Unravelling little mysteries in the genome of Atlantic salmon

Salmon have always been part of my life. I grew up along the Miramichi River in New Brunswick, Canada, which is a river that is famous for its Atlantic salmon fishing. Even my high school mascot was a salmon named Samoo the Plamu (Mi’kmaq for Atlantic salmon). People travel from far and wide for the opportunity to catch a salmon on the Miramichi, and I have been lucky enough to catch at least one grilse (a salmon that spends only one winter at sea) when fly fishing with my dad. Although my grad studies took me to the west coast to study Pacific salmon, I was glad to have had the opportunity to move back to the east coast and work on a species that has always been close to my heart.

Catching my first salmon on the Miramichi River.

In 2017, I moved to Halifax, Nova Scotia, and started a postdoc with the Bradbury lab. I was excited to get some experience in genomic projects involving Atlantic salmon. Although, I was quickly reminded that Atlantic salmon are complicated. They exhibit a wide range of life history strategies (see Fig. 1), and unlike Pacific salmon, they don’t die when they spawn (they are iteroparous), making things even more complex (in my opinion). Nonetheless, the amount of diversity that exists makes them an exciting species for exploring never ending evolutionary questions. 

Fig. 1. The wonderful and complicated life of Atlantic salmon (Fig. 1 from Gibson and Haedrich, 2006)

 

One interesting part of the Atlantic salmon story is that Atlantic salmon occupy rivers on both sides of the Atlantic Ocean – in Europe and North America. Salmon from these continents diverged >600,000 years ago, and this divergence has occurred primarily in allopatry, although opportunities for gene flow have occurred. In one of my recent papers, we investigated genomic differences between Atlantic salmon from these two continents.

  

Atlantic salmon returning to a river to spawn. Copyright: Nick Hawkins Photography

As populations begin to diverge from each other, the genome can start to show variable levels of differentiation, resulting in peaks and valleys of differentiation. Regions of high differentiation between populations can occur through different mechanisms, one being divergent selection acting in opposite directions in each population. These regions often called ‘genomic islands of speciation’ have attracted a lot of attention as these regions may be important for initiating speciation. 

But some question whether these islands of speciation are real…

 

It is now clear that other mechanisms can produce the same signals of differentiation without divergent selection (Wolf and Ellegren, 2017). This can include purifying background selection, which acts to remove deleterious mutations. In regions of low recombination, this type of background selection can reduce diversity and lead to signals of increased differentiation between populations.

 

This means that two very different processes can lead to similar signals, so it’s important to consider what mechanisms might be operating in the genome to better understand the speciation process. In our study, we attempted to do just that with Atlantic salmon.

 

I will admit that the role of purifying background selection is not something that I had given much attention to before this paper. Luckily, another postdoc in the lab at the time, Tony Kess, had spent some time thinking about it already. Tony and I spent a lot of time drinking a lot of coffee and discussing the role of background selection in salmon. Admittedly, as I read more papers and became more caffeinated, I sometimes got more confused. One question that I kept coming back to is ‘how will we know if it’s divergent selection or if it’s just background selection?’. One answer we seemed to settle on was maybe we won’t know for sure, but by using multiple approaches, we can provide evidence that is more consistent with one of these processes.

 

Tony and me at Moominworld for ESEB 2019 asking Moominpapa about his thoughts on background selection. 

Fortunately, a lot of other scientists have focused their efforts on understanding and identifying the signals associated with background selection, and their work has been a tremendous help for understanding how such processes can shape the genomic landscape. For a nice example on the role of linked selection and recombination in driving regions of high differentiation, I suggest Burri et al. (2015), which investigates this across flycatcher species.

 

To try to disentangle these different mechanisms in Atlantic salmon, we took note of methods used in other studies. We expected regions under divergent selection (rather than just background selection) to show: 

1) high differentiation

2) high linkage disequilibrium

3) no reduction in recombination rate

4) no increase in gene density

5) signals of positive selection

 

The question about these differences between European and North American salmon is interesting from an evolutionary perspective, but also important for conservation and management. Atlantic salmon are moved all around the world for aquaculture purposes. The historical use of European salmon for aquaculture in eastern North America has posed problems to some recent conservation efforts in Canada (see CBC article).

