Species introductions serve as a natural laboratory to study introgression and selection. In a recent paper in Molecular Ecology, Rachael Bay and colleagues use introduced rainbow trout and native cutthroat trout to study hybridization, introgression, and selection. Bay et al. find evidence that some alleles have repeatedly introgressed from rainbow trout into cutthroat trout in independent populations. Their results suggest that selection has played an important role in this introgression, and highlight the usefulness of species introductions for understanding the predictability of evolution. Below, get a behind the scenes look at this work from author Rachael Bay.

What led to your interest in this topic / what was the motivation for this study? 
This study combined two of my primary research interests. The first is: How do humans alter the evolutionary trajectories of species? By introducing rainbow trout, we have provided access to an extended gene pool for native cutthroat trout species. Previous studies have shown that hybrids have lower fitness, but with hybridization and recombination continuing over decades we can investigate whether particular rainbow trout alleles might be adaptive in westslope cutthroat trout. This study also speaks to the predictability of evolution. The stocking of rainbow trout has resulted in a highly replicated evolutionary experiment. Do we find the same alleles repeatedly under positive selection in independent watersheds?

What difficulties did you run into along the way? 
One of the main difficulties was trying to understand the null expectation. How much introgression should we expect between the two species and what fraction of that introgression is a result of selection? This depends on not only the strength of selection, but also on other demographic factors like population size, and stocking history. Ultimately, we decided to use simulations in order to understand the level of selection necessary to produce the patterns of introgression we were seeing in hybrid populations.

What is the biggest or most surprising finding from this study? 
We found that across multiple independent locations, the same rainbow trout alleles rose to high frequency in hybrid populations, suggesting they were under positive selection. This is somewhat surprising because previous studies have suggested that hybrids have reduced fitness and have found broad signals of purifying selection against rainbow trout alleles. However, hybridization and backcrossing has been occurring for many generations, allowing plenty of time for recombination and allowing different parts of the rainbow trout genome to segregate more independently. So despite the fact that hybrids have lower fitness, there seem to be a few regions of the rainbow trout genome that may be advantageous to westslope cutthroat trout.

Moving forward, what are the next steps for this research?
While our results suggest that some rainbow trout alleles provide an adaptive advantage we still have yet to identify the selective force. Is there some component of the abiotic environment to which these alleles are better adapted? Do these alleles confer higher reproductive success or fecundity? Rainbow trout have been successfully introduced to many different environments across North America – do alleles at high frequency in hybrid populations also explain the invasion success of rainbow trout?

What would your message be for students about to start their first research projects in this topic? 
I think it’s really important to choose your system carefully. We didn’t start out thinking about this as a project on trout, we started thinking about human-induced evolution and repeatability. It took a long time and a lot of thought to realize that a broadly introduced species was the perfect natural experiment for the questions we had.

What have you learned about science over the course of this project?
One of the cool things about this project is that it is a demonstration of how science evolves as technology evolves. Through a collaboration with Rick Taylor, we were able to learn something new from samples that had been sitting in a freezer for many years. Previous researchers had used these samples to analyze rates of hybridization across British Columbia and Alberta, but the increasing ease of high-throughput sequencing allowed us to take a deeper dive and look at genome-wide signals of introgression. So you never know how experiments you are doing now will contribute to knowledge in the future!

Describe the significance of this research for the general scientific community in one sentence.
Some genes from introduced rainbow trout can confer an adaptive advantage in native cutthroat trout species.

Describe the significance of this research for your scientific community in one sentence.
Rainbow trout alleles show consistently high levels of introgression into the westslope cutthroat trout genome across multiple independent watersheds.

Read the full article:
Bay RA, Taylor EB, Schluter D. Parallel introgression and selection on introduced alleles in a native species. Mol Ecol. 2019;28:2802-2813. https://doi.org/10.1111/mec.15097

Many insects have lost their wings at high altitude, and this might have contributed to their diversification. In their recent paper, Graham A. McCulloch and colleagues study loss of wings in stone flies from New Zealand and found that the alpine tree line, rather than altitude alone better explains the ecotypic distribution of these morphs across geography. Their system paves the way for powerful studies of convergence and adaptation and how, like in other systems, loss of a trait, might be intrinsically related to many cases of deterministic evolution in nature. Below, we learn about their journey in discovering some of the causes of diversification of alpine insects and their assemblages.

