Interview with the authors: Which software is best to use for de novo assembly?

Reduced representation sequencing (e.g. RAD and GBS) is becoming ever more popular, but for species which lack a reference genome, little work has been done to assess which software may be best suited to building de novo assemblies from this data. Here, we speak to Melanie LaCava of the University of Wyoming about her recent Molecular Ecology Resources article, which explores the accuracy of de novo assemblies built by various software programs using DNA generated from double-digest libraries. Melanie and her co-authors found highly variable degrees of accuracy of assemblies built by six different software programs, and discuss which programs are best suited to this application. They also highlight the importance of optimising parameter settings within any given software. Read on to get a behind-the-scenes view of this study.

The completeness of assemblies in simulations of unmutated genomes (a, d), in simulations of an equal number of SNPs and indels (b, e), and simulations of 1–5 base pair indels (c, f). Values are reported for five assemblers: CDHIT (green), STACkS (blue), STACkS2 (purple), VelVeT (pink) and VSeARCH (orange). The hue of each color corresponds to the percent match parameter setting used in the assembly. For more information on this figure go to Figure 1’s caption here.

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

This study began as a research project in a graduate-level course on computational biology at the University of Wyoming led by the senior author on the paper, Alex Buerkle. Dr. Buerkle initiated the project and worked with the rest of the coauthors to pursue this de novo assembly software comparison. As reduced representation genotyping-by-sequencing has become more popular, new and repurposed software programs have been applied to each step in the bioinformatics pipeline. When a reference genome is unavailable for a study species, de novo assembly is essential, yet we recognized a gap in the evaluation of software used for this important step.

What difficulties did you run into along the way? 

Technology and software associated with genotyping-by-sequencing and de novo genome assembly are rapidly changing. During the course of our project, some of the software programs we tested were significantly updated, so we chose to rerun our analyses using the new software versions to ensure we were providing up-to-date information in our manuscript.

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

We were surprised to find such a substantial difference in performance among these assembly programs. We were especially surprised at the variation in performance among software for our first simulation where no mutations were introduced. In this scenario, we made many identical copies of genome fragments and then performed de novo assembly using each software program. Without any mutations introduced, the job is basically to generate a list of unique sequences – it should be very straightforward. In some cases, however, these genome fragments were broken into shorter sequences and rearranged beyond recognition, leading to incorrect reconstruction of the simple, unmutated data.

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

For our study, we selected a sample of assemblers from peer-reviewed literature that use different assembly algorithms, are freely available, and have updated user resources available online. However, this was not a comprehensive evaluation of all software capable of de novo assembly. Therefore, the evaluation of other programs would be valuable. Additionally, as new software programs are introduced or existing programs are updated, continued efforts to evaluate de novo assembly performance is warranted.

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

Reduced representation genotyping-by-sequencing is becoming less expensive and more accessible, making it a viable option for more research projects. While it is exciting to apply these emerging technologies and methods, it is important to recognize that approaches to filter and analyze these large datasets are still in development. Doing your background research to ensure you are applying the best available tools and using the most appropriate methods for your study is essential to doing good research in this field and in any field of research.

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

Doing this study has reaffirmed the importance of simulations to test how software works. Testing analyses on simulated data and altering parameters of the simulation or analysis can provide immense insight into how the software works and how variation in real data may affect software performance. Larger simulation projects like our study can provide information that many people can use, but I also find it incredibly helpful to run a simulated dataset through an analysis before analyzing my own data to ensure I understand what the software is doing. Taking advantage of simulated datasets available in vignettes for software is a great tool to get acquainted with the analyses you plan to do.

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

Our study demonstrates the importance of ensuring that software you use is really doing what you think it is supposed to do; and simulations can help evaluate software performance.

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

Researchers who need to perform de novo assembly of reduced representation genotyping-by-sequencing data can use our study as a guide for which software to use and the importance of different parameter settings for assembly.

