Interview with the authors: utilising GT‐seq for minimally invasive DNA samples

Minimally-invasive sampling is commonly used to obtain samples from rare, elusive or dangerous animals. However, this sampling technique often results in samples that are too low in quality or quantity for successful use with most high-throughput sequencing methods. Using cloacal swabs from the threatened Western Rattlesnake (Crotalus oreganus), Danielle Schmidt and colleagues show that Genotyping-in-Thousands by sequencing (GT-seq) can successfully be used to generate high-throughput sequence data from low-quality, low-quantity samples. We interviewed Danielle Schmidt (first author) and Professor Michael Russello (last author) to find out more about what went on behind-the-scenes of this study.

The Western Rattlesnake (Crotalus oreganus), a threatened species in British Columbia, Canada. Photo credit: Marcus Atkins

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

Conservation genomics has become an increasingly common term in the literature, yet many study systems that involve elusive or at-risk species must rely on minimally- or non-invasive sampling to meet research and management objectives. Although a valuable source of biological material, DNA extracted from minimally- or non-invasive samples is typically of low quantity, poor quality, and contaminated with exogenous DNA, all of which may be incompatible with modern sequencing technologies. Implementing leading-edge genetic and genomic tools to study conservation-related questions has been a long-standing interest in the Russello Lab.

What difficulties did you run into along the way?

Based on earlier work that came out of our lab (Russello et al. 2015 PeerJ), we suspected that employing a non-targeted sequencing approach like RADseq would not be efficient for collecting genotypic data from minimally-invasive samples. Therefore, we decided to test the efficacy of GT-seq (Campbell et al., 2015), as it is a targeted method that could help circumvent the typical issues involved with sequencing and genotyping lower quality DNA. Our biggest challenge was designing a GT-seq SNP panel that minimized ascertainment bias to ensure our downstream estimates of within- and among-population variation would be accurate. Also, given the number of samples and loci we planned to analyze simultaneously, optimizing the workflow for data collection took some time.

Library designs for A) RADseq and B) GT-seq. Included samples selected to facilitate within- and among-method genotype comparisons

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

One of the most surprising findings was the exceptionally high genotype consistency between paired blood and cloacal swab samples genotyped with GT-seq, and those blood samples genotyped with both RADseq and GT-seq. We even found that samples with initial concentrations as low as ~0.5 ng/uL successfully amplified, which is promising for future applications of GT-seq with minimally- and non-invasive DNA samples.

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

We are now exploring the application of GT-seq on a host of species to provide rapid, cost-effective genetic information to support research in molecular ecology and to assist wildlife and fisheries management. We are also testing the performance of this workflow with other non-invasive sample types, including feces and hair. Moving forward, we will be exploring ways of deploying these tools in the field to inform management decisions in real-time.

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

An important message we would like to convey is to think carefully about potential biases when designing a panel of markers to target, as the composition of your panel must be tailored to your research questions. For example, some applications of GT-seq may seek to intentionally maximize the among-population component of genetic variation in order to identify individuals of unknown origin to a particular fish stock with high confidence. In other cases, as with our study, we wanted a panel that could be used to most accurately reconstruct population structure and connectivity, which we were able to subsequently validate relative to a larger RADseq dataset.

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

This project highlighted the benefits of taking a new approach to address a long-standing challenge. In molecular ecology and conservation genetic studies, minimally-invasive sampling is commonly employed as either a required or a preferential approach for obtaining sufficient sample sizes. Yet, it has been recognized since the advent of non-invasive genetic sampling in the 1990’s that issues associated with DNA quality and quantity require careful consideration and extra quality control steps. Today, these considerations also apply to the use of modern DNA sequencing technologies from suboptimal starting material; however, GT-seq provides a versatile approach for overcoming DNA quality issues and providing the population-level data needed to address research and management objectives.

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

Multiplexed, amplicon DNA sequencing, such as that employed in GT-seq, is compatible with the minimally-invasive sampling often required for obtaining population-level data to inform biodiversity conservation.

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

GT‐seq offers an effective approach for genotyping minimally-invasive samples, providing accurate and precise estimates of within‐ and among‐population diversity metrics relative to genome-wide approaches such as RAD-seq.