What led to your interest in this topic / what was the motivation for this study? 
An astonishingly large proportion (20%) of New Zealand’s stonefly species (freshwater insects) have reduced wings as adults, with most of these low-dispersal lineages found in alpine environments. Our research group is interested in the ecological drivers and genomic basis of this fascinating pattern: how and why do insect wings become reduced/lost, and what are the consequences of this? The Zelandoperla fenestrata complex in particular is an outstanding system for studying these questions, as this widespread polymorphic stonefly has full-winged populations at low altitudes, but vestigial-winged populations up in the mountains. When we started looking within individual streams, we were amazed to find strikingly clear altitudinal clines within this ‘species’, where wings become dramatically smaller as we move a few hundred metres up a mountainside. This was an exciting finding, so understanding the ecological and genomic basis of these clines became the focus of our research.

What difficulties did you run into along the way?
Our study involved working in rugged mountain landscapes, which can be both exciting and spectacular – but also carries challenges. You need to be fit, and to persist in sometimes uncomfortable conditions. It also can be notoriously difficult to find adults of these stoneflies in the wild, even in the right season, so it was a big logistical breakthrough to be able to successfully rear large nymphs to adulthood in the lab. This breakthrough really allowed us to get a good handle on the system morphologically.

What is the biggest or most surprising finding from this study?
There were several unexpected and exciting discoveries from this project: (i) We were particularly excited to find a tight association between wing-reduction and the alpine treeline, both at a fine scale (within our transects), and at a broader scale (using distributional analyses). These results suggest that harsh conditions above the alpine treeline (most likely high winds) are a major driver of insect wing loss. (ii) The genome-wide divergence between the full-winged and vestigial-winged ecotypes within each stream was particularly surprising, as these ecotypes have overlapping distributions (and interbreed in the wild). Likewise, the genome-wide divergence between the two independent vestigial-winged alpine populations, from streams less than 3km apart, was astonishing given the small distance involved.

Moving forward, what are the next steps for this research?
We plan to examine additional ecotypic clines in this species across the lower South Island. This next step will allow us to assess whether the genome-wide differentiation we observe between ecotypes in the Rock and Pillar range is found in other regions. By genetically characterising geographically (and phylogenetically) independent clines we can then test for independent wing loss events (associated with the alpine treeline) more broadly. We are also in the process of using genomic (GWAS) and transcriptomic approaches to identify the key genes underpinning wing-reduction in Z. fenestrata (and potentially other alpine stonefly species). We are really keen to find out whether the wing reduction events in different streams and mountains involve the same genes dispersed by winged populations (e.g. transporter hypothesis), or whether they are completely independent.

What would your message be for students about to start their first research project in this topic?
Discovering this fascinating research system took time – including quite a bit of ground-work exploring out in the field, background research to understand the species complex, including discussions with entomologists, taxonomists, looking at museum collections, and then putting a team together with the skills to get the work done. So it didn’t happen overnight, and it took time to set the stage and to know for sure that we’d found a good system. But it really started from curiosity about evolution, as well as our ongoing interest in New Zealand’s landscapes and natural history. So our most important advice is to follow your curiosity, and keep exploring the natural world.

What have you learned about science over the course of this project?
We have learned that there are still plenty of exciting discoveries yet to be made about nature, evolution and ecology – and that the more we discover, the more new questions emerge. It might take five or ten years to really understand a system to the extent that you can begin to answer the most exciting questions. We certainly expect to keep working on this project for many years to come.

Describe the significance of this research for the general scientific community in one sentence.
We have discovered what we believe to be a ‘textbook example’ of speciation in action – one that makes evolutionary biology easier to teach and understand.

Describe the significance of this research for your scientific community in one sentence.
This story brings together ecological and genomic tools to reveal clear-cut cases of parallel ecological speciation over surprisingly small spatial scales.

