Sunday, June 28, 2015

Speciation, genomes, and pancakes

A decade ago, I began my PhD at Vanderbilt University in Nashville, Tennessee, where I was interested in studying the evolutionary process of speciation (or how new biological species evolve). I was very lucky during my PhD to be surrounded by great people. Case in point, I shared an office for part of the year with a visiting collaborator, Patrik Nosil, who studied speciation in a group of stick insects called Timema. Second, my PhD advisor encouraged me to invite great thinkers on speciation to be part of my dissertation committee – enter Jeff Feder from the University of Notre Dame, who studied speciation in a group of fruit-feeding flies called Rhagoletis and served as my external committee member. These connections made during the beginning of my PhD last to this day.

Figure 1. The Pancake Pantry in Nashville, TN, USA.
During a fateful visit to a common grad student hangout (circa 2007), the Pancake Pantry (Fig. 1), Patrik Nosil and I and a group of graduate students started discussing the age-old debate about the number of genes involved in adaptation (and speciation): few versus many? And whether the traits responsible for adaptation and speciation were polygenic traits or traits with a simple genetic basis? One way we thought to test this was to use as many molecular markers as you could survey, distributed across the genome, and ask the question: how many of these gene regions exhibit significant population differentiation, but are restricted to populations adapting to different environments? We came up with ideas of how to test it, and what type of tools we would need, right over our plates of pancakes! I think we even had a budget by the time we walked back in our calorie coma from lunch.  My major takeaway from this lunch was that I now considered the genome as an active player, not a passive mediator, in the speciation process and I would never think about speciation in the same way again!

What emerged initially from this pursuit were two comparative AFLP genome scans of two different study systems, each undergoing speciation driven by divergent ecology, that were published in the journal Evolution (Nosil et al. 2008; Egan et al. 2008).  These studies were very informative in highlighting the proportion of gene regions (AFLPs) in the genome exhibiting strong differentiation between divergent populations, and possibly addressed the repeatability of gene regions associated with adaptation to two environments (in our case, host plants).  But we were also left with many more questions than answers. How were these divergent loci distributed and arrayed across the genome? And were the loci exhibiting strong differentiation driven by selection or other evolutionary phenomena?

Fast-forward to 2010 – I finished my PhD and I was awarded a Faculty Fellowship at the University of Notre Dame, which came with some seed money for research and the chance to work more closely with my external committee member, Jeff Feder. Almost immediately upon arriving in South Bend, IN, Patrik (now in Sheffield, UK), Jeff, and I had a set of conference calls and email exchanges that started the project that would result in the Ecology Letters MS I will summarize below. (Jeff and Patrik had just finished a sabbatical in Berlin the year before where they spent much of their time ruminating on the genome-level phenomena influencing the speciation process.) We recruited other evolutionary biologists well trained in Rhagoletis biology (Tom Powell, Glen Hood, and Greg Ragland), as well as two computer scientists (Scott Emrich and his PhD student Lauren Assour) with the ability to process the large amount of data we would gather.

Our interests were to better understand the role the genome might play in the evolution of new species. We were inspired by a paper published over 30 years ago by Joe Felsenstein (1981), where he described the difficulty of building up many-locus differences between populations if gene flow was ongoing and recombination was breaking up associations. This conflict between selection and gene flow would form the basis for our project. How is it that populations can diverge in the face of ongoing gene flow? What are the properties or characteristics of species that are suspected of speciation-with-gene-flow which facilitated their divergence?

Figure 2. Rhagoletis pomonella exploring the fruit of the hawthorn tree (Crataegus mollis). Photo credit: Hannes Schuler
Rhagoletis pomonella offered a great study system to test these ideas, as it is a well-documented case of speciation-with-gene-flow (Fig. 2). Rhagoletis pomonella is a member of a sibling species complex containing numerous geographically overlapping taxa proposed to have radiated in sympatry by adapting to many new host plants from several different plant families. Rhagoletis flies infest the fruits of their host plants, where host fruits are typically available for a discrete window of time over the growing season and each fly species completes one generation per year. Adult flies meet exclusively on or near the host fruits to mate; females oviposit into the host fruit; larvae consume the fruit, then burrow into the soil to pupate, entering a pupal diapause that lasts until the following year. Thus, phenological matching of fly to host-plant fruiting is critical to fly fitness.