Read the full study here:
Schmidt, Danielle A., et al. “Genotyping‐in‐Thousands by sequencing (GT‐seq) panel development and application to minimally invasive DNA samples to support studies in molecular ecology.” Molecular ecology resources (2020). https://doi.org/10.1111/1755-0998.13090

Interview with the author: Using host transcriptomics to sample blood parasites

Hosts offer diverse habitat for an incredibly rich array of microbial groups. Genomic resources for many groups residing within hosts (‘infra-communities’) are poor often due to the difficulty in isolating the DNA from the microbe from that of the host, particularly for species living within host cells. In this interview we go behind the scenes with Spencer Galen as he guides us through his transcriptomic approach he developed with colleagues to sample blood parasites such as malaria. Given how ubiquitous and important these parasites can be for animal health, this resource has the potential to pave the way for important advances in disease ecology. Read the paper here.

Avian blood transcriptomes revealed that hosts often have far more complex parasite communities than traditionally thought. For instance, the transcriptome of this Baltimore oriole (Icterus galbula) revealed at least six malaria parasite infections from three malaria parasite genera. The blood smear image from this bird shows the three genera in close contact within the host bloodstream. L: Leucocytozoon, PL: Plasmodium, PA: Parahaemoproteus.
Credit: Spencer Galen

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

This study began with two classic ingredients of scientific discovery: a lot of frustration mixed with a bit of inspiration from other researchers. The frustration was born from a lack of available genetic resources for malaria parasites and other blood parasites, which I felt was hindering the kind of research that I wanted to do. The inspiration came during the first year of my PhD, when several papers were published within a span of just a few months showing that researchers were passively generating large quantities of blood parasite genomic data by sequencing the transcriptomes of their vertebrate hosts. My PhD advisor Susan Perkins and I thought that designing a study to explore this approach in more detail could solve some of my frustrations and help the field of blood parasite research at large.

What difficulties did you run into along the way? 

When we started this project there was always the looming possibility that we would sequence a number of host transcriptomes that were infected with blood parasites and simply not recover any useful parasite data. Even a small-scale transcriptomic project is not a trivial matter financially, and so I will admit that I lost some sleep wondering if this project was a bad idea. Fortunately, field and lab work went quite smoothly, and the results of my first scan for parasites within our initial test transcriptomes exceeded my wildest expectations. And so in reality the biggest challenge was my own self-doubt – if I had paid too much attention to those thoughts, this project might not have gotten off the ground.

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

We were astounded by just how prevalent blood parasite transcripts can be within host transcriptomes. For instance, in one bird (Vireo plumbeus sampled in the mountains of New Mexico) we found that nearly 17% of all contigs generated from the initial Trinity assembly were derived from a parasite that was infecting just 0.75% of all blood cells. A second surprising finding was the degree to which many of the birds that we sampled were infected with complex communities of parasites that we did not detect using traditional microscopic and DNA barcoding methods. Across all samples we found that transcriptomes revealed about ~20% more infections than the methods that are typically used to study these parasites. This included one individual bird that was infected by three different genera and at least six species of malaria parasite.

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

While it is exciting to find that a transcriptomic approach can improve our ability to study the genomic diversity and abundance of wildlife blood parasites, it still remains a rather inefficient approach – at the end of the day, the majority of transcripts from each sample came from the host organism that was not the focus of our study. The next step will be to apply single-cell and other advanced RNA sequencing techniques that have successfully been applied to model systems to provide greater resolution to studies of blood parasite gene expression and host-parasite interactions.   

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

At risk of sounding overly pessimistic, be prepared for things to fail the first time around and have a plan B in place. It is wonderful to have a lot of confidence, but pessimism does tend to favor preparedness. Small actions within this frame of mind can save you a lot of grief in the long run, and can be as simple as testing a new method on a sample that isn’t important before you start your project or taking the time to visit a lab to learn a technique before you try it yourself. I naturally assume everything I try in the lab will fail, so each time things work (and they actually often do!) it is a pleasant surprise.

