Interview with the authors: Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads

Understanding how species form, and the factors that contribute to reproductive isolation has been a long-standing goal of evolutionary biology. Cryptic radiations can provide insight into these questions. Dufresnes and colleagues investigate these questions in a cryptic radiation of Eurasian spadefoot toads (Genus Pelobates). They find a correlation between the amount of time spent in allopatry and the level of reproductive isolation between lineages experiencing secondary contact. Get a behind-the-scenes look at the research below with first author Christophe Dufresnes.

Aquatic portrait of the Common Spadefoot Pelobates fuscus. This species belongs to a cryptic speciation continuum new to science, deciphered through a fine-scaled genomic phylogeography. Credit: Edvárd MIZSEI.

What led to your interest in this topic / what was the motivation for this study? 

This study was part of my post-doc efforts to compare geographic and genomic patterns of introgression across hybrid zones from several amphibian radiations, in order to understand the pace and the genetic mechanisms of allopatric speciation. For Pelobates, we originally intended to focus only on the P. fuscus/vespertinus hybrid zone in Ukraine/Russia, but inadvertently discovered that our outgroup taxon (the traditionally-recognized P. cf. syriacus) consisted of a cryptic diversification involving several phylogeographic transitions. Instead of one contact, it turned out we could study as many as six within a single radiation. We thus seized that rare opportunity to understand the relationship between genetic divergence and reproductive isolation under natural settings, which had only been attempted in a handful of systems so far.

What difficulties did you run into along the way? 

Not much actually, except perhaps time constrains and taxonomic issues. Because we were willing to describe the new Pelobates species/subspecies discovered and use the appropriate names in our Mol. Ecol. article for clarity, we had to synchronize the peer-reviewing and publication of an accompanying paper (ZooKeys 859: 131–158). This was successful thanks to the support of the two journals and both papers were released just a few hours apart. The scientific stages per se went remarkably smoothly. Colleagues from many countries were enthusiastic to send us samples, the RAD-seq wet lab and bioinformatics performed admirably, and data analysis was straightforward. At the end, it took only about a year and a half from project initiation to publication.

What is the biggest or most surprising finding from this study? 

We were astonished by the strong divergence (>5My) between the Asian and European populations of P. cf. syriacus, and their lack of interbreeding despite parapatric (and perhaps even sympatric?) distributions near the Bosphorus. While they clearly represent two different species (coined P. balcanicus and P. syriacus), no phenotypic differences have been reported (despite several morphometric surveys), so this was not suspected. But more globally, our big finding is the very neat link between genetic divergence and admixture across phylogeographic transitions. It was beyond expectations since hybridization at contact zones often depends on local factors (dispersal constraints, etc.), which blurs the link. Such a clear relationship supports the Darwinian view of a gradual and dynamic speciation continuum, and extends it to cryptic radiations of eco-morphologically similar species.

Moving forward, what are the next steps for this research? 

We have several major follow-ups ongoing. Our Pelobates speciation genomic framework is now being implemented into multi-system comparative analyses aiming to understand how the genetic architecture of reproductive isolation evolves as speciation progresses, by re-analyzing transitions at the locus scale. In parallel, we are characterizing the homomorphic sex chromosomes of Pelobates to gauge their importance in hybrid incompatibilities. Following this research, our co-author Ilias Strachinis has now just started a PhD on Pelobates from the Balkan Peninsula to study the diversity and distribution of the new lineages unraveled. It should provide significant insights for their biogeography and conservation, notably of the mysterious Peloponnese endemic P. balcanicus chloeae.

What would your message be for students about to start their first research projects in this topic? 

Even in supposedly well-studied biogeographic regions like the Western Palearctic, do not take the established phylogenies, species delimitations and taxonomies for granted! Significant diversifications may have been overlooked, especially since previous work relied mostly on mitochondrial and poorly-informative nuclear markers, which can be deceptive to disentangle among closely-related lineages. Nowadays, RAD-seq provides affordable, powerful and straightforward resources to address many questions with a combination of population genetic and phylogenetic analyses, so it appears a tool of choice to study the phylogeography of speciation in many taxonomic groups.

