Friday, March 31, 2017

Dissecting phases of speciation in stick-insects

I have worked on many different study systems over the years, including killer whales, livebearing fishes (family Poeciliidae) and Timema stick insects (for more detail please see my homepage). Originally, I started my research career with a Diploma in Biology in Germany (equivalent to a Masters in other countries), with my thesis work focusing on whistle communication in diverging killer whale populations around Vancouver Island in British Columbia, Canada. However, I soon realized that there are only so many questions you can ask using a study system that largely precludes running controlled experiments. Thus, for my PhD thesis and subsequent postdoctoral work, I focused on population divergence and speciation in livebearing fishes (Poeciliidae) living along various environmental gradients (e.g., gradients of predation, toxicity, and access to light). It was during this time that I realized that my main interest was not so much in one particular study system, but rather in discovering the mechanisms that create, maintain, and sometimes constrain, biodiversity. That interest eventually led me to add yet another study system to the mix in 2012: the system of Timema stick insects I am writing about here. More specifically, I wanted to use that particular system to study the potential role chemical communication might have on population divergence and speciation.

As the above paragraph suggests, each of my study systems has their own system-specific peculiarities [e.g., cultural differences seem to play a prominent role in driving population divergence in killer whales (Riesch et al. 2012), but not in stick insects or livebearing fishes]. However, they also have a lot in common. For example, selection from predation is integral to both, the livebearing-fish and stick-insect systems I study, while foraging specialization plays a prominent role in population divergence of both, stick insects and killer whales. Thus, the three systems simply constitute different examples of how ecologically-based divergent selection drives population divergence and ultimately (ecological) speciation.
A rain shower moving through the chaparral near Santa Barbara, California. Timema are often found in this biome of dense thickets and thorny bushes.
The idea that speciation can be thought of as a continuum is yet another concept that dates back at least to Charles Darwin’s world-changing On the origin of species. The concept in its modern form posits that pairs of populations move along a continuum between panmixis on one extreme end and complete reproductive isolation on the other. Progress can be towards speciation or towards collapse, the latter showcased by studies on speciation reversal in European whitefish Coregonus spp. (Vonlanthen et al. 2012), three-spined stickleback Gasterosteus aculeatus (Tayler et al. 2006), and cichlid fishes (Seehausen et al. 1997).

This concept of a speciation continuum has gained traction again in recent years (e.g., Hendry et al. 2009). Consequently, studies across closely related taxa at different phases of speciation are beginning to illuminate the processes and genetic changes underlying the formation of new species (Seehausen et al. 2014). It is well-known, of course, that speciation involves genetic differentiation, and that, in the absence of gene flow, genome-wide differentiation can readily build up by selection and drift. If speciation is to happen in the face of gene flow, however, the picture gets more complex. According to the genic model of speciation (Wu 2001), speciation is initiated by a few genetic regions that become resistent to gene flow before others. This results in a localized pattern of genetic differentiation, which becomes more genome-wide as speciation progresses.

In a recent study just published in the April issue of Nature Ecology and Evolution (, we took a closer look at the transitions between phases of genomic differentiation during speciation of Timema stick insects. Like other studies on the speciation continuum (including my other study systems), we were faced with a key problem: speciation is often slow enough that we cannot simply follow a single lineage through time to see in real-time how the process unfolds. The solution then is to take as many different snapshots of the process from different pairs of natural populations as possible, and to then start to reconstruct a bigger picture of what might be happening across different moments in time. This is exactly what we did using data from >100 populations of 11 species of Timema stick insects. Our work suggests that speciation can be initiated by few genetic changes associated with natural selection on few loci, but the overall process is multi-faceted and involves mate choice and genome-wide differentiation.