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

I think that there is a difference between producing a resource, and producing a resource that is easily accessible to the broader research community in practice. As a result, I spent a lot of time thinking about how my colleagues would most directly benefit from the data that we had generated. In the end we made the data from this study available in as many formats as we thought might be useful to other researchers (raw sequences, assemblies from before and after parasite identification, curated alignments, DNA barcodes, etc.). The amount of time that it took to prepare these datasets was extremely small relative to the length of the entire project, and I think will go a long way towards making these data as useful as possible.

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

This study improves our ability to research the ecology and evolution of wildlife blood parasites, a cosmopolitan and ubiquitous group that is widely relevant to global health.

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

The methodological framework that we present in this study profoundly improves the genomic resource base that is available to research understudied blood pathogens of wildlife, as well as better detect multi-species parasite communities within hosts.

Interview with the authors: response to amphibian-killing fungus is altered by temperature

Recently, Drs. Ellison, Zamudio, Lips, and Muletz-Wolz published their work focused on some of the ways amphibians respond to an infection by Batrachochytrium dendrobatidis (Bd). Bd is a fungus that is causing devastating worldwide decline of amphibians, meaning that understanding how some species manage the infection is important for conservation of myriad species. Using an elegant experimental set up and subsequent RNA sequencing data, Dr. Ellison and co-authors suggests that the variation in amphibian susceptibility to the fungus, which is related to temperature, occurs due concurrent temperature-dependent shifts in immune system function; lower temperatures were associated with an inflammatory response while higher temperatures with an adaptive immune response. Understanding exactly how and when this fungus alters wild amphibian populations is important for conservation of these often imperiled species. For more information, please see the full article and the interview with Dr. Ellison below. 

Eastern red-backed salamander (Plethodon cinereus). Photo credit: Alberto Lopez.

What led to your interest in this topic / what was the motivation for this study? I have always been fascinated with how parasites and pathogens influence fitness and shape host populations, particularly generalists infecting a wide range of host species. The pathogenic chytrid, Batrachochytrium dendrobatidis (Bd), is arguably one of the most generalist pathogens known to science, capable of infecting hundreds of amphibian species globally. However, even within a single host species, disease outcome (e.g. succumbing to or clearing infection) is highly variable and is often temperature-dependent. Given the devastating impacts Bd has already had on amphibian populations, the recent discovery of another amphibian-killing chytrid (B. salamandivorans), and the ever-pressing threat of climate change, we were driven to uncover how amphibian gene expression responses to chytrid infections vary under different temperatures.

What difficulties did you run into along the way? For me, it was the sheer scale of the sequencing dataset. Plethodon salamanders, notorious for their large genome sizes, had yet to have a published genome or transcriptome to use as a reference for RNAseq studies such as ours. Therefore, we had to ensure sufficient sequencing to de novo assemble the transcriptome, and enough per-sample depth to capture potentially subtle but important changes in gene expression due to temperature and infection. With multiple temperature treatments and multiple disease outcomes at each temperature, this resulted in relatively large RNAseq dataset of over 2 billion reads. Thankfully, having returned to Wales from the US by the time we received our sequence data, I had access to Supercomputing Wales, a nationwide high-powered computing initiative that allowed me to handle the computationally intensive analyses. More importantly, without the hard work of the other authors to carefully design and execute the highly-controlled animal experiments to generate the tissue samples, this study would simply not be possible.

What is the biggest or most surprising innovation highlighted in this study? I think that, within a relatively narrow thermal range, the substantial shifts in the types of immune genes being expressed in response to infection is really important to our understanding chytrid infection dynamics. The finding that adaptive immune transcripts (particularly those involved in MHC pathways) are more highly expressed at warmer temperatures – where amphibians tend to survive infection better – is most exciting. Given the growing evidence for the importance of certain MHC allele variants in Bd resistance, our results suggest it is not only be what MHC genotype amphibians possess, but how they express them during infection that dictates survival.

Moving forward, what are the next steps in this area of research? This study, while providing new insights into how temperature influences Bd-amphibian interactions, has generated many further questions. Some of the authors on this study have recently shown both temperature and Bd has a significant impact amphibian skin microbiome communities, a potentially critical line of defense against infections. It is currently unknown whether temperature-dependent host immune expression responses to Bd shapes skin microbiomes during infection or if skin bacteria are influencing host responses (or a combination of both). Work to directly assess host gene expression under different microbial community compositions would be an exciting future avenue of research. In addition, further investigation of both MHC genotype and expression phenotype simultaneously could be highly relevant to understanding intraspecific variation in chytrid resistance. Finally, we have previously developed methods to quantify Bd gene expression in vivo; it would be fascinating to couple our current findings with how Bd genes are expressed in-host under different temperatures.

