Monthly Archives: February 2020

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

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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. 

Thomas Maigret sits beside a copperhead (Agkistrodon contortrix) in Breathitt County, Kentucky, USA. Photo credit: Jocelyn Fortier.

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.

Two copperheads (Agkistrodon contortrix) sit inside a plastic bucket before having tissue collected for a study on spatial genomic patterns of copperheads across eastern Kentucky; Breathitt County, Kentucky, USA. Photo credit: Neva Williams.

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 authors: utilising GT‐seq for minimally invasive DNA samples

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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

Summary from the authors: Individualized mating system estimation using genomic data

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Mimulus guttatus

Hermaphroditic species of plants and animal can produce a mixture of outcrossed and self-fertilized offspring. Estimating the relative frequency of these two outcomes, i.e. the outcrossing rate, has been a major focus in the evolutionary study of reproductive strategies. Outcrossing rate is also a key parameter for plant breeding and for conservation efforts. This paper generalizes a Bayesian method to estimate outcrossing rate (BORICE) using genomic data. Application of the program to an experimental study of Mimulus guttatus illustrates estimation (10% of progeny were selfed), and also how inference of mating system parameters can set up “downstream” evolutionary studies. In the Mimulus study, these downstream analyses included pollination biology (the genetic composition of pollen changed over the season) and local adaptation (inversion polymorphisms exhibit unique patterns of micro spatial structure within the population).

-Professor John K Kelly, University of Kansas

Full article: Colicchio, J., Monnahan, P. J., Wessinger, C. A., Brown, K., Kern, J. R., & Kelly, J. K. (2020). Individualized mating system estimation using genomic data. Molecular ecology resources. https://doi.org/10.1111/1755-0998.13094

Interview with the chief editor: Molecular Ecology Resources (MER)

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In this special new-years post we interview the Chief Editor of MER Shawn Narum. Shawn, based at the Columbia River Inter-Tribal Fish Commission and the University of Idaho, has been chief editor for over 5 years. In this interview we get his perspective on the journal and the field in general as well as his advice for early career researchers.

See this link for a past interview with Shawn all the way back in 2014 with the Molecular Ecologist and this link for his 2020 editorial.

Image result for shawn narum

What are some of the main changes you have witnessed in the field of molecular ecology since you became Chief Editor of MER?

The advancement of molecular and statistical methods have driven the field of molecular ecology to new heights. Questions that were previously out of reach can now be addressed for most non-model species with careful study design.

What methods and resources do you think the field needs in the future?

Advances in sequencing methods have lead to fascinating discoveries of candidate genes associated with local adaptation and phenotypic variation many species, but development of candidate markers for intensive testing and validation is lacking. For example, bioinformatic resources are needed that efficiently and accurately develop primers/baits for specific subsets of markers that can be genotyped cost effectively in many individuals (e.g., Meek & Larson, 2019).

What are some of your favourite scientific discoveries from the past two decades?

Genomic islands of divergence are real! These islands often occur as inversions with low recombination that drive life history variation in organisms ranging from plants (Hoffmann & Rieseberg, 2008), birds (Lamichhaney et al., 2016), and fish (Jones et al., 2012)

As a fish geek, I also very much enjoyed the discovery that there is a warm blooded fish! It has long been known that some species like tuna and swordfish exhibit partial endothermy in brain tissue, but discovery of whole body endothermy in Opah living in cold, deep seas makes me smile (Wegner et al., 2015).

What advice would you give students wanting to develop a career in science?

Establish close collaborations with colleagues that you trust and nurture those relationships for the long-term.

What advice would you give to your younger-self about science and life?

Seize opportunities to work with others in a team environment, but it is OK to turn down some opportunities when there is already too much on your plate. “Too much” is when you can’t keep up with expectations that you have for yourself or projects substantially interfere with spending time with the people you love

What is your writing style like? Do you have some favourite writers that inspired you earlier on during your career?

My writing tends to be structured following a mental or written outline for clearly defined study questions. I have always been inspired by papers coming from Louis Bernatchez and have been grateful to have co-authored a few recent articles with him.

What are some of the aspects of your job as a scientist that you enjoy the most?

Two of the most rewarding aspects of my work are being involved with the development of young scientists and making new genomic discoveries that contribute towards conservation and recovery of naturally occurring species.

Outside of sequencing, what is your favourite methodological advance in the last five years?

Statistical advances that improve signal to noise in order to reduce false positives are critical to our field. One such approach called “Local score” was developed by Fariello et al (2017) to account for linked SNPs from high density genome scans to yield strong candidates (after Bonferroni correction). This is a powerful approach to detect adaptive genetic variation.

References

Meek, M. H., & Larson, W. A. (2019). The future is now: amplicon sequencing and sequence capture usher in the conservation genomics era. Molecular ecology resources. 19, 795–803.
https://doi.org/10.1111/1755-0998.12998

Hoffmann, A. A., & Rieseberg, L. H. (2008). Revisiting the impact of inversions in evolution: from population genetic markers to drivers of adaptive shifts and speciation?. Annual review of ecology, evolution, and systematics, 39, 21-42.
https://doi.org/10.1146/annurev.ecolsys.39.110707.173532

Lamichhaney, S., Fan, G., Widemo, F., Gunnarsson, U., Thalmann, D. S., Hoeppner, M. P., … & Chen, W. (2016). Structural genomic changes underlie alternative reproductive strategies in the ruff (Philomachus pugnax). Nature Genetics, 48(1), 84.
https://doi.org/10.1038/ng.3430

Jones, F. C., Grabherr, M. G., Chan, Y. F., Russell, P., Mauceli, E., Johnson, J., … & Birney, E. (2012). The genomic basis of adaptive evolution in threespine sticklebacks. Nature, 484(7392), 55.
https://doi.org/10.1038/nature10944

Wegner, N. C., Snodgrass, O. E., Dewar, H., & Hyde, J. R. (2015). Whole-body endothermy in a mesopelagic fish, the opah, Lampris guttatus. Science, 348(6236), 786-789.
https://doi.org/10.1126/science.aaa8902

Fariello, M. I., Boitard, S., Mercier, S., Robelin, D., Faraut, T., Arnould, C., … & Gourichon, D. (2017). Accounting for linkage disequilibrium in genome scans for selection without individual genotypes: the local score approach. Molecular ecology, 26(14), 3700-3714.
https://doi.org/10.1111/mec.14141

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

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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.