LaCava, M. E., Aikens, E. O., Megna, L. C., Randolph, G., Hubbard, C., & Buerkle, C. A. (2019). Accuracy of de novo assembly of DNA sequences from double‐digest libraries varies substantially among software. Molecular ecology resources. https://doi.org/10.1111/1755-0998.13108

Interview with the authors: How does invasiveness evolve? A look at feral pigs

Understanding how and why some species readily invade new habitats is an interesting view into the myriad ways species evolve. Limiting the expansion of such introduced species can be important for managing ecosystems, particularly when the invasive species is as ecologically destructive and economically costly as the feral swine in the US south. In a paper published recently in Molecular Ecology, researchers led by Dr. Tim Smyser investigated the origins of the invasive feral swine populations to determine how much the expanding footprint of this species was a due of recently escaped domesticated pigs. Surprisingly, they found that the expanding range was largely attributable to range expansion by the established invasive swine population. Read on to for more details from Dr. Smyser into this very interesting work!

Invasive feral swine originated from a combination of European feral pigs and domesticated stock. Photo by Dr. Mirte Bosse, dvdwphotography.

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

Invasive feral swine have expanded rapidly throughout the United States over the past 30 years. The impetus for the study was to identify the drivers for that expansion, to ask: where are new feral swine populations coming from? Prior to our work, there was a hypothesis that domestic pigs had sufficient phenotypic plasticity that they would revert to a wild phenotype, resembling a wild boar, if living in the wild. Under this hypothesis, any pig farm could have served as a viable source population for invasive feral swine. With this study, we revealed that there is very little direct contribution to invasive feral swine populations from domestic pigs, potbellied pigs, or wild boar. Rather, the rapid expansion observed over the past 30 years has been driven by incremental range expansion of established invasive feral swine, which overwhelmingly represent animals of mixed European wild boar-heritage domestic breed ancestry, and long-distance translocation of feral swine from established populations to uninvaded habitats.

What difficulties did you run into along the way? 

The challenges were largely computational. We had amassed over 9,000 genotypes by the time we compiled the reference set and generated genotypes from invasive feral swine genotypes for this study. Such a large dataset required that we do everything we could to optimize runtime efficiency. Even with these efforts, the analysis still took about 4 months of runtime while using 30 CPUs with 60 threads.

What is the biggest or most surprising innovation highlighted in this study? 

I would say the most surprising result was the very high proportion of invasive feral swine that had a significant ancestry association to European wild boar. The historical record suggests wild boar releases have been far more limited than the potential for domestic pig releases, yet 97% of feral swine had significant European wild boar ancestry. This might suggest hybrid wild boar-domestic pig ancestry is biologically important for feral swine to establish self-sustaining populations and become invasive.

Moving forward, what are the next steps in this area of research?

Descending from this work, our next steps are multifaceted. With this analysis, we have identified the drivers of range expansion at a broad-scale with ancestry results pointing to the expansion of established populations. We are now interested in adding a fine-scale understanding of expansion to identify the specific sources of newly emergent populations and map the patterns of feral swine expansion. Also, this analysis has provided an understanding of the ancestral composition of invasive feral swine. Given the hybrid origin of these animals, we will identify elements of the genomes from their ancestral groups, that is heritage breeds of pig and European wild boar, that have been selectively retained in feral swine. By describing selective sweeps relative to ancestral groups, this analysis will allow us to describe the evolution of invasiveness among feral swine.   

What would your message be for students about to start developing or using novel techniques in Molecular Ecology?

The field of Molecular Ecology is changing so quickly that it is hard as a scientist to keep up, from both a computational/statistical standpoint and with all the new molecular techniques and analyses that allow us to dive deeper into the genome than we had previously imagined. My recommendation for students would be to not let the lack of a specific skill deter you from asking interesting questions – take the time to develop the needed skill sets or develop collaborations to facilitate your learning or use of those skills. Also, keep asking questions – don’t be content with the answers we are able to resolve today.

What have you learned about methods and resources development over the course of this project? 

Reflecting back on my answer immediately above, when I started asking the question of what are the drivers of invasive feral swine range expansion, I did not have the data or the skills to meaningfully address that question. Through the development of a great team of collaborators and independent learning, I was able to assemble the needed skills and then the data to pose this question and reveal interesting results. Through this project, I learned about the statistical tools used in the analyses, developed the coding skills necessary to execute those analyses, and identified strategies to maximize computational efficiency as was needed for working with such a large dataset.