Dr Carly Muletz-Wolz field sampling. Photo credit: Karen Lips.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? Many others on this blog have already highlighted the importance of well thought out experimental designs, and the need to grips with the theory before embarking on a project, that I can only echo. Although having now worked on many transcriptomic datasets in non-model organisms, I still sometimes get overwhelmed with the amount of information that could be potentially conveyed in a manuscript, particularly with more complex experimental designs such as this study. I recommend periodically taking a step back from your analyses, share it with colleagues to gauge the most important “headline” results, and finally, don’t worry that some things have to go as supplemental material; they can still be gems of information that kick-off an exciting new line of inquiry for someone!

What have you learned about methods and resources development over the course of this project? With high-throughput sequencing methods becoming ever more accessible and the explosion of innovative ways to analyse and present NGS data, it is all too easy to feel your project is not “cutting-edge” enough. It’s all very well having billions of sequences and a slick set of figures, but a research team most importantly needs to be able to provide meaningful biological/ecological interpretation. That’s why it has been great to be part of a collaborative team of amphibian ecologists and geneticists, which was critical to the development of this new resource of information on salamander transcriptomic responses to temperature and infection.

Describe the significance of this research for the general scientific community in one sentence. The thermally-altered transcriptional responses of salamanders to fungal pathogen infection is an important component to understanding observed seasonal and climatic patterns of chytrid disease outbreaks. 

Describe the significance of this research for your scientific community in one sentence. Our results suggest shifts from inflammatory to adaptive immune gene expression responses to Bd infection at warmer temperatures are a key component to thermal and/or seasonal patterns of amphibian chytridiomycosis.

Eastern red-backed salamander (Plethodon cinereus). Photo credit: Dr Carly Muletz-Wolz.

Ellison A, Zamudio K, Lips K. Muletz-Wolz C. 2019. Temperature-mediated shifts in salamander transcriptomic responses to the amphibian-killing fungus. Molecular Ecology 28:50586-5102.

Interview with the author: Creating the SPIKEPIPE metagenomic pipeline

Reliable abundance estimates is a significant challenge for eDNA metagenomic studies. One important issue is that sequencing introduces multiple sources of noise that can significantly alter the accuracy of abundance estimates. Here we interview Douglas Yu, a professor at the University of East Anglia, about the SPIKEPIPE pipeline recently published in Molecular Ecology Resources. This method is particularly exciting as it can use either short read barcodes or mitogenome data to estimate species abundances by accounting for sequencing noise using correction factors. They test this eDNA pipeline on arthropod samples taken from the High Arctic in Greenland and show that this approach can produce remarkably accurate species abundance estimates compared to samples of known composition. Read the full article here and get the code to run this pipeline here.

image
The 5 steps of SPIKEPIPE.

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

We very much want to know how a heating climate is affecting biodiversity. Greenland is a direct window into this, both because heating has progressed very fast here, and because local species richness is manageable for study:  375 known aboveground arthropod species at the Zackenberg research station. Equally important, the Danish research station at Zackenberg had had the foresight to systematically collect arthropods starting in 1996, and those samples were sitting in ethanol in a warehouse in Denmark. The main obstacle to using them had been that no one could identify the hundreds of thousands of individuals to species level. Luckily, Helena Wirta and Tomas Roslin had in parallel carried out a DNA barcoding campaign at Zackenberg. Put together, we had in our hands a complete time series of community dynamics over a stretch of time during which summer had almost doubled in length. 

What difficulties did you run into along the way? 

When we started, we were all set to use metabarcoding. However, we soon learned (not surprisingly) that the sample-handling protocols had not been designed with molecular methods in mind:  the trap water was reused across time periods, the collecting net was used across traps, and the sorting trays were not bleached between samples. We thus needed a protocol that would be robust to cross-sample contamination and would ideally return quantitative information, since we wanted to detect change in population dynamics. This is why we turned to mitochondrial metagenomics (Tang et al. 2015, Crampton-Platt et al. 2016) and came up with SPIKEPIPE, which combines read-mapping, a percent-coverage detection threshold, and a spike-in to correct for pipeline stochasticity. 