A male Timema cristinae on one of its host plants (genus Ceanothus).
Photo: Moritz Muschick
This study is the culmination of almost 30 years of research into this system, and consists of data collected between 1996 and 2014, including >1000 re-sequenced whole genomes. In fact, research in this system began when two of our coauthors, Cristina Sandoval (University of California in Santa Barbara, USA) and Bernie Crespi (Simon Fraser University, Canada), recognized this group harbours variation in phases of speciation. Patrik Nosil then entered the system in 2000 and eventually wrote his PhD on it, using experiments to estimate reproductive isolation. An important component of the current paper was the chemical ecology of stick insects. This part of the project was born ~2009, emerging out of initial discussions between Patrik and I, with additional input from Bernie Crespi and Gerhard Gries at Simon Fraser University. Fast-forward to the year 2012, where the alignment of different projects getting funded finally enabled us to team up at the University of Sheffield in the UK (key components were a European Research Council Grant to study the genomics of speciation to Patrik, a Human Frontier Science Program Postdoctoral Fellowship to study the role of chemical communication in speciation to myself, and a burgeoning collaboration with Zach Gompert, a statistical population geneticist from Utah State University).

The emphasis of the chemical ecological aspect of the project was on cuticular hydrocarbons (CHCs), the oily/waxy chemicals on the cuticle of insects that can function to prevent desiccation and physical injury (Drijfhout et al. 2013 in Behavioral and Chemical Ecology, pp. 91-114), but that have also been repeatedly implicated as integral to mate choice (e.g., Blows and Allan 1998; Chung et al. 2014). For stick insects, we found that populations that differed more strongly in their CHC profiles also had higher degrees of sexual isolation and stronger genome-wide differentiation. We confirmed the causal role of CHCs in mate choice by means of a perfuming experiment.

Evaporating hexane samples in Santa Barbara, California, as part of the perfuming experiment on Timema mate choice.
Preparing another CHC-hexane sample for analysis with the gas chromatograph in the Gries lab at Simon Fraser University in 2014. Photo: Sean McCann

When combining this CHC-data with other phenotypic data and genomic analyses, we uncovered that, consistent with early phases of genic speciation, colour-pattern loci that confer camouflage to particular host plants reside in localised genetic regions of accentuated differentiation between populations experiencing gene flow. Transitions to genome-wide differentiation are also observed with gene flow, but appear to have little to do directly with differentiation in color. Rather, genome-wide differentiation is associated with divergence in CHCs, which we show to be polygenic, modestly heritable traits. Thus, intermediate phases of speciation are not associated with growth of a few peaks or ‘islands’ in the genome. Finally, we show that complete reproductive isolation was associated with a conspicuous increase in the overall degree of genomic differentiation. Thus, although speciation is perhaps continuous, this does not mean it always proceeds in a strictly uniform fashion (this component was led by our collaborators Moritz Muschick, now a postdoctoral fellow at EAWAG in Switzerland, and Victor Soria-Carrasco, who is now a Leverhulme Early Career Fellow at the University of Sheffield). Overall, the results suggest that substantial progress towards speciation may involve the alignment of multi-faceted aspects of differentiation. We suspect similar conclusions may apply to other systems where strong reproductive isolation involves many traits and evolves in a polygenic fashion.

A female Timema bartmani, cryptic against the needles of white fir. Photo: Moritz Muschick.

In conclusion, although many questions remain unanswered, it seems clear that speciation in this group involves more than divergence in cryptic coloration, and the results point to mating isolation and other reproductive barriers, as well as geographic separation, as being important. Thus, the striking example of crypsis in Timema that has been the focus of many previous studies (e.g., Sandoval 1994; Nosil and Crespi 2006; Comeault et al. 2015) represents only one aspect of the multi-faceted speciation process.

Friday, March 24, 2017

Roads go ever ever on

As a grad student—at a time when journals were rapidly disappearing from print in favor of online-only formats—I set two pseudo goals for myself: one was to publish a paper (period!) in an actual, printed journal, and the other was to have a paper featured on a cover. As a tremendous amount of luck would have it, Jonathan Richardson and I recently managed to pull off this feat. This past month, in a paper in Frontiers in Ecology and the Environment, we advocate for evolutionary perspectives in the ever-growing field of road ecology.