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

We have demonstrated that the recent and rapid expansion of feral swine, an ecologically destructive and economically costly invasive species distributed throughout much of the US and the world, has been facilitated by movement (in many cases anthropogenic movement) from established populations to uninvaded habitats as opposed to novel introductions of either domestic pigs or wild boar.

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

In identifying the admixed origins of invasive feral swine, descending from heritage domestic pig breeds and European wild boar ancestry, we can begin to gain an understanding of the evolution of invasiveness for this species and invasive species more broadly. 

Feral Swine are not native to the U.S. They are the result of recent and historical (1500’s Spanish explorers) releases of domestic swine and Eurasian boar. USDA APHIS photo Laurie Paulik.

Smyser TJ, Tabak MA, Slootmaker C, Robeson MS, Miller RS, Bosse M, Megens H-H, Groenen MAM, Rezende Paiva S, Assis de Faria D, Blackburn HD, Schmidt BS, Piaggio AJ. 2020. Mixed ancestry from wild and domestic lineages contributes to the rapid expansion of invasive feral swine. Molecular Ecology. https://doi.org/10.1111/mec.15392

Interview with the authors: can we identify the acting selective regime in evolution experiments?

Rapid adaptation to novel conditions is an exciting and growing area in evolutionary research due, at least in part, to our desire to understand the effects of climate change, introduced species, and other conservation-related concerns. However, our ability to detect this evolution is fraught with both biological realities and technical difficulties. A recent paper by Drs. Pfenninger and Foucault, published in Molecular Ecology, illustrate how deep resequencing of replicated experimental populations can fail to provide evolutionary insights, even with extreme selective pressures, due to adaptation to unintentional environmental conditions that overwhelm the genomic signals of the intended selection. This rapid adaptation, in this case to captivity, is an interesting phenomenon that is almost certain to alter other experimental systems, including those that take place in the field. In addition to more details on this fascinating study, the interview with Dr. Pfenninger below also provides an interesting view into technical issues the research team faced: a short time after the initial publication of their manuscript, they discovered a bug in allele frequency calling software that they used! 

Swarming flight of Chironomus midges over a small puddle. Photo credit: Markus Pfenninger.

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

I wanted to know whether rapid adaptation of a natural population to an environmental stressor, in this case temperature, is possible and, if so, by which processes in detail. Apart from this being a fundamental question in population genetics, it is a crucial issue for biodiversity in the ongoing global change.

This is the official and completely true answer – but, to be completely honest, not the entire story: I wanted to see, analyse and prove evolution by natural selection hands-on. Because it’s one thing to teach something gleaned from literature and another to have seen it with your own eyes.

What difficulties did you run into along the way? 

There were actually quite a few: finding a suitable PhD student, technical difficulties with the experimental facilities that almost killed the long term experiment after some months, to mention only the most important ones.

And finally, of course, the almost detrimental issue with a bugged software tool: A few days after the official publication in January, a student reanalysing the data from a different angle, stumbled over unexplainable inconsistencies between the raw data and the allele-frequencies inferred from them. You can imagine the shock it gave me!

When I looked into the problem, I quickly found out that the allele frequencies had little to do with the raw data for most, but perfidiously not all positions in the genome, in particular not the first few on the first scaffold – that’s why the error escaped my attention during a cursory check. The allele frequencies were extracted by a software tool which indeed produced consistently wrong results – a task in principle so simple that systematic checking would have required to write a second tool that exactly did what the first should have done in the first place.

I immediately contacted the authors of the tool and they promptly confirmed that the version we used contained this bug. They did nothing wrong, though. Once they discovered the bug a few months ago, they had promptly updated the tool and documented the error in the release notes. In fact, it appears that the wrong version was on the server for a few days only. Unfortunately it was exactly during the time when we downloaded it – and who looks into the release notes after a tool seemingly did without a hitch what it was supposed to?

I had no choice but to contact the editorial office of Molecular Ecology, informing Genevieve Horn that parts of the publication were flawed and should probably be retracted. At the same time, I started reanalysing the complete data set with a correct version of the tool. Fortunately, after a hard week of number crunching, it turned out that the wrong values were highly correlated in terms of location and allele frequencies to the true values so that some numerical values, but none of the study’s conclusions, needed to be revised. The journal agreed that in this case, a correction article would be sufficient and here it is.