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

The individual elements of SPIKEPIPE were reasonably well known, but what we hadn’t anticipated is just how accurate the results were when combined in a single pipeline. With mock samples, we found no false-positive species detections (when the percent-coverage threshold is applied) and recovered highly accurate estimates of intraspecific abundances (in terms of DNA mass). With resequenced environmental samples, we found high repeatability of abundance estimates across sample repeats, even though DNA extraction and Illumina library prep, sequencing, and base-calling all inject stochasticity into datafile sizes.

Also very gratifying was finding that SPIKEPIPE returned useful data even when mapping reads only to short DNA barcodes, as originally presaged by Xin et al. (2013). This means that we can make use of the existing vast DNA-barcode reference library.

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

SPIKEPIPE is of course only the means to an end, and our next goal is the statistical analysis of community change in a rapidly heating ecosystem. Nerea Abrego and Otso Ovaskainen are now applying joint species distribution modelling (with the R package Hmsc, Tikhonov et al. 2019) to the dataset of 712 pitfall-trap samples. One important question is to quantify how much of the year-to-year variation in species abundances can be attributed to species interactions, as opposed to climate variables. 

More broadly, the result that SPIKEPIPE can be used with DNA barcodes makes possible an intriguing strategy:   one may now generate both the species reference database and the sample-by-species table from the same set of samples. We are using Greenfield et al.’s (2019) Kelpie software to carry out targeted assembly of DNA barcodes from shotgun-sequenced bulk samples, which we compile into a single DNA-barcode reference database, against which we then map reads from each sample to generate the data table. 

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

Build in a lot of testing:  multiple, complex mock samples for pipeline development, repeat environmental samples to measure repeatability, realistically complex positive controls, many negative controls, and many sanity checks as you work through your bioinformatic code. 

You are likely to be learning to code at the same time that you write your first pipelines. Take the extra time *now* to learn and apply robust coding techniques, even if there are easier but less robust methods available. 

Read Jenny Bryan’s tutorial on file naming:  https://speakerdeck.com/jennybc/how-to-name-files

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

A great way to inspire new methods is to talk with non-molecular researchers about their scientific questions, currently used methods, and available sample types. Our team includes arctic ecologists, molecular ecologists, and a mathematician.

For one’s method to have impact, it will need to be useful for years after one first thinks of it. Stay up to date with technology trends, including costs, to avoid rapid obsolescence.

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

We can use DNA sequencing to quantify how insect and spider communities respond to environmental change.

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

Mitochondrial metagenomics is a viable alternative to amplicon sequencing for characterising arthropod communities. 

Interview with the authors: Massive introgression of major histocompatibility complex (MHC) genes in newt hybrid zones

Hybridization is a mechanism by which adaptive alleles can cross species boundaries and possibly boost the adaptive potential of hybridizing species. This may be especially true for alleles that confer a selective advantage when rare, which is common among major histocompatibility complex (MHC) genes involved in pathogen defense. We therefore would expect MHC genes to introgress across hybridizing species relatively easily, though there exists relatively few examples supporting this hypothesis. In this paper from Molecular Ecology, Katarzyna Dudek, Tomasz Gaczorek, Piotr Zieliński, and Wiesław Babik document the extent of introgression in MHC variants across two hybridizing European newts across replicated transects. Read below for a behind-the-scenes look at their paper!

Link to the study: https://onlinelibrary.wiley.com/doi/full/10.1111/mec.15254

F1 hybrid male. Photo from M. Niedzicka

What led to your interest in this topic / what was the motivation for this study? 
The evolutionary significance of adaptive introgression is increasingly appreciated and many examples have been described, but few generalizations are available. There is a relatively well understood mechanism – novel/rare allele advantage – which should promote introgression of genes evolving under balancing selection (a prime example of these are MHC genes). However balancing selection itself produces signatures resembling introgression, so convincing demonstration of introgression in genes under balancing selection is difficult. Hybrid zones, especially in the form of replicated transect, are among the best tools you can imagine for such a project. And we’ve been studying these newts for some time – in a way this study was motivated by our long standing interest in adaptive introgression, but it’s an off-shoot of another project (see the paper in the same issue of Mol. Ecol.).