The most recent example of a host shift driving speciation is the shift of R. pomonella from its native host hawthorn to introduced, domesticated apple, which occurred in the mid-1800’s in the eastern United States. Genetic and field studies have shown that apple and hawthorn flies represent partially reproductively isolated host races and that gene flow has been continuous between the fly races since their origin. One key trait that differs between the races is the timing of diapause termination, which varies between the races to match the 3–4 week earlier fruiting time of apple versus hawthorn trees (Fig. 3). Rhagoletis emerge from their fruits as late-instar larvae and overwinter in the soil in a facultative pupal diapause. The earlier fruiting time of apples therefore results in apple flies having to withstand warmer temperatures for longer periods prior to winter. As a result, natural selection favors increased diapause intensity, or greater recalcitrance to cues that trigger premature diapause termination in apple flies.
Figure 3. Fruit on apple trees ripens 3-4 weeks earlier than hawthorn fruit (dashed lines). Apple flies eclose earlier as adults (solid lines) and are exposed to warmer temperatures as pupae in the soil for a longer period of time before winter.
Jeff had the perfect experiment frozen in his freezer from 20 years ago. Previously, his lab had reared the ancestral haw race of Rhagoletis under the phenological conditions of both host plants it attacks in nature. He had previously looked at changes in a set of allozymes and microsatellites, but did not have the ability at the time to look across the genome at tens of thousands of SNPs.  Specifically, he exposed ancestral hawthorn fly pupae to warm temperatures for a short 7-day (‘hawthorn-like’ control) vs. long 32-day (‘apple-like’ experimental) period prior to winter (Fig. 4).

Figure 4. In the selection experiment, hawthorn flies were exposed to a short (7-day) versus long (32-day) prewinter period to emulate the time difference experienced by hawthorn versus apple-fly pupae in nature. 
We also had a specific hypothesis we wanted to test that integrated Jeff’s selection experiment with sampling from natural populations. We tested whether the changes across the genome induced by the lab experiment on divergent host-plant phenology would predict the genome-wide differences observed at these same loci between natural sympatric populations. In this experiment, we stressed that we were quantifying the total genome-wide impact of selection, which involves both direct effects, where natural selection favors the causal variants underlying selected traits, and indirect effects, where additional loci respond because they are correlated due to linkage disequilibrium with these causal variants. Thus, the ‘total’ impact of divergent selection (i.e. direct + indirect effects) that we quantify here can involve changes at many loci (Gompert et al. 2014; Soria-Carrasco et al. 2014).

Quantifying the impact of selection genome-wide is important because, as populations diverge, the effects that individual genes have on reproductive isolation (RI) can become coupled, strengthening barriers to gene flow and promoting speciation (Barton 1983, Bierne et al. 2011). If predicated solely on new mutations, this transition could take a long time and populations could go extinct or conditions change without speciation, which may explain why sympatric speciation is difficult to observe and test. Thus, a prediction for systems with the potential for speciation-with-gene-flow is that they exhibit large stores of standing variation and consequently, show extensive, genome-wide responses to selection when challenged by divergent ecology.

In our selection experiment, about 6% of the SNPs showed significant frequency shifts between the short and long prewinter periods. However, because of extensive linkage disequilibrium (LD) in Rhagoletis, these SNPs did not provide an estimate of the independent number of gene regions influenced by selection. Thus, we assessed the pattern of LD between SNPs to delimit independent sets of loci.  We determined that the 6% of responding SNPs represented 162 different sets whose members were in LD with each other, but in equilibrium with all other SNPs. After accounting for the table-wide null expectation of 52 significant sets due to type I error, using a modeling approach we detail in our Supplemental material, a lower bound estimate of 110 gene regions responded to selection. To determine how physically widespread the response was across the genome, we constructed a recombination linkage map for Rhagoletis that contained 2,352 SNPs. About 13% of mapped SNPs showed significant frequency shifts in the selection experiment and were dispersed widely across the five major chromosomes of the R. pomonella genome (Fig. 5). Thus, numerous independent gene regions responded to selection and they were distributed throughout the genome.

Figure 5. Genome-wide comparison of allele frequency shifts in the selection experiment (red line; left axis) versus divergence between field-collected sympatric host races (blue line; right axis) along chromosomes 1-5. Circles above panels denote SNPs showing statistically significant response in the selection experiment (open red) or difference between the host races (solid blue). Correlation coefficient (r) is reported independently for each chromosome.
Now we tested our main hypothesis: does the genomic response in the selection experiment reflect nature?  The answer is yes. The direction and magnitude of allele frequency changes for all 32,455 SNPs in the selection experiment was highly predictive of genetic differences between the sympatric hawthorn and apple host races at the Grant, MI, site (r = 0.39, P < 10-6). Most strikingly, for the SNPs showing significant responses in both our selection experiment and host divergence in nature, the allele that increased in frequency in the hawthorn race after selection was the exact same allele in higher frequency in the apple race in nature (P = (½)154 = 4.4x10-47).