Roads as drivers of evolutionary change! With a paper hot off an actual press and featured on a cover, the author of this post is overly giddy to tick off two bucket list items.

The motivation for this paper (besides our personal view that road-induced evolution is really fascinating and probably pervasive the world over) is that evolutionary perspectives have generally been lacking in road ecology. While road ecologists have furnished a wealth of knowledge about the vast suite of road effects impacting the planet, insights into evolutionary consequences have typically been overlooked. And from what we can tell, that vast suite of effects is a recipe for evolutionary change.

But before we dive into the topic of road-induced evolution, let's first consider why roads matter from a conservation perspective. As always, it seems prudent to turn to a passage from Tolkien for an inspiring prelude. Thankfully, in The Hobbit, Bilbo Baggins returns from his journey singing a song about roads:

       Roads go ever ever on,
       Over rock and under tree,
       By caves where never sun has shone,
       By streams that never find the sea… (Tolkien 1938)

True to form, Tolkien's words speak as much about present day as days of yore. Roads most certainly do go ever ever on, and indeed can be found among the most remote and picturesque places. Said to be the largest human artifact, roads traverse some 64,000,000 km across the planet. When we consider the numerous negative ecological effects caused by roads (see below), it is easy to see why this global network of pavement and dirt has earned the nickname 'the giant embracing us'. 

The global road network -- an intertwining matrix of highways and byways -- is well described by its (underutilized) nickname 'the giant embracing us'.

In the United States (which hosts about 10% of the global road network), one percent of the landscape is occupied by road infrastructure. At first blush, this may not sound like much; after all what could one percent really amount to? Well, in this case, quite a lot. In terms of shear magnitude, this one percent constitutes 14,000,000 lane km of roads. Said another way, the U.S. has enough road materials to pave a 25-lane highway (with a 12-lane unpaved shoulder) from here to the moon.

In the United States alone, there are enough roads to pave a 25-lane highway (and 12-lane soft shoulder) from Earth to the Moon.

Across this massive network, roads cause numerous, negative ecological effects. From road kill to contaminant laden runoff to fragmentation effects; from the spread of invasive species to the spearheading of land development to impacts on gene flow; roads are something of a one-stop shop for human impacts on the environment.

Among the various ways we utilize and modify the landscape – from agriculture to urbanization to forest harvest – roads are rather unique in that their effects are far-reaching despite having a relatively small footprint. Indeed, road effects extend well beyond the road surface and verge. For example, in the United States, that one percent of the area covered by roads is estimated to ecologically influence 20% of the landscape.

Roads are also unique in that their impacts can be substantial in ares that are otherwise undisturbed. Whether in contexts of highways bisecting large tracts of undeveloped land between distant destinations, or forest roads zigzagging through protected landscapes, roads and their impacts can be found among the most beautiful places on the planet. Indeed, while the impacts of roads may seem obvious in urban places, the aesthetic of our beloved country roads may belie their insidious nature (sorry, John Denver). All of this is to say that there are an awful lot of roads out there causing an awful lot of ill effects in an awful lot of places.

Road effects are many. And many of those effects likely act as agents of natural selection (making roads ripe for evolutionary study!). 

Before I go any further on this road-bash, I have to make a confession: I love roads (and John Denver, just to be sure). As much as there is to hate about roads and their nasty ecological effects, there are easily as many things to love about them. Roads are the great journey-makers, the great soul-soothers, the great inspirers. Roads welcome all traveler alike. Roads do not judge, they do not play favorites, and they rarely let us down. Roads have shaped our economies, our cultures, and arguably our collective conscience. I might even go so far as to say that roads are the veins on the planet through which we humans course—with all of our hopes, dreams, and love.