What difficulties did you run into along the way? 
The most difficult part was the design and justification of simulations that we used to rule out explanations alternative to introgression. Because MHC in newts is multi-locus and shows extensive copy number variation, it’s been difficult to design simulations that would at the same be time realistic and feasible. This may sound surprising, but genotyping and interpretation of MHC variation has not been a major problem, although the system is quite complicated. It seems that the field has matured enough that exon-based genotyping of MHC variation has become a standard. Another frontier would be population genetic analysis of entire MHC haplotypes, extremely interesting but currently beyond reach in non-model (and most model) taxa.

Field sampling. Photo from M. Liana

What is the biggest or most surprising finding from this study? 
The scale of apparently adaptive introgression. It’s not only that MHC variants introgress – we have suspected this before. One could expect that a single or a handful of novel, introgressed MHC haplotypes would be favoured in the recipient species, but we found massive introgression, apparently involving tens or more haplotypes, most likely in both directions. It’s been quite a surprise for us – this suggests that introgression can really remodel MHC variation in hybridizing species – an influx of large amount of variation may cause species to share, at least locally, pool of MHC variation.

Moving forward, what are the next steps for this research?
A natural next step is to test generality of our findings. The mechanism of novel/rare allele advantage should operate rather universally. If so, we expect that MHC genes will be among the last genes to stop introgressing between species that still hybridize, but are strongly reproductively isolated genome-wide. In other words we expect MHC introgression should be detectable (and perhaps strong) in systems, where despite hybridization, there is very little genome-wide introgression. We’ve been lucky to obtain funding for a collaborative project, in which we are going to test this prediction using over twenty hybrid zones from major vertebrate groups. We’d also like to look at the process at the level of entire haplotypes, but this would need to wait until technologies mature.

Albino L. montandoni male. Photo from W. Babik

What would your message be for students about to start their first research projects in this topic?
The most important would probably be: have your questions worked out and if you find a system that is good to address them – go for it. Try to understand the available theory, there’s nothing more practical than good theory to guide you and to save countless hours of your precious time. And finally, start writing before you think you’re ready. Writing is the best way to have your ideas clear, to spot weak points and see things you didn’t realized before.

What have you learned about science over the course of this project? 
Over and over again – that science is unpredictable. That reality mocks your well laid out ideas and plans, twisting and turning your paths, but if you recognize and follow the opportunities that appear on the way, everything will be fine :). For example something that appears as an offshoot of a major project may turn out at least equally interesting and important. Two key components are good and diverse collaboration and the scale of research appropriate to your question – that is just large enough to provide sound answers, but not necessarily larger.

Field sampling pt 2. Photo from W. Babik

Describe the significance of this research for the general scientific community in one sentence.
Our research suggests that MHC introgression may be a widespread process that introduces novel and restores previously lost variation, boosting the adaptive potential of hybridizing taxa.

Citation
Dudek, K., Gaczorek, T.S., Zieliński, P. and Babik, W., 2019. Massive introgression of MHC genes in newt hybrid zones. Molecular Ecology. 28(21). 4798-4810. https://onlinelibrary.wiley.com/doi/full/10.1111/mec.15254

Interview with the authors: RAD‐sequencing for estimating genomic relatedness matrix‐based heritability in the wild: A case study in roe deer

Working on non-model organisms comes with both challenges and rewards. While the joy and satisfaction of uncovering knowledge in wild populations drives many scientists, the lack of genomic resources can be a roadblock for many important research themes, such as determining the extent of evolutionary potential and response to selection. In this paper from Molecular Ecology Resources, Laura Gervais and co-authors demonstrate the potential for RAD-sequencing to overcome these challenges and estimate heritability and evolutionary potential in wild populations, even for non-model organisms without many existing genomic resources. Read below for a behind-the-scenes look at their paper!