Professor Deborah Charlesworth discusses her research on the origin of mitochondrial male sterility mutations. Some hermaphroditic plant species have a fraction of individuals with flowers that are male sterile and only express the female function. This reproductive system is known as gynodioecy, and it can create conflicts between genes favouring male or female function. In a paper recently published in Molecular Ecology, Bergero et al. explore the persistence of gynodioeccy in the Plantago system and test two fundamental hypothesis for the maintenance of gynodioeccy in nature. Deborah Charlesworth chatted with us and gave us some wonderful insights as to how this research started and where it is going now. Don’t miss it!

What led to your interest in this topic / what was the motivation for this study? 
The phenomenon of male-sterility in plants has been a puzzle for a long time, and the discovery that mitochondrial mutations are involved only deepened the mysteries further, because it became clear that the mutations were not the usual kinds, such as point mutations in or deletions of coding sequences. I hoped that getting sequences of the mitochondrial genome might be helpful

What difficulties did you run into along the way? 
There were no major difficulties in getting the sequence data, though because plant mitochondrial genomes are very larger, we could sequence only coding regions of genes. Of course, some genes proved not to include any sequence variants, so the number of genes with useful data was reduced, compared with our initial hoped.

What is the biggest or most surprising finding from this study? 
A surprising finding was that some populations that contain females, and therefore certainly have male-sterility mutations present, did not have variants in the mitochondrial genes we sequenced. Because male-sterility is generally cytoplasmically inherited, it seems unlikely that in this species it is due to nuclear male-sterility mutations (especially as earlier excellent genetic studies in our study species strongly support cytoplasmic inheritance in populations in the Netherlands). It was also unexpected to find that, in most European populations sampled, the only variants were pretty rare, so that the entire mitochondrial genome behaved almost as a single genetic “allele” or “haplotype”, whereas populations from one region in northern UK had two different haplotypes, with variants seen in several genes.  

Moving forward, what are the next steps for this research? 
The next steps will have to be taken by others, as I am too old to continue (I now work on sex chromosome evolution in a fish, and one project is enough at my age). But I hope that others will add mitochondrial sequence data to see whether other plants with cytoplasmic male-sterility have mitochondrial haplotypes that are associated with the femaleness factors. It will also be good to test whether, as in our study species, multiple sequence variants are associated into haplotypes, or not. It is known that mitochondrial genomes can undergo a process of genetic recombination, and studying sequence variants can tell us whether this occurs often in natural populations, or not. If it occurs often, then it should be possible to identify sequence variants that are associated with male-sterility. If, however, exchanges between haplotypes occur very rarely, it could prove to be very difficult to find the actual mutations involved. Another intriguing possibility is that male-sterility is caused by parts of the genome other than the protein-coding genes, and that, even if those genes do not undergo recombination, other parts of the genome might do so, perhaps producing the variants that lead to male-sterility. It has long been known that mitochondrial genome rearrangements seem to be involved in male-sterility, rather than point mutations, and I have sometimes wondered whether perhaps this phenotype occurs when parts of the genome are duplicated, which might lead to down-regulation of genes that suddenly have an extra copy, or partial copy, as occurs in transgenic plants.

What would your message be for students about to start their first research projects in this topic? 
Find something easier unless you are really clever. On the other hand, my experience has been that one often gets puzzling results, and they often prompt one to think about the question in a different way. In fact, I would say that one almost never starts by asking the right questions, but that these tend to emerge as one sees the data and thinks about them.

What have you learned about science over the course of this project? 
You never know what you will turn up, and whatever it is, it often raises more puzzles than it solves.

Describe the significance of this research for the general scientific community in one sentence.
This was the first multi-gene sequence data collected to study mitochondrial male sterility in a plant, and it showed that, in our study species, this genome recombines rarely, at least in the parts we could study, and, by extension, across the entire mitochondrial genome.

Describe the significance of this research for your scientific community in one sentence.
This study is just a small step in attempts to understand the puzzle of how mitochondrial male sterility mutations may arise.