Personally, I’ve put more miles on my cars than the ecologist in me cares to admit (although decidedly fewer miles than the road-tripping climber in me would prefer). Few feelings can compete with the first glimpse of an iconic destination after hours or days on the road. Whether rounding the corner to that first view of a massive granite cliff or cresting a hill to that first sight of the ocean, roads are a path to something that resonates deep inside us. Roads can also be great places to think. I find few things as mind-cleansing or idea-provoking as a long, quiet drive (or a bike ride for that matter). Undoubtedly (and surely out of necessity at times), some of my best ideas have come to me on long drives.

Yet another hazard of roads: the out-the-window selfie. The author, boat in tow, blazes across the country for the umpteenth time, chasing the sun, the open road, and the promise of possibility that lies ahead. 

Long before roads appealed to my adventurous side (and about the time when my siblings and I lived in fear of the phrase 'scenic route'), I was struck by the potential for roads to impact nature. I can remember the uneasy feeling I would get as a child watching a plow truck pass by, leaving a blanket of salt in its wake.

At that time (mid 1980s), ecologists had not paid much attention to road effects. In fact, despite the long history of roads on our landscapes, it was not until the mid 1990s that the ecological impacts of roads drew the eyes of many ecologists. As the story goes, it was 1994 when Richard T. T. Forman (the 'father of road ecology') was driving to the annual ESA meeting (via the scenic route, no less!). On that fateful drive, Forman—impressed by the scale of the canyon road before him—envisioned the critical need for an ecological understanding of road effects. (A perfect example of what roads can inspire!)

Contrasting Forman's vision, the ESA meeting left him disappointed: of the more than 2000 presentations that year, only one had the word ‘road’ in its title. This disappointment was apparently converted rapidly into inspiration and action. Less than a decade later, Forman and colleagues had amassed enough information on the ecological effects of roads to write the formative book on the subject (Forman et al. 2003). Since that time, the field has advanced steadily, unveiling the diverse ecological consequences of the net of roads blanketing modern landscapes. Road ecology institutes have formed, reviews have been published (e.g. Formanand Alexander 1998, Trombulak and Frissell 2000), and practical guides have been written (Andrews et al. 2015, van der Ree et al. 2015). Yet amid this wealth of knowledge, one insight remains conspicuous by its absence; namely, evolutionary change caused by roads. Indeed, my experience at a recent ESA meeting left me feeling disappointed much like Forman; among the dozens of talks on road effects, only one mentioned evolution.

The field on road ecology has grown steadily since its inception in the 1990s (inset). While many effects are well described, road ecologists have only scratched the surface of evolutionary consequences of roads.

This absence of the evolutionary consequences of roads is surprising for a couple of reasons. First, other contexts of conservation have been hot on the trail of human-induced evolution for some time now. And second, some of our earliest knowledge of human induced evolution was actually discovered in roadside habitats. Studies by Briggs (1972), Wu and Antonovics (1976), Atkins et al. (1982), and Kiang (1982) all demonstrated rapid, adaptive evolutionary responses to road runoff contaminants (road salt and lead). Strikingly, all of these studies occurred back in the 1970s and early 1980s! But as far as we can tell, there was little evolutionary road activity after this early group of studies.

Until now. In 2013, Brown and Bomberger Brown reported that during a 30-year period, the number of road-kill cliff swallows decreased despite an increase in overall population size. At the same time, wing length of road-kill swallows increased while that of the overall population decreased. Together, these patterns suggest an evolved response to selection favoring increased maneuverability and vertical takeoff achieved by shorter wings.

At about the same time as the cliff swallow paper, I was finding evidence in my own work suggesting that roadside populations of amphibians were evolving in response to road-adjacency and road salt. In reciprocal transplant experiments, roadside spotted salamander populations showed a local adaptation pattern whereas roadside wood frog populations (in the same suite of ponds as the salamanders!) showed a maladaptation pattern (Brady 2013; 2016). From my perspective, evidence for maladaptation in roadside wood frogs was both confusing and surprising (detailed in another Eco-Evo Evo-Eco post), particularly since it occurred in counterpoint to the adaptive response of the co-inhabitant salamanders. 