Link to the study: https://onlinelibrary.wiley.com/doi/full/10.1111/1755-0998.13031

Image result for Capreolus capreolus
Photo of male and female roe deer (Capreolous capreolus) from Wikimedia Commons

What led to your interest in this topic / what was the motivation for this study? 
We are interested in how natural populations adapt to environmental changes. These changes occur rapidly and there is an urge to accumulate results on wild populations’ capacity of adaption for a wide range of species. Traditionally, measuring the evolutionary potential of a trait required long-term field surveys of phenotypic data and genetic relatedness obtained from a multi-generational pedigree. This is challenging to obtain because many free-ranging populations are hard to sample with the intensity required for pedigree reconstruction. We believe that genome-wide data and in particular RAD-sequencing data might be an opportunity to overcome this issue but we still lack an accessible practical framework to go from genomic data to the estimation of a population’s evolutionary potential.

What difficulties did you run into along the way? 
We had to overcome two main methodological difficulties. First, to investigate the effects of the sequencing strategy and the SNP calling/filtering procedure ultimately on GRM-based heritability, we had to run a considerable amount of bioinformatic and quantitative genetic analyses, which both proved to be time consuming. Secondly, there was not much methodology available on how to implement genomic relatedness matrix in a quantitative genetic linear mixed model. We hope that our work will make this approach more easily accessible.

What is the biggest or most surprising finding from this study? 
When we started the study, we did not expect that it would be possible to run genomic quantitative genetic analyses with only a few hundred individuals. Most of our colleagues were skeptical when we mentioned that we found significant heritability (at the beginning with only 170 genotyped individuals). Our results give hope that evolutionary potential studies in the wild might be virtually accessible for any natural population when using the appropriate sampling and sequencing design.

Moving forward, what are the next steps for this research?
We are working to combine genome-wide data with intensive bio-logging technology (data on animal movement) and high-resolution habitat information. The synergy between these three high-density data technologies offers a great opportunity to understand how species adapt to environmental changes across complex landscapes.

What would your message be for students about to start their first research projects in this topic?
Our message would be to never hesitate to contact people and surround yourself with all the necessary help. This is a domain that evolves rapidly and that is very exciting but may be quite disconcerting. It seems essential to remain informed and open-minded. Lastly, I would say that self-learning is really rewarding but that there is always the opportunity to ask for help to learn and get over a problem efficiently.

What have you learned about science over the course of this project? 
We have learned that more interdisciplinary exchanges between ecologists, molecular biologists and bioinformaticians are useful and can help to build such an integrative approach. This may be challenging as they often have different views on different issues that need to be conciliated. There is a need to meet and exchange ideas to get the most out of this type of projects.

Describe the significance of this research for the general scientific community in one sentence.
This study sheds light on a unique opportunity to evaluate whether species have the genetic potential to adapt to environmental changes, and this for virtually any non-model organism.

Citation
Gervais, L., Perrier, C., Bernard, M., Merlet, J., Pemberton, J. M., Pujol, B., & Quéméré, E. RAD‐sequencing for estimating genomic relatedness matrix‐based heritability in the wild: A case study in roe deer. Molecular Ecology Resources. 19(5). 1205-1217. https://onlinelibrary.wiley.com/doi/abs/10.1111/1755-0998.13031

Interview with the authors: testing the role of ecological selection on color pattern variation

The color variation that exists among individuals has lent itself to the study of selection since Darwin. Recently, Zaman, Hubert, and Schoville (2019) investigated the effects of selection on the diversity of the wing color pattern in the butterfly Parnassius clodius across a large portion of its range. These researchers found evidence supporting the idea that coloration may serve as a warning signal to predators, providing some predator avoidance benefits to individuals. In addition, the variation of solar radiation and precipitation observed geographically across sites was negatively correlated with the amount of melanin observed at each site. This suggests that the occurrence of melanin may provide a selective advantage in the form of thermoregulatory function. For more information on how selection influences butterfly wing coloration, please see the full article and the interview with Dr. Schoville below. 

Parnassius clodius mating, male located below female. Photo by Sean Schoville.