I have to say that everyone, from the software authors to the editor in chief, I have dealt with in this affair has responded greatly and I want to express my deep gratitude here. Given this experience with Molecular Ecology, I can only encourage everyone to address such unfortunate as perhaps unavoidable mistakes immediately and openly.

What is the biggest or most surprising innovation highlighted in this study? 

The rather unsettling major result of the study was the realisation that it is nearly impossible to experimentally manipulate the selection regime of a natural population in a targeted, predictable manner. I think, however, that such “failures” finally advance science by showing which approaches are worth pursuing and which not. Besides this more philosophical aspect showed the study the impressive power of rapid polygenic adaptation.

Moving forward, what are the next steps in this area of research?

I am currently moving into analysing population genomic time series from the field to get an idea on the selective forces acting on natural populations.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology?

Have a good plan, be ready to revise it once the plan meets reality and be prepared for setbacks, remain critical about your results and incorporate appropriate controls. But perhaps most importantly, always take your time to think what you are currently doing and what should be the next steps.

What have you learned about methods and resources development over the course of this project? 

Obviously to even more thoroughly back-check every single analysis. Beyond this, I realised the value and potential of population genomic time series analysis.

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

An evolutionary experiment tells you something about the experiment – not necessarily about nature.

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

The acting selective regime in evolutionary experiments is difficult to predict and to manipulate – but perhaps it may be inferred from the results.

Pfenninger M and Foucault Q. 2020. Genomic processes underlying rapid adaptation of a natural Chironomus ripariuspopulation to unintendedly applied experimental selection pressures Molecular Ecology 59:536-548. https://doi.org/10.1111/mec.15347

Summary from the authors: inbreeding and management in captive populations

Pacific salmon hatcheries aim to supplement declining wild populations and support commercial and recreational fisheries. However, there are also risks associated with hatcheries because the captive and wild environments are inherently different. It is important to understand these risks in order to maximize the success of hatcheries. Inbreeding, which occurs when related individuals interbreed, is one risk that may inadvertently be higher in hatcheries due to space limitations and other factors. Inbred fish may have reduced fitness and survival compared to non-inbred fish. We quantified inbreeding and its effect on key fitness traits across four generations in two hatchery populations of adult Chinook salmon that were derived from the same source. We utilized recent advancements in DNA sequencing technology, which provide much more precise estimates of inbreeding and its potential effects on fitness. Our results indicate that inbreeding may not be severe in salmon hatcheries, even small ones, provided that appropriate management practices are followed. However, we documented an influence of inbreeding on the phenology of adult spawners, which could have biological implications for individual fitness and population productivity. Our findings provide a better understanding of changes that may occur in hatchery salmon and will further inform research on “best” hatchery practices to minimize potential risks. 

Article: Waters CD, Hard JJ, Fast DE, Knudsen CM, Bosch WJ, Naish KA. 2020. Genomic and phenotypic effects of inbreeding across two different hatchery management regimes in Chinook salmon. Molecular Ecology https://doi.org/10.1111/mec.15356.

Interview with the authors: barriers to fox gene flow in urban and rural settings

In an article published recently in the latest issue of Molecular Ecology, researchers from Researchers from the Leibniz Institute for Zoo and Wildlife Research and the Luxembourg National Museum of Natural History investigated differences between urban and rural red fox populations. They found that physical barriers in both habitats, such as a river or road, limited fox movement, and also that human activities influenced where foxes moved. This is important because it means that the interaction between human activity and other structures on the landscape may negatively alter the fox populations. For more information, please see the full article and the interview with lead author Sophia Kimmig below. 

A red fox (Vulpes vulpes) moving along rail roads in the city centre of Berlin, Germany. © Jon A. Juarez.

What led to your interest in this topic / what was the motivation for this study? Human population growth and land use are altering ecosystems worldwide and although continuing urbanization results in dramatic environmental changes, some species seem to cope well with the anthropogenic pressure. Foxes are distributed over the entire metropolitan area of Berlin, therefore it is usually assumed that they cope well with human presence. However, city life can affect key aspects of wildlife ecology and have substantial impact on the movement ecology and dispersal ability of populations. Dispersal in urban areas may be influenced by physical barriers, but also by behavioural barriers that we cannot directly see. Thus species that are physically capable of crossing the urban matrix may nevertheless face behavioural barriers due to avoidance of man-made objects (with their artificial structure, scents etc.) as well as human presence per se. We therefore wanted to understand how the landscape influences gene flow patterns in red foxes across the urban-rural matrix.