These outcomes of adaptation and maladaptation occurring side by side are part of our argument for the need to consider evolutionary change in the context of road ecology. In essence, the severity of the consequences of roadside dwelling appear to be decreasing for some species but increasing for others. These contrasting patterns occurring in response to the same form of habitat conversion (ie roads) highlight the complexity induced by road effects that are only unveiled through the lens of evolution.

Our goal in writing this paper is to advocate for a new way of thinking about road effects. It is our hope that questions aimed at understanding road effects will be guided by a recognition of the evolvable nature of populations, both in adaptive and maladaptive directions. Indeed, shortly after our paper was published, I was heartened by an influx of emails by researchers who were prompted to share their anecdotal evidence of evolutionary change in a variety of roaded contexts. If those emails any indication, we appear to be on the right track toward our stated goal of shifting gears in road ecology. Hopefully, road ecology will soon yield insights into adaptation and maladaptation across suites of species and contexts, the genetic variation underlying those responses, and the likelihood for adaptive versus maladaptive outcomes.

The future of road (evolutionary) ecology is wide open.

As I wrap up this blog entry, something else occurs to me. That is, this call to shift gears should go out not only to road ecologists, but also to evolutionary ecologists by and large. So here is my pitch to those researchers already steeped in evolutionary biology: come over to the roadside! Roads are natural (ish) laboratories for studying evolution. The suite of potential selective agents associated with roads is diverse and seemingly strong. And, selection appears to be 'hard' in many cases, where populations decline from pressures such as road kill. Further, as Mary Rogalski keenly points out in a recent Eco-Evo Evo-Eco post, selection from pollution may pose a unique set of challenges compared to more typical forms of selection (such as from predation and competition). Not to mention, roads acting as impediments to gene flow can hasten responses to selection.

All in all, roads are ripe for evolutionary study. Hopefully, a decade from now, we will have a handle not only on the giant's embrace, but also the way that life squeezes back.

Saturday, March 18, 2017

Over-citation: my papers that should be cited less often

Ever write a great paper – an important one – and publish it to great expectations? “Surely everyone will love this paper,” you think. It is going to be a barn-burner. It is going to bust Web of Science – maybe even Google Scholar – with citations. Then, as the weeks and months and years go by, pretty much nothing happens. The paper gets a few citations (mostly from your own group), a few people seem to have read it, but not much else. And you think, “How did this happen”? “That was one of my best papers ever – it should be more widely cited.” Perhaps you start to think, “Maybe folks just missed it. If I could only get it in front of them again, people would recognize its greatness and it would go viral.” So you write a blog about “hidden gems” or you emphasize the paper on your website or you send out a few tweets or all of the above. And …. nothing happens. So you carry a (mild) resentment to your retirement, where you give your “exit seminar” and talk about your great work that just didn’t get the attention it deserved. (Yes, I have seen this happen.) Well, this post is about the exact opposite situation – papers that get way more attention than they deserve.

When one applies for a research grant, one usually has to talk about how wonderful one is – at least partly in relation to publications and citations. This need usually takes one to Web Of Science or Google Scholar to find out numbers of citations and H-indices and so on. Whenever I do this (such as yesterday while preparing a grant application), I see my top cited papers. I look at some of them and think, “Well, yeah, that paper was indeed useful and influential” but, about the same amount of time, I think “What the hell, why does THAT paper have so many citations?” So, I thought I would here take the opposite tack to the usual “papers of mine that should be cited more” and write about “papers of mine that should be cited less.” In doing so, I first need to point out that there isn’t anything wrong with these papers, they simply seem to have received more attention (or at least citations) than their content might deserve – or that we, as authors, expected.