What led to your interest in this topic / what was the motivation for this study? Khuram and I were both interested in butterfly color pattern variation, and in particular, cases where there might be competing selective pressures acting on wing pattern phenotypes. Most work on butterfly wing patterns focuses on predator-prey interactions and aposematic colors, but butterfly wings are essential to flight performance and important in thermal regulation. A number of recent papers have shown that butterfly color pattern appears to be responding to climate warming, and then there are well-known cases (such as alpine Colias, thanks to Ward Watt) where thermoregulation has been linked to basking behavior and the pigment on wings. Thus, in examining variation in Parnassius clodius (which occurs over a broad elevation and latitudinal range), we hoped that we could decouple environmental signals that might act on different wing color elements. 

What difficulties did you run into along the way? Sampling our butterflies across this large region was a major challenge, particularly as adult flight times are rather short (two to three weeks). And then we were surprised by the strong difference in adult phenology across sites (some adults are active in May, others in late July). This is evident regionally (Utah versus Washington), and across elevation within a region. In the end, it required three years of effort, with some very long road trips from Wisconsin.

What is the biggest or most surprising innovation highlighted in this study? After some initial efforts to link aposematic variation (red eyespots) indirectly to predator communities (through climate variables that might covary with predator abundance), we realized this was too tenuous. So, we were delighted to discover publicly available data on bird abundance. While this did not solve the problem (perhaps due to lack of spatial resolution in the bird data), I think using this data to analyze butterfly wing patterns was one of the more innovative aspects of our paper. We had a much easier time linking spatial climate data to melanization (dark pigmentation). As an aside, this raises the important point that some data, i.e. abiotic environmental data, is much easier to come by than biotic data. This is unfortunate, as we expect biotic selective forces to be equally or more important drivers of microevolution in some cases.

Lead author on this study, Khuram Zaman, after a day of collecting samples. Photo by Sean Schoville.

Moving forward, what are the next steps in this area of research? We’d like to extend this work in two directions. First, we’d like to connect color pattern traits to underlying genes and measures of heritability. Although the genes controlling butterfly color pattern are well studied, to date no representative of the snow Apollo subfamily Parnassinae have been included in these efforts. Members of the family are tremendously variable and quite stunning in their dramatic contrasts of color. Second, experiments are needed to link our inferences of ecological selection to fitness differences, as well as performance in the field. Physiological assays of melanic variants, coupled with mechanistic thermodynamic models, have been developed for Colias butterflies (see Joel Kingsolver and Lauren Buckley’s work). This type of modeling could provide important connections to conservation of Parnassius clodius populations under changing climates, and might perhaps extend to conservation work on other highly threatened Parnassius species.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? The development of novel approaches is a key part of advancing biological knowledge, but it can be a daunting endeavor given the breadth and scope of the scientific literature nowadays. Integrating multiple approaches, on the other hand, can equally help to advance our knowledge and provide opportunities to address long-standing questions. This is the direction we took in this paper. My personal view is that students should to try to master multiple techniques (assemble a toolkit, so to speak) and apply those techniques to fundamental problems. Hopefully, it’s a lot of fun in the process and leads to interesting collaborations!

What have you learned about methods and resources development over the course of this project? We are entering a golden age of data-rich resources, in terms of spatial environmental data and genomics data. These increasingly provide the power to test refined hypotheses about evolutionary and ecological processes, and are becoming more accessible to all researchers. One of my favorite accomplishments in the paper is using genetic covariance data among populations (relatedness data) as a covariate in fitting morphology ~ environment models. The use of such population contrasts is important in controlling for non-independence in the data due to ancestry. While we have known about the importance of genetic covariance in hypothesis testing for some time (thanks to Joseph Felsentstein’s work), it is only recently possible to use genome-wide data. This provides very precise measures that are highly informative, and enabled us to rule out the role of genetic drift as a driver of wing pattern variation. 

Describe the significance of this research for the general scientific community in one sentence. Our research demonstrates that butterfly wing color patterns evolve in response local climate conditions, as a way to regulate body temperature.

Describe the significance of this research for your scientific community in one sentence. Our work demonstrates that elements of butterfly wing pattern phenotypes respond independently to different sources of selection, with climate variation acting on thermoregulatory ability as an important driver of butterfly color pattern.

Parnassius clodius basking to thermoregulate. Photo by Sean Schoville.

Zaman K, Hubert MK, Schoville SD. 2019. Testing the role of ecological selection on color pattern variation in the butterfly Parnassius clodius. Molecular Ecology 28:50586-5102.