What difficulties did you run into along the way? With an increasing number of population genetic clustering approaches and R packages that differ in their precise working mechanisms, it becomes more challenging to interpret diverging results and recognize biological patterns. Further, the promising and fascinating possibilities of modelling gene flow through the landscape also come with uncertainties in how to deal with certain circumstances or type of data. For example, we discuss the issue of dealing with overlapping landscape features in the studied environment i.e. linear landscape elements (such as roads or rivers) that cross a surface structured landscape element (e.g. a forest or park). Especially in urban areas, the habitat has such a high level of complexity that you could easily spend years modelling and testing different land use layers.

What is the biggest or most surprising innovation highlighted in this study? Regarding the fox in the Berlin Metropolitan area: Foxes are quite common in urban areas, so we presumed that there would be few dispersal barriers in the urban environment. Our results have nevertheless shown that foxes disperse preferentially along linear landscape elements such as motorways and railway lines despite the inherent mortality risk. We interpreted this to mean that even urban foxes avoid the presence of humans if possible. 
Regarding a broader, biological perspective: Although we have to further improve our methods (for our study, for example, by including data on population densities, road traffic or other proxies of human presence and activity), it is fascinating that molecular genetic methods may enable us to answer more questions in behavioural ecology in the future. 

Moving forward, what are the next steps in this area of research? Now that we have familiarised ourselves with the landscape genetic techniques, we are looking forward to applying the approaches to a broad range of taxa to better understand how animals move through the landscape. This is not just of academic interest, but may help to identify and protect dispersal corridors for endangered species in a scientifically robust way.
For the Berlin foxes we are going to analyse data from a radio tracking study and research their movement patterns and space use – it will be interesting to compare those results with the ones from landscape genetics. We are looking forward to hopefully adding some more pieces to the puzzle of the city as a wildlife habitat.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? From a beginners’ perspective: For our project, we greatly benefitted from the exchange with other researchers working in this field. For example, we contacted William Peterman, who created the ResistanceGA package that we used for our landscape resistance analysis, with some methodological questions and he provided very helpful advice. I would therefore recommend getting in touch with people who work with the same methods and discussing your ideas and obstacles. Also, our work greatly benefitted from the thorough review process that it underwent. Although the requested changes and suggestions sometimes may come with a lot of re-thinking and -working effort and we usually do not always agree with every single given comment, it is crucial to take constructive criticism to improve our scientific work.

What have you learned about methods and resources development over the course of this project? Molecular genetic methods and the inherent potential to study complex ecological contexts have been changing a lot in the last decades. This is a field of frequent on-going development and improvement. Especially regarding the analytical methods, for an ecologist it is difficult to keep on track with all the latest approaches. Also, due to big data involved in the landscape analysis and the resulting time for computational analysis, the computational effort for a model becomes a real issue in landscape genetics. It is really a pity when more thorough analysis are theoretically possible and even free data is available but the analysis can just not be conducted in a feasible amount of time.

Describe the significance of this research for the general scientific community in one sentence. Assessing the impact of the habitat on (urban) wildlife beyond the physical properties of the landscape may help us to more deeply understand dispersal, behaviour and population genetic structure of populations.

Describe the significance of this research for your scientific community in one sentence. Methodological advancement due to more in depth comparisons of different genetic measures used in resistance modelling.

Kimmig ES, Behinde J, Brandt M, Schleimer A, Kramer-Schadt S, Hofer H, Börner K, Schulze C, Wittstatt U, Heddergott M, Halczok T, Staubach C, Frantz A. 2020. Beyond the landscape: resistance modeling infers physical and behavioral gene flow barriers to a mobile carnivore across a metropolitan area. Molecular Ecology. https://doi.org/10.1111/mec.15345.