What have you learned about science over the course of this project? 

That unexpected results are worth exploiting and may lead to fascinating scientific discoveries. An unthinkable amount of biodiversity still lies unnoticed right under our noses. Moreover, our study was only possible (especially in such an efficient timeframe) thanks to a great collective effort bringing together renowned teams of herpetological researchers. From my personal perspective, this human aspect emphasizes how science is best appreciated collaboratively rather than through competitive emulation, and I look forward to reiterate the experience.

Describe the significance of this research for the general scientific community in one sentence.

In cryptic diversifications, whether the continuous nature of speciation leads to discrete, reproductively-isolated entities is mostly dependent on the time they spent in allopatry.

Describe the significance of this research for your scientific community in one sentence.

Our study provides empirical evidence within a single radiation that speciation is a dynamic and reversible process where phylogeographic lineages can merge together upon secondary contact, unless a threshold of evolutionary divergence is reached (>3My in amphibians), in which case they can quickly build up reproductive isolation and become incipient species.

Read the full article here: Dufresnes C, Strachinis I, Suriadna N, et al. Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (Pelobates). Mol Ecol. 2019;28:3257–3270. https://doi.org/10.1111/mec.15133

Summary from the authors: Geography best explains global patterns of genetic diversity and post-glacial co-expansion in marine turtles

A hawksbill turtle (Eretmochelys imbricata). Photo Credit: Banco de Imagens Projeto Tamar.

Marine turtle species exhibit differences in characteristics that could affect their sensitivity to climate change, such as size, generation time, diet, and thermal preferences. Research on nesting turtles has also shown that there are often multiple maternal lineages within a species, some spanning whole ocean basins and others much more restricted. These geographic differences could also have influenced past responses to climate change. We compiled data from 23 marine turtle lineages and compared the observed data to many simulated datasets to determine whether lineages were stable, expanding, or contracting over time. We then looked at which factors best predicted past population history and genetic diversity. We found evidence for population expansion in 60% of the lineages, with the remaining lineages stable over time. A co-expansion model showed that the lineages that expanded did so in a highly synchronous manner after the last Ice Age. Geographic factors (ocean basin and range extent) were much better predictors of population history and genetic diversity than species traits. So, where you were mattered more than who you were in determining response to global warming. This can inform conservation planning for these species and other marine organisms in the face of climate change.

For the full article: Reid BN, Naro‐Maciel E, Hahn AT, FitzSimmons NN, Gehara M. Geography best explains global patterns of genetic diversity and postglacial co‐expansion in marine turtles. Mol Ecol. 2019;28:3358–3370. https://doi.org/10.1111/mec.15165

Summary from the authors: Plant DNA-barcode library and community phylogeny for a semi-arid East African savanna

A DNA-barcode library is provided for the plant community of Mpala Research Centre’s semi-arid savanna ecosystem. Photo Credit: Tyler Kartzinel

African savannas represent iconic ecosystems comprising diverse plants and animals. Despite their importance to nature and people, the species that live in these ecosystems are relatively underrepresented in global biodiversity databases. To facilitate studies on the ecology and evolution biodiversity in East Africa, this international team of researchers developed a plant DNA-barcode library. We collected and identified 460 plant species from habitats across the ~200-km2 Mpala Research Centre in Laikipia, Kenya. These voucher specimens are archived at the National Museums of Kenya and the Smithsonian Institution. Based on these collections, we constructed a DNA-barcode library by sequencing 5 molecular markers from 1,781 vouchered plant specimens and generated 4,696 DNA sequences. This library increased the representation of plant DNA sequences from Africa within the Barcode of Life Database by nearly 10%. We demonstrated that these DNA barcodes are capable of discriminating between the vast majority of plant species present in this semi-arid savanna community and we used these sequences to infer a robust community phylogeny. We believe that this collection of plant voucher specimens, DNA barcodes, and the community phylogeny will support further research occurring both within this savanna ecosystem and across global biodiversity databases.