 

In our study (recently published in Molecular Ecology), we utilized genomic data for 26 populations in North America and 54 populations in Europe, which cover a wide range of latitudes within each continent (Fig. 2). The study was ‘spawned’ partly out of curiosity when my postdoc supervisor, Ian Bradbury, asked if any loci were fixed between Europe and North America in our dataset. Upon a quick inspection of the genome, I found over a hundred loci that showed almost fixed differences between continents. But what really got our attention was that a large number of these loci (almost 40%) were localized in one large genomic region. This was news to us, and this led to a more formal investigation of where these large regions of differentiation were located in the genome, and what processes were shaping them. Identifying these regions would also be useful for developing markers that can be used to detect salmon of European origin (or with recent European ancestry) in Canadian waters.

 

Fig. 2. Location of Atlantic salmon sampling sites in (A) North America and (B) Europe.

In our study, we first found large genomic regions (>1 to 3 million base pairs) showing consistent signals of high differentiation across multiple methods. These were found on four chromosomes in the salmon genome.

Next, for these four chromosomes, we went back to our check list to see if these regions showed patterns consistent with divergent selection. With these data, we confirmed in these regions (see Fig. 3):

1) high differentiation: yes, we found highly divergent regions

2) high linkage disequilibrium: yes, we showed high linkage disequilibrium 

3) no reduction in recombination rate*: yes, we found no significant reduction in recombination rate relative to the rest of the chromosome

4) no increase in gene density: yes, we found no significant increase in gene density

5) signals of positive selection: yes, we found signals of positive selection

Fig. 3. Example of one region showing high differentiation (high F_ST) between continents on chromosome Ssa06. This region showed no significant reduction in recombination rate and no significant increase in gene count relative to other regions of the chromosome, lending support to the role of divergent selection in driving these differences

Together, these results support that differentiation is not likely due to background selection alone, which is more likely to produce signals of differentiation in regions of low recombination.

 

*Side note: Originally, I may have calculated recombination rate incorrectly. Before the paper was published, I uploaded my R scripts for the analyses to my GitHub. Arne Jacobs (postdoc at Cornell University – who I’ve met a few times at conferences) kindly reached out to let me know that my calculations were wrong. Turns out, it is not as simple as just centimorgans divided by base pairs. Who would have thought? (probably everyone else!) But, to be fair, there are many different ways to calculate recombination rate. While this was a bit embarrassing, I was happy to have the chance to correct this mistake before the paper was officially published. I was even more happy to find out that this did not change the results/interpretation of the paper. So thank you to Arne for reaching out in a kind and respectful manner, we can always use more kindness in academia! 

 

Overall, our results were consistent with the role of divergent selection acting to drive patterns of differentiation between continents rather than just purifying background selection. One question remains as to what traits/genes may be under selection at the continental level. As I think about salmon from each of these continents, I think about the diverse landscapes that they live in and the different conditions that they encounter. But we know salmon from Europe and North America are not morphologically distinct, and generally populations are expected to be adapted to local river conditions rather than at a large scale. So one question that weighed on me was ‘what could be showing adaptive signals across such a broad scale?’. We found genes and biological processes that could potentially relate to differences in ocean navigation/migration and immunity. One hypothesis could be that while salmon from each continent migrate to shared feeding grounds in the ocean, they have to travel in different directions to get there, so perhaps differences in ocean navigation may have evolved. I think this is a cool idea that would be interesting to study in the future.

 

Of course, there are caveats to any study, and we address these limitations in our paper. Future studies using genome sequencing and experimental work would help to better understand the adaptive differences between continents. 

 

Our study found differences between European and North American Atlantic salmon that may be contributing to early stages of speciation. These differences may explain some partial incompatibilities that exist between continents, and highlight the potential risk associated with the trans-Atlantic movement of salmon provided the currently limited data, high genome-wide differentiation, and largely unknown consequences. More focus on understanding these differences may help inform management decisions in the future as more plans develop to move salmon across the ocean. Luckily, I have recently started a job as a scientist with Fisheries and Oceans and can continue to concentrate on questions related to Atlantic salmon management and conservation.