One choice for an over-cited paper might be a short note we published in Conservation Biology about how species distribution models that predict massive extinction under climate change generally ignore evolution and are therefore probably often wrong. Models of this sort look at the abiotic conditions where a species is currently found, ask how the geographical distribution of those conditions is expected to change into the future, and then – if the conditions currently occupied by a given species in a given area shrink excessively – make a prediction of likely extinction. The problem, of course, is that species might evolve to occupy the changing abiotic conditions as selection forces them to do so – which is the only point we made in this paper. This point is certainly correct and many papers have now shown that such modelling is likely to be wrong much of the time, partly because of evolution. Yet it just seems so obvious as to not warrant a citation and – really – all our note did was point out that evolution could be rapid and that it could cause a mismatch between predicted and realized future species distributions. Does this rather obvious insight in a very small note really deserve 200+ citations in 7 years?

And the third most cited paper on Eco-Evolutionary Dynamics is ....
(coauthors redacted to protect the innocent)
Another choice for an over-cited paper might be the introduction we wrote to a Philosophical Transactions of the Royal Society special issue on Eco-Evolutionary Dynamics. The introduction simply pointed out that evolution could be rapid and that evolution could influence ecological process, before then summarized the papers in the special issue. Again, nothing wrong with the paper, but a summary of papers in a special issue is hardly cause for (soon) 300+ citations, nor is that typical of such a summary. I here assume that people are citing this paper mainly for the first two general points we make as listed above. This is fine, but excellent papers that treat eco-evolutionary dynamics as a formal research subject, rather than a talking point, are out there and should be cited more. Indeed, several papers in that special issue are precisely on that point, and yet our introduction is cited more. Similar to this example of over-citation, I could also nominate the introduction to another special issue (in Functional Ecology) – which is my fourth most cited paper (437 citations).

Why are these “OK, but not that amazing” papers so highly cited? My guess is that two main factors come into play. The first is that these papers had very good “fill in the box” titles. For instance, our PTRSB paper is the only one in the literature with Eco-Evolutionary Dynamics being the sole words in the title. Thus, any paper writing about eco-evolutionary dynamics can use this citation to “fill in the citation box” after their first sentence on the topic. You know the one, that sentence where you first write “Eco-evolutionary dynamics is a (hot or important or exciting or developing) research topic (REF HERE)” The Functional Ecology introduction has much the same pithy “fill in the box” title (Evolution on Ecological Time Scales) and, now that I look again, so too does the Conservation Biology paper (Evolutionary Response to Climate Change.) The second inflation factor is likely that citations beget citations. When “filling in the box”, authors tend to cite papers that other authors used to fill in the same box – perhaps partly because they feel safe in doing so, even if they haven’t read the paper. (In fact, I will bet that few people who cite the above papers have actually read them.) One might say these are “lazy citations” – where you don’t have to read anything but can still show you know the field by citing the common-cited papers.

Of course, I too sometimes take the lazy citation strategy. Sometimes when I am busting out an introduction and initially write “This [topic here] is a (hot or important or exciting or developing) research area (REF HERE)”, I simply fall back to my usual set of citations that I haven’t looked at for years and years. Doing so is a quick, easy, and safe way to simply move on to the more interesting stuff that really requires reading papers. Or, if I don’t know what to cite, but I know I am stating a well-known fact, I will simply search for the topic on Google Scholar to see what is most cited and then check the title and abstract to make sure citing it is safe. Perhaps this is a bad scholarship – or perhaps it is clever efficiency in the sense that these citations don’t really matter. They are generally known phenomena that have been discussed before and for which detailed additional reading would simply be a waste of time – so I am not exactly condemning “lazy citations” here.

My final closing point is that numbers of citations to a paper don’t always reflect the originality, importance, and quality of the paper. Sometimes papers are dramatically under-cited given their quality and potential importance. Sometimes papers are dramatically over-cited given their quality and importance. Of course, this point isn’t a new one but perhaps I am making it in a slightly novel way.


1.       Patrick Nosil first pointed out to me the “fill in the box” citation-inflation phenomenon.
2.       While writing this post, I noticed that the Google Scholar link for the Conservation Biology paper doesn’t even list me as an author – irony!
3.       No disrespect to my co-authors on the papers discussed above. In fact, my favorite part of all of the above papers was the collaborative writing efforts they involved. Clearly, we did a great job in the writing!