For the full article, see:
Gill, BA, Musili PM, Kurukura S, et al. Plant DNA-barcode library and community phylogeny for a semi-arid East African savanna. Mol Eco Resour. 2019;19:838-846. https://doi.org/10.1111/1755-0998.13001.

Interview with the authors: Parallel introgression and selection on introduced alleles in a native species

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.

West slope cutthroat trout. Photo by Ernest Keeley.

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

Summary from the authors: Divergence with gene flow is driven by local adaptation to temperature and soil phosphorus concentration in teosinte subspecies

A central question to evolutionary biology is how separate species form. Speciation is thought to occur after one population splits into two that then diverge over time. Divergence between the populations can be slowed by some evolutionary forces, such as when migrants share genes between populations, but it can be reinforced by others.  One reinforcing force is local adaptation; when a migrant from one population is not adapted to the environment of the second population, it may not reproduce successfully and share its genes.  To study the conflicting forces of migration and local adaptation, we assayed populations of two subspecies of Mexican wild maize, the teosinte, with genome-wide DNA.  Our results suggest that the two subspecies have diverged genetically despite continuous gene migration between them and that their divergence has been fueled by adaptation to contrasting temperatures and soil phosphorus concentrations. Genetic divergence between the two subspecies is particularly marked for five chromosomal regions that are enriched for genes that contribute to local adaptation. These regions have low recombination rates between populations, suggesting they could be chromosomal inversions. We conclude that Mexican teosintes may be undergoing the initial steps of the process of speciation, despite ongoing gene flow.

Male inflorescences of teosinte (wild corn) individuals from Huilotepec, Morelos, México. This population has intermediate genotypes between Zea mays ssp. mexicana and Zea mays ssp. parviglumis possibly as a result of gene flow between the subspecies.

Aguirre‐Liguori JA, Gaut BS, Jaramillo‐Correa JP, et al. Divergence with gene flow is driven by local adaptation to temperature and soil phosphorus concentration in teosinte subspecies (Zea mays parviglumis and Zea mays mexicana). Mol Ecol. 2019;28:2814–2830. https://doi.org/10.1111/mec.15098

Summary from the authors: Standing genomic variation within coding and regulatory regions contributes to the adaptive capacity to climate in a foundation tree species

Photos clockwise: a fully mature marri tree (Corymbia calophylla; by R. Mazanec), the characteristic large gumnut (by R. Davis), and the flowers (by R. Davis).

Marri, an economically and ecologically important tree in the southwest Australian floristic region, is under pressure from the effects of climate change, notably heatwaves and droughts. In this study we focus on understanding how this species is adapted to climate, providing information to develop evidence-based forest management policy, which aims to improve the tree’s persistence in a future climate. We used genomic techniques to explore genetic variation along climate gradients that are indicative of adaptation. We found adaptive genomic variants within and immediately up stream of functional genes that play key roles under temperature and water stress. Genomic variants within genes can lead to changes to the function of the protein themselves, providing opportunities for selection, while genomic variants upstream of genes may alter regulation, producing a varying amount of proteins. Both of these are important ways in which organisms are able to adapt to different climatic conditions. The amount of adaptive genetic variation within and around genes suggests the species can persist under the pressures and pulses of climate change with the help of evidence-based, proactive forest management.

Ahrens CW, Byrne M, Rymer PD. Standing genomic variation within coding and regulatory regions contributes to the adaptive capacity to climate in a foundation tree species. Mol Ecol. 2019;28:2502–2516. https://doi.org/10.1111/mec.15092

Interview with the author: Sex allocation plasticity on a transcriptome scale: Socially sensitive gene expression in a simultaneous hermaphrodite

Morphological evidence has long supported that simultaneous hermaphrodites invest more into the male sexual function at larger group sizes. In their recent work, Steven Ramm and colleagues use transcriptomic data link this morphological response to gene expression. Learn more about their study below, and in the full paper.