 

Our recent paper:

Lehnert, S.J., Kess, T., Bentzen, P., Clément, M. and Bradbury, I.R. (2020) Divergent and linked selection shape patterns of genomic differentiation between European and North American Atlantic salmon (Salmo salar). Molecular Ecology 29:2160-2175.

References:

Burri, R. et al. (2015) Linked selection and recombination rate variation drive the evolution of the genomic landscape of differentiation across the speciation continuum of Ficedula flycatchers. Genome Research 25:1656-1665.

Gibson, J. and Haedrich, R. (2006) Life history tactics of Atlantic salmon in Newfoundland. Freshwater Forum 26:38-45.

Wolf, J.B. and Ellegren, H. (2017) Making sense of genomic islands of differentiation in light of speciation. Nature Reviews Genetics, 18:87-100.

Dissecting phases of speciation in stick-insects

I have worked on many different study systems over the years, including killer whales, livebearing fishes (family Poeciliidae) and Timema stick insects (for more detail please see my homepage). Originally, I started my research career with a Diploma in Biology in Germany (equivalent to a Masters in other countries), with my thesis work focusing on whistle communication in diverging killer whale populations around Vancouver Island in British Columbia, Canada. However, I soon realized that there are only so many questions you can ask using a study system that largely precludes running controlled experiments. Thus, for my PhD thesis and subsequent postdoctoral work, I focused on population divergence and speciation in livebearing fishes (Poeciliidae) living along various environmental gradients (e.g., gradients of predation, toxicity, and access to light). It was during this time that I realized that my main interest was not so much in one particular study system, but rather in discovering the mechanisms that create, maintain, and sometimes constrain, biodiversity. That interest eventually led me to add yet another study system to the mix in 2012: the system of Timema stick insects I am writing about here. More specifically, I wanted to use that particular system to study the potential role chemical communication might have on population divergence and speciation.

As the above paragraph suggests, each of my study systems has their own system-specific peculiarities [e.g., cultural differences seem to play a prominent role in driving population divergence in killer whales (Riesch et al. 2012), but not in stick insects or livebearing fishes]. However, they also have a lot in common. For example, selection from predation is integral to both, the livebearing-fish and stick-insect systems I study, while foraging specialization plays a prominent role in population divergence of both, stick insects and killer whales. Thus, the three systems simply constitute different examples of how ecologically-based divergent selection drives population divergence and ultimately (ecological) speciation.

A rain shower moving through the chaparral near Santa Barbara, California. Timema are often found in this biome of dense thickets and thorny bushes.

The idea that speciation can be thought of as a continuum is yet another concept that dates back at least to Charles Darwin’s world-changing On the origin of species. The concept in its modern form posits that pairs of populations move along a continuum between panmixis on one extreme end and complete reproductive isolation on the other. Progress can be towards speciation or towards collapse, the latter showcased by studies on speciation reversal in European whitefish Coregonus spp. (Vonlanthen et al. 2012), three-spined stickleback Gasterosteus aculeatus (Tayler et al. 2006), and cichlid fishes (Seehausen et al. 1997).

This concept of a speciation continuum has gained traction again in recent years (e.g., Hendry et al. 2009). Consequently, studies across closely related taxa at different phases of speciation are beginning to illuminate the processes and genetic changes underlying the formation of new species (Seehausen et al. 2014). It is well-known, of course, that speciation involves genetic differentiation, and that, in the absence of gene flow, genome-wide differentiation can readily build up by selection and drift. If speciation is to happen in the face of gene flow, however, the picture gets more complex. According to the genic model of speciation (Wu 2001), speciation is initiated by a few genetic regions that become resistent to gene flow before others. This results in a localized pattern of genetic differentiation, which becomes more genome-wide as speciation progresses.

In a recent study just published in the April issue of Nature Ecology and Evolution (http://www.nature.com/articles/s41559-017-0082), we took a closer look at the transitions between phases of genomic differentiation during speciation of Timema stick insects. Like other studies on the speciation continuum (including my other study systems), we were faced with a key problem: speciation is often slow enough that we cannot simply follow a single lineage through time to see in real-time how the process unfolds. The solution then is to take as many different snapshots of the process from different pairs of natural populations as possible, and to then start to reconstruct a bigger picture of what might be happening across different moments in time. This is exactly what we did using data from >100 populations of 11 species of Timema stick insects. Our work suggests that speciation can be initiated by few genetic changes associated with natural selection on few loci, but the overall process is multi-faceted and involves mate choice and genome-wide differentiation.