4.       Of course, I have my own papers that I think are way under-cited, particularly several awesome ones published in PLoS ONE (an analysis here). Check it how Humans are less morphologically variable (within populations) than are other animals and Bear predation drives the evolution of salmon senescence in unexpected ways. (And, no, I didn’t write this post simply to plug these under-cited papers.)

Friday, March 3, 2017

Maladaptation to chemical exposure – what may be happening and where do we go from here?

Guest post written by Mary Rogalski

Industrial, residential, commercial, and agricultural development greatly benefit human populations, but with the unintended and widespread consequence of increasing the release and availability of chemical pollution.  Surface runoff and atmospheric deposition introduce a complex mixture of heavy metals, pesticides, pharmaceuticals, and other contaminants into our water bodies. While some pollutants are regulated in an effort to protect human and environmental health, we have very little knowledge of how pollution exposure affects organisms over the long-term. To effectively manage pollution risk, we need to have a better understanding of these consequences.

Exposure to chemical pollution can have a host of negative effects on organisms, including reduced reproductive output, and at high enough doses, death. Individuals in the wild have been found to vary in their sensitivity to pollution exposure, both within and among populations. Based on these negative effects on individuals and the variation in sensitivity, most evolutionary biologists would likely predict that pollution exposure should select for more tolerant individuals. In other words, populations should be able to adapt to exposure.

This was my hypothesis when I set out to investigate the evolutionary consequences of long-term exposure to heavy metals in Daphnia populations in New England lakes (Rogalski 2017). Daphnia (aka “water fleas”) are tiny crustacean zooplankton that are extremely efficient at grazing algae in lakes but are also pretty sensitive to contaminant exposure. To my surprise, I found the opposite of my predicted trend. Daphnia had evolved to become more sensitive to metal exposure over decades of increasing contamination.

 Image of a Daphnia ambigua mother (approx. 1 mm in length). Photo: Eric Lazo-Wasem
 When I first started to see this result, I thought there must be some mistake. However, the pattern was repeated. I saw the same increase in sensitivity to copper following increasing historic exposure in two different populations, and also in response to cadmium in a third population.  In one lake, thirty years after peak copper levels, the sensitivity remained.

Grey points show copper or cadmium contamination through time. Black points show copper or cadmium sensitivity of individual Daphnia clones hatched from different time periods. LC50 is a measure of acute sensitivity, with lower LC50s indicating greater sensitivity. Further details on the study.

I tried to think of what could explain my unexpected result. Surely adaptation must really be happening here, right? But my study is clearly showing the opposite trend. Perhaps there’s an evolutionary trade off at play? Or some other reason why the populations have not only failed to adapt to metal exposure but also became more sensitive?

Alexander Lake, the study site where Daphnia have become more sensitive to rising cadmium concentrations

While the evolutionary pattern is striking and repeated, at this point I just don’t have enough information to understand the mechanism underlying the pattern. I certainly wouldn’t rule out the possibility that these Daphnia populations are adapting to their changing environmental conditions but just happen to be getting more sensitive to acute copper and cadmium exposure. In particular, I am curious to know if the acute and chronic toxicity responses might be inversely correlated. My assays measured acute toxicity – is it possible that being good at chronic chemical exposure makes a Daphnia worse at dealing with acute exposure? At least one study looked at this question in Daphnia with mixed results, finding no evidence of such a trade-off in response to cadmium, and no obvious pattern in response to copper (Barata et al. 2000).

Yet after having spent a lot of time reflecting on my results, I no longer find the trend so unexpected.
First of all, while maladaptation has received relatively little attention by evolutionary biologists, a metaanalysis by Hereford (2009) suggests that maladaptation happens fairly frequently. Of all reciprocal transplant studies examined in this metaanalysis, Hereford found that local maladaptation (defined as foreign population advantage) happened in 29% of cases. If we see evidence of maladaptation when we expect to see local adaptation nearly a third of the time, my result of increasing sensitivity to metals seems much less unexpected.