Ramm SA, Lengerer B, Arbore R, et al. Sex allocation plasticity on a transcriptome scale: Socially sensitive gene expression in a simultaneous hermaphrodite. Mol Ecol. 2019;28:2321–2341. https://doi.org/10.1111/ mec.15077

Sex allocation plasticity in Macrostomum lignano from a morphological perspective. The investment into testes (Te, blue) and ovaries (Ov, orange) can be readily quantified in these transparent flatworms (as illustrated in the inset) and used to derive a proxy for sex allocation (as testis area/[testis area + ovary area]). We confirmed that sex allocation varies significantly with the group size treatment (see main text for details). This represents a by now well‐established phenotypically plastic response that we here investigate further from a transcriptional landscape perspective 

What led to your interest in this topic / what was the motivation for this study? 
We’ve long since known about the ability of many simultaneous hermaphrodites to adjust their sex allocation at a morphological level, fine tuning their investment into their male and female sex functions according to cues in their social environment so as to maximise their total fitness returns. More specifically, at larger group sizes, individuals have to compete more to gain fertilisations and it therefore pays to shift investment from their female to their male sex function. The exciting prospect with this study was to be able to link this morphological response to the underlying plasticity in gene expression in organs such as the testis and ovary.

What difficulties did you run into along the way? 
One difficulty in switching from a morphological to a transcriptomic level of analysis was simply the sheer amount of data that we generated. We were measuring gene expression in tens of thousands of transcripts and found thousands of differentially expressed candidates that differed in expression according to the social environment, making it initially difficult to decide how best to focus our follow-up studies of the transcriptomic data.

What is the biggest or most surprising finding from this study? 
For me, one of the biggest surprises was that such a large proportion of the M. lignano genome is differentially regulated in its expression according to the social environment. There are different ways we measured that, but at least 10% of the transcriptome showed evidence for variable expression depending on something as simple as the number of other flatworms they regularly encountered. That’s of course both a blessing and a curse, since we’ve still got a big task ahead figuring out the functional roles of all those genes, and in particular the key gene expression changes within the subset of differentially expressed transcripts that really drive the plasticity.

Moving forward, what are the next steps for this research? 
One thing we’ve already been following up on in some detail is that alongside many transcripts which we expected to be plastically expressed in the testis and ovary (since these are the key organs for sex allocation), we found an additional large class of genes that were also highly plastic in their expression and are predominately or exclusively expressed in the tail of the flatworms. We’ve now found that many of these are expressed in the prostate gland cells responsible for seminal fluid production, another key component of male allocation, opening up the possibility of studying their functional and adaptive significance for sperm competition and sexual conflict.  

What would your message be for students about to start their first research projects in this topic? 
Be realistic about what that project can achieve. The ability to measure gene expression on a transcriptome scale has been a huge boon for the field, and opens up many exciting possibilities. But because we can now measure everything at once, there’s always a risk of drowning in data. If, for example, we would now follow up on all the plastically expressed genes we found in our study, that could easily fill several PhD projects. Clear questions and good experimental design become even more important as technology advances, not less.

What have you learned about science over the course of this project? 
The importance of collaboration. I’ve benefited from working with a great team of people spread across four different countries, which allowed us to combine several different techniques (morphological assays, RNA-Seq, in situ hybridisation, RNAi) in a single study. 

Describe the significance of this research for the general scientific community in one sentence.
Our project has begun to show how the dynamic allocation of resources to producing either sperm or eggs in hermaphroditic organisms occurs at the underlying level of the genes responsible for spermatogenesis and oogenesis, respectively. 

Describe the significance of this research for your scientific community in one sentence.
I hope our research can move sex allocation research forward on a couple of fronts: primarily, it offers a first – though still far from complete – glimpse into the mechanisms of phenotypic plasticity in simultaneous hermaphrodites; and second, it provides the starting point for deciphering the functions of a vast swathe of genes now implicated as being of adaptive significance for either the male or female sex function.