Interview with the authors: historical barriers to gene flow in a fragmenting landscape

In a recent issue of Molecular Ecology, Drs. Maigret, Cox, and Weisrock published their work focused on copperhead snake response to habitat fragmentation. Interestingly, these researchers detected population structure putatively resulting from a historically important highway, even though most traffic has been shuttled to an alternative route for the last 50 years. Understanding the complexities of movement patterns in response to barriers is of increasing importance as our landscape becomes more and more fragmented. For more information, please see the full article and the interview with Dr. Maigret below. 

What led to your interest in this topic / what was the motivation for this study? The immense and rapid shift from forest to barren land and grassland which accompanies surface mining in central Appalachia is striking, especially when viewed from the air. Upwards of 20% of the land surface of some counties has been mined since 1980 through a process often termed “mountaintop removal”. The lack of research on the implications of this fragmentation was curious to me: why had such a major driver of forest loss garnered so little attention? Moreover, if we use next-generation sequencing, could we detect any effects of this land-use change on wildlife populations? It seemed like a nice natural experiment waiting to be investigated.

What difficulties did you run into along the way? Fieldwork was challenging: on top of the issues one deals with when trying to capture large numbers of secretive venomous snakes, nearly all the land in our study area is privately held, and thus gaining access to properties to collect tissue samples was time consuming. In terms of generating our data, obtaining enough DNA from our tissues (mainly scale clips) proved to be a challenge, though DNA quality was fortunately not an issue. Finally, given the diverse array of methods and subsampling protocols we used, optimizing our software pipeline took a little extra time. Thankfully, our university’s computing resources – including our associated staff and faculty – were more than adequate for the task at hand.

What is the biggest or most surprising innovation highlighted in this study? We found no evidence for an effect of mining or the current array of high-traffic roads on genetic differentiation; both of these features were hypothesized to be barriers to movement. But the most surprising part was what we did detect: a break in population similarity spatially coinciding with the path of a road which was a major highway for most of the 20th century. Previous research has suggested that highways can cleave populations of herpetofauna, and modeling work has suggested that these effects could persist for many years. We seem to have found evidence for a combination of these hypotheses, and subsampling suggested that we could have come to a similar conclusion with fewer markers and more missing data.

Moving forward, what are the next steps in this area of research? It will be interesting to see what unfolds as more genomic data is integrated into landscape genetics studies, and especially in landscapes with putative barriers of different ages or permeabilities. Re-analysis of existing data sets using (possibly) more sensitive methods, like the spatially-informed methods we used, might reveal barriers where none were detected using other approaches. As for surface coal mining, more study of the consequences of forest fragmentation – ideally, using species which might be more sensitive – could be very informative.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? Try to keep abreast of the new programs coming out. It seems like every month new approaches are being developed, and while the deluge of methods can be overwhelming at times, employing an assortment of different approaches can help enlighten one’s interpretation of genomic patterns.

What have you learned about methods and resources development over the course of this project? I’ve learned about the importance of integrating methods within an ecological framework. While a new method for analyzing genomic data is usually developed to fill a particular analytical gap, translating that goal into an ecological framework can make the method much more accessible to a broader range of researchers. And in general, doing one’s best to stay on top of the new methods coming online is important, if a little overwhelming at times.

Describe the significance of this research for the general scientific community in one sentence. Our results seem to suggest that the genomic legacy of human settlements and infrastructure can persist in wildlife populations beyond the lifespan of the infrastructure itself.

Describe the significance of this research for your scientific community in one sentence. With genomic data and statistical approaches that integrate spatial information, it might be possible to detect relatively weak genetic structuring in wild populations, and it may not require large amounts of the highest-quality data.

Maigret TA, Cox JJ, Weisrock DW. 2020. A spatial genomic approach identifies time lags and historical barriers to gene flow in a rapidly fragmenting Appalachian landscape. Molecular Ecology. https://doi.org/10.1111/mec.15362.

Interview with the author: Landscape genomic signatures indicate reduced gene flow and forest‐associated adaptive divergence in an endangered neotropical turtle

The Dahl’s Toad-headed turtle (Mesoclemmys dahli) is one of the 25 most endangered turtle species on Earth and one of the unlucky few that is constantly being compared to a toad. A previous study has shown that extensive habitat conversion and fragmentation in its native range in Colombia has led to genetic differentiation among populations. However, critical questions remain for the conservation and management of this species: how does habitat type and quality in this rapidly changing region influence gene flow? How can genomic data inform the management of this endangered species?