My study is not the first evidence of maladaptation to pollution conditions in wild animal populations. Researchers found that barnacles (Balanus amphitrite) in polluted estuarine environments were more sensitive to exposure to an antifouling biocide, copper pyrithione, compared with animals from less polluted conditions (Romano et al. 2010).  Rolshausen et al. (2015) found that Trinidadian guppy (Poecilia reticulate) populations have failed to adapt to crude oil pollution, despite devastating effects of exposure to oil. Also, a PhD student in the lab where I did my dissertation work, Steve Brady, found that wood frog (Rana sylvatica) populations in Connecticut ponds were more sensitive to road side environments in general, and road salt in particular compared with salamanders from forested ponds (Brady 2013). Steve found that there were also some overall fitness consequences of this increasing sensitivity to roadside environments. Interestingly, he found the opposite trend of adaptation to roads and road salt in another amphibian species, spotted salamanders (Ambystoma maculatum), inhabiting the exact same ponds (Brady 2012).

Results from Brady’s 2013 study of wood frogs.
In addition, just because pollution can have fitness consequences, I don’t think we should expect chemical exposure to act like other forces of selection such as predation, parasitism, or changing temperatures. Pollution exposure can also lead to increasing rates of developmental malformations, cause changes in sex ratios, and cause cancer. Some pollutants can bioaccumulate in tissues, including those of offspring. When the cadmium chloride that I had ordered for my toxicity trials arrived in the mail, the hazards listed on the safety sheet sounded pretty scary. In particular, cadmium exposure “may cause heritable genetic damage”. The other metal I studied, copper, has been linked with increasing mutation rates in exposed Daphnia populations. It’s not hard to imagine how some of these toxicological impacts could have accumulating consequences over the course of many generations of exposure.

One thing that is valuable about my study is that it tracks evolution through time. While local adaptation studies have provided valuable insight into how populations have evolved in response to contaminant exposure, we are missing three critical pieces of information. 1) We don’t know how pollution conditions have changed in the past in a given habitat. We can only compare organisms in polluted and unpolluted conditions; 2) we don’t know what the historical evolutionary trajectories of these populations look like; and, 3) in most cases the phenotypic responses that we observe may include genetic, plastic, epigenetic, and/or maternal effects.

In my study, I was able to address these issues. I used lake sediment archives to track both environmental and evolutionary trajectories over time. I measured metal contaminants in dated sediments to put together the history of exposure experienced by these populations over the past century. I hatched Daphnia from resting egg banks buried in sediments from high and low metal time periods. I then tested these Daphnia for sensitivity to copper or cadmium to see if the populations had evolved in their tolerance for these stressors. Since we can raise Daphnia clonally in the lab I was able to minimize any maternal effects that might have been present.

Extracting metals from lake sediments using hot block acid digestion. Photo: Sara Demars
In closing, I’ve come away with two key points from this study. First, maladaptation appears to be fairly common but our theoretical understanding of why it happens is pretty limited. This leaves us trying to explain seemingly counterintuitive results with a bit of hand waving and throwing around terms like “genetic drift”, “trade offs”, and “dispersal rates”. As evolutionary biologists we need to do more to understand what drivers may lead to maladaptation to improve our ability to both explain and predict evolutionary trends. Second, if species as different as Daphnia, barnacles, and wood frogs are becoming maladapted to pollution, we should think critically about the risk associated with multi-generational pollution exposure. How common is maladaptation to pollution exposure, and how does this affect the ability of organisms to adapt to other stressors? In what contexts might we expect to see adaptation vs. maladaptation to a contaminant? Could pollution exposure be having long-term damaging impacts on human populations? Those who oppose pollution regulation focus on the financial costs today, but the cost of inadequate pollution control for humans and other species could be much greater over the long-term.

Daphnia resting egg cases from one of the study lakes. Photo: Eric Lazo-Wasem.