represents the ESU. To make this easier to visualise, the solid lines indicate the boundaries of ESUs while the dashed lines represent MU boundaries. You’ll notice that MUs are subsets of ESUs, and that Population 6 actually fits into two different ESUs: see below.
YPP_Structure
An example of the output of an analysis (STRUCTURE) that determines population boundaries for Yarra pygmy perch using genetic data, generously taken from Brauer et al. (2013). Structure is an ‘assignment test’; using the input genetic information, it tries to make groups of individuals which are more similar to one another than other groups. In the graphs, each small column represents a single individual, with the colour bars representing how well it fits that (colour) population. The smaller numbers at the bottom and the labels above the graphs represent geographic populations (see the figure above). A) Shows the 4 major ESUs of Yarra pygmy perch, with some clear mixing between the Eastern ESU and the Merri/Curdies ESU in population 6. The rest of the populations fit pretty well entirely into one ESU. B) The MUs of Yarra pygmy perch, which shows the genetic structure within ESUs that can’t be seen well in A). Notice that some ESUs are made of many MUs (E.g. Central) while others are only one MU (e.g. MDB).

The two can be thought of as part of the same hierarchy, with ESUs reflecting more historic, evolutionary groups and MUs reflecting more recent (but not necessarily evolutionary) groups. For conservation management, this has traditionally meant that individuals from one ESU were managed independent of one another (to preserve their ‘pure’ evolutionary history) whilst translocations of individuals across MUs were common and often recommended. This is based on the idea that mixing very genetically different populations could cause adaptive genes in each population to become ‘diluted’, negatively affecting the ability of the populations to evolve: this is referred to as ‘outbreeding depression’ (OD).

Coffee comic
Sometimes, adding something can make what you had even worse than before. The most depressing analogy of outbreeding depression; a ruined coffee.

However, more recent research has suggested that the concerns with OD from mixing across ESUs are less problematic than previously thought. Analysis of the effect of OD versus not supplementing populations with more genetic diversity has shown that OD is not the more dangerous option, and there is a current paradigm push to acknowledge the importance of mixing ESUs where needed.

Adaptive potential and future evolution

Understanding the genetic basis of evolution also forms an important research area for conservation management. This is particularly relevant for ‘adaptive potential’: that is, the ability for a particular species or population to be able to adapt to a variety of future stressors based on their current state. It is generally understood that having lots of different variants (alleles) of genes in the total population or species is a critical part of evolution: the more variants there are, the more choices there are for natural selection to act upon.

We can estimate this from the amount of genetic diversity within the population, as well as by trying to understand their previous experiences with adaptation and evolution. For example, it is predicted that species which occur in much more climatically variable habitats (such as in desert regions) are more likely to be able to handle and tolerate future climate change scenarios since they’ve demonstrated the ability to adapt to new, more extreme environments before. Examples of this include the Australian rainbowfishes, which are found in pretty well every climatic region across the continent (and therefore must be very good at adapting to new, varying habitats!).

Rainbowfish both.jpg
Left: The distribution of rainbowfish across Australia, with each colour representing a particular ecotypeRight: A photo of a (very big) tropical rainbowfish taken from a recent MELFU field trip. Source: MELFU Facebook page. He really got around after that one stint in that children’s story.

Genetics-based breeding programs and pedigrees

A much more direct use of genetic information for conservation is in designing breeding programs. We know that breeding related individuals can have very bad results for offspring (this is referred to as ‘inbreeding depression’): so obviously, we would avoid breeding siblings together. However, in complex breeding systems (such as polygamous animals), or in wild populations, it can be very difficult to evaluate relationships and overall relatedness.

That’s where genetics comes in: by looking at how similar or different the DNA of two individuals are, we can not only check what relationship they are (e.g. siblings, cousins, or very distantly related) but also get an exact value of their genetic relatedness. Since we know that having a diverse gene pool is critical for future adaptation and survival of a species, genetics-based breeding programs can maximise the amount of genetic diversity in following generations. We can even use a computer algorithm to make the very best of breeding groups, using a quirky program called SWINGER.

Cats DNA dating
If You Are the One, conservation genetics edition.

Taxonomy for conservation legislation

Another (slightly more complicated) application of genetics is the designation of species status. Large amounts of genetic information can often clarify complex issues of species descriptions (later issues of The G-CAT will discuss exactly how this works and why it’s not so straightforward…).

Why should we care what we call a species or not? Well, much of the protective legislation at the government level is designed at the species-level: legislative protections are often designated for a particular species, but doesn’t often distinguish particular populations. Thus, misidentified species can sometimes but lost if they were never detected as a unique species (and assumed to be just a population of another species). Alternatively, managing two species as one based on misidentification could mess with the evolutionary pathways of both by creating unfit hybrid species which do not naturally come into contact together (say, breeding individuals from one species with another because we thought they were the same species).

Cryptic cats comic
Awkward.

Additionally, if we assume that multiple different species are actually only one species, this can provide an overestimate of how well that species is doing. Although in total it might look like there are plenty of individuals of the species around, if this was actually made of 4 separate species then each one would be doing ¼ as well as we thought. This can feed back into endangered status classification and thus conservation management.

 

These are just some of the most common examples of applied genetics in conservation management. No doubt going into the future more innovative and creative methods of applying genetic information to maintaining threatened species and populations will become apparent. It’s an exciting time to be in the field and inspires hope that we may be able to save species before they disappear from the planet permanently.