Dr. Natalia Gallego García (@NataGalle) and colleagues set out to answer these questions. Read below for an interview with Natalia about the challenges of working with such a rare species and how this study provides the foundation for a genetic rescue program for the Dahl’s Toad-headed turtle.

The critically endangered Dahl’s Toad‐headed turtle (Mesoclemmys dahli). Photo by N. Gallego García

What led to your interest in this topic / what was the motivation for this study? 
We conducted a previous population genetics study using microsatellite loci on this highly endangered species, in which we found significant genetic evidence of population fragmentation. In this follow-up study, we wanted to know how the current landscape, now composed of open grasslands for cattle instead of tropical dry forest, might be restricting gene flow and thus causing the observed fragmentation. We also wanted to know how this new anthropogenic environment was potentially driving local adaptation.

What difficulties did you run into along the way? 
The main difficulty was finding this rare species across its range to get the samples. Another difficulty was standardizing the RADseq protocol, as our research was the first genomic-level study on any side-necked (pleurodiran) turtle. Also, there is no reference genome for this species or for any closely related one, making the analysis of our data difficult. We had to assembly a de novo genome, which prevented us from running other analyses that could have allowed us to learn more about adaptive mechanisms to new environments.

What is the biggest or most surprising finding from this study? 
First of all, we were able to show that population fragmentation was related to habitat loss. However, we were expecting movement through grasslands to be costly, given that this is a forest species, but we found that from a scale of 1 (easy) to 1000 (hard) the cost of traversing grassland was only 13. We believe that this low cost is associated with the presence of water ponds built in the pastures for the cattle to drink, which are increasingly being used by this species. These ponds might be serving as a sort of stepping stone array of lower quality but still usable aquatic habitat, enabling movement over an otherwise hostile matrix. Our second surprising finding was observing possible adaptive divergence between populations occupying areas with more forest than populations in areas with almost no forest. This result suggests that the populations might be adapting to this new transformed environment. However, adaptation alone is not rescuing this species from the negative effects of fragmentation, and currently the species is facing a high risk of extinction.

Moving forward, what are the next steps for this research?
The next steps can be divided in terms of management and research. In terms of management, we are currently designing a genetic rescue program to reduce inbreeding and increase population genetic diversity, without disrupting the potential ongoing adaptation that we observed. In terms of research, we are currently assembling the genome of a closely related species, which will allow us to map the putatively adaptive loci found, and better understand how this species is adapting to its new transformed environment. This will also allow us to design a field and/or laboratory experiment to further explore the possibility of adaptation to altered, and degraded habitat.

What would your message be for students about to start their first research projects in this topic? 
Working with non-model, rare, and threatened organisms, although challenging, can lead to valuable information that is vital in their conservation. So, accept the challenge and stand up for those forgotten species. Any new information on a data deficient species will increase its chance of survival, which in itself already makes the research worthwhile.

What have you learned about science over the course of this project? 
Science always comes with exciting surprises that do not always comply with our expectations, and it usually leaves more questions than answers. But it is gratifying to contribute, even in a small way, to the understanding of complex processes that can eventually be applied to solve difficult problems, such as the conservation of an endangered species.

Describe the significance of this research for the general scientific community in one sentence.
Adaptation to habitat change can happen, but perhaps not quickly or completely enough to overcome the negative effects of population reduction and fragmentation.

Describe the significance of this research for your scientific community in one sentence.
Landscape genomics analyses provide evidence of reduced gene flow in a fragmented habitat, leading to harmful effects on a critically endangered neotropical turtle, despite its possible adaptation to the new anthropogenically created environment.

Citation
Natalia Gallego‐García, Germán Forero‐Medina, Mario Vargas‐Ramírez, Susana Caballero, & Howard Bradley Shaffer. (2019). Landscape genomic signatures indicate reduced gene flow and forest‐associated adaptive divergence in an endangered neotropical turtle. Molecular Ecology, 28(11), 2757-2771. https://onlinelibrary.wiley.com/doi/10.1111/mec.15112