A male Timema cristinae on one of its host plants (genus Ceanothus).
Photo: Moritz Muschick

This study is the culmination of almost 30 years of research into this system, and consists of data collected between 1996 and 2014, including >1000 re-sequenced whole genomes. In fact, research in this system began when two of our coauthors, Cristina Sandoval (University of California in Santa Barbara, USA) and Bernie Crespi (Simon Fraser University, Canada), recognized this group harbours variation in phases of speciation. Patrik Nosil then entered the system in 2000 and eventually wrote his PhD on it, using experiments to estimate reproductive isolation. An important component of the current paper was the chemical ecology of stick insects. This part of the project was born ~2009, emerging out of initial discussions between Patrik and I, with additional input from Bernie Crespi and Gerhard Gries at Simon Fraser University. Fast-forward to the year 2012, where the alignment of different projects getting funded finally enabled us to team up at the University of Sheffield in the UK (key components were a European Research Council Grant to study the genomics of speciation to Patrik, a Human Frontier Science Program Postdoctoral Fellowship to study the role of chemical communication in speciation to myself, and a burgeoning collaboration with Zach Gompert, a statistical population geneticist from Utah State University).

The emphasis of the chemical ecological aspect of the project was on cuticular hydrocarbons (CHCs), the oily/waxy chemicals on the cuticle of insects that can function to prevent desiccation and physical injury (Drijfhout et al. 2013 in Behavioral and Chemical Ecology, pp. 91-114), but that have also been repeatedly implicated as integral to mate choice (e.g., Blows and Allan 1998; Chung et al. 2014). For stick insects, we found that populations that differed more strongly in their CHC profiles also had higher degrees of sexual isolation and stronger genome-wide differentiation. We confirmed the causal role of CHCs in mate choice by means of a perfuming experiment.

Evaporating hexane samples in Santa Barbara, California, as part of the perfuming experiment on Timema mate choice.

Preparing another CHC-hexane sample for analysis with the gas chromatograph in the Gries lab at Simon Fraser University in 2014. Photo: Sean McCann

When combining this CHC-data with other phenotypic data and genomic analyses, we uncovered that, consistent with early phases of genic speciation, colour-pattern loci that confer camouflage to particular host plants reside in localised genetic regions of accentuated differentiation between populations experiencing gene flow. Transitions to genome-wide differentiation are also observed with gene flow, but appear to have little to do directly with differentiation in color. Rather, genome-wide differentiation is associated with divergence in CHCs, which we show to be polygenic, modestly heritable traits. Thus, intermediate phases of speciation are not associated with growth of a few peaks or ‘islands’ in the genome. Finally, we show that complete reproductive isolation was associated with a conspicuous increase in the overall degree of genomic differentiation. Thus, although speciation is perhaps continuous, this does not mean it always proceeds in a strictly uniform fashion (this component was led by our collaborators Moritz Muschick, now a postdoctoral fellow at EAWAG in Switzerland, and Victor Soria-Carrasco, who is now a Leverhulme Early Career Fellow at the University of Sheffield). Overall, the results suggest that substantial progress towards speciation may involve the alignment of multi-faceted aspects of differentiation. We suspect similar conclusions may apply to other systems where strong reproductive isolation involves many traits and evolves in a polygenic fashion.

A female Timema bartmani, cryptic against the needles of white fir. Photo: Moritz Muschick.

In conclusion, although many questions remain unanswered, it seems clear that speciation in this group involves more than divergence in cryptic coloration, and the results point to mating isolation and other reproductive barriers, as well as geographic separation, as being important. Thus, the striking example of crypsis in Timema that has been the focus of many previous studies (e.g., Sandoval 1994; Nosil and Crespi 2006; Comeault et al. 2015) represents only one aspect of the multi-faceted speciation process.