To what extent did the single bout of selection on hawthorn flies genetically create the derived apple race?  The answer is a good deal. For all 32,455 SNPs, the mean SNP frequency for hawthorn flies surviving the long prewinter treatment shifted 38.9% of the difference between the host races toward apple flies. For the 154 SNPs showing significant responses in the selection experiment and host divergence, the shift was 84.1%.

Why is the impact of divergent ecological adaptation so pronounced and pervasive in Rhagoletis?  One contributing factor is the extensive LD in the fly, some of which is due to inversions, requiring additional DNA sequence analysis to resolve. A second factor is the presence of substantial standing genetic variation in R. pomonella, which supports the hypothesis that such stores may define taxa having a greater capacity for speciation-with-gene-flow. Finally, when ecological adaptation involves traits like diapause that can be highly polygenic, selection may more often have genome-wide consequences. In this regard, microarray studies of R. pomonella have revealed hundreds of loci varying in expression during diapause breakage that are potential targets of selection (Ragland et al. 2011).
Figure 6. Rhagoletis pomonella fly exploring apple fruit. Photo credit: Andrew Forbes
Interestingly, this work shares some important similarities and differences with other recent studies combining selection experiments with surveys of genome-wide genetic variation in natural populations, including the Timema ecotypes that are the mainstay of the Nosil lab. In both a within-generation (Gompert et al. 2014; similar to the Rhagoletis study here) and a between-generation study of selection in the field (Soria-Carrasco et al. 2014), a genome-wide response involving many loci was observed. However, LD was much lower in the Timema ecotypes, and thus the association between genetic differences induced in those selection experiments did not match natural genetic variation as closely as in the Rhagoletis experiment.

In summary, divergent ecological selection can have genome-wide effects even at early stages of speciation. Large stores of standing variation in Rhagoletis flies may potentiate the evolution of genome-wide reproductive isolation and their adaptive radiation with gene flow. As the study of speciation genomics expands, it will be possible to test the degree to which other taxa prone to ecological sympatric speciation share similar characteristics as R. pomonella, and to assess the relationship between standing variation and clade richness.

That was one productive plate of pancakes!


Barton, N.H. 1983. Multilocus clines. Evolution 37, 454471.

Bierne, N., Welch, J., Loire, E., Bonhomme, F. & David, P. 2011. The coupling hypothesis: why genome scans may fail to map local adaptation genes. Molecular Ecology 20, 2044–2072.

Egan, S.P., P. Nosil, & D.J. Funk. 2008. Selection and genomic differentiation during ecological speciation: isolating the contributions of host-association via a comparative genome scan of Neochlamisus bebbianae leaf beetles. Evolution 62: 1162-1181.

Egan, S.P., G.R. Ragland, L. Assour, T.H.Q. Powell, G.R. Hood, S. Emrich, P. Nosil & J.L. Feder. 2015. Experimental evidence of genome-wide impact of ecological selection during early stages of speciation-with-gene-flow. Ecology Letters, online early. (doi: 10.1111/ele.12460)

Felsenstein J. 1981. Skepticism towards Santa Rosalia, or why are there so few kinds of animals? Evolution 35:124 – 138.

Gompert, Z., A.A. Comeault, T.E. Farkas, J.L. Feder, T.L. Parchman, C.A. Buerkle, and P. Nosil. 2014. Experimental evidence for ecological selection on genome variation in the wild. Ecology Letters 17: 369-379

Nosil, P., S.P. Egan, & D.J. Funk. 2008. Divergent selection plays multiple roles in generating heterogeneous genomic differentiation between walking-stick ecotypes. Evolution 62: 316-336.

Ragland, G.J., S.P. Egan, J.L. Feder, S.H. Berlocher, & D.A. Hahn. 2011. Developmental 
trajectories of gene expression reveal regulatory candidates for diapause termination, a key life history transition in the apple maggot fly, Rhagoletis pomonella. Journal of Experimental Biology 214: 3948-3960.

Soria-Carrasco, V., Z. Gompert, A.A. Comeault, T.E. Farkas, T.L. Parchman, J.S. Johnson, C.A. Buerkle, J.L. Feder, J. Bast, T. Schwander, S.P. Egan, B.J. Crespi, & P. Nosil.  2014. Stick insect genomes reveal natural selection's role in parallel speciation. Science 344: 738-742. 

Monday, June 15, 2015

Old Monkeys in New Habitats: The Biogeography of Terrestrial Biotas.

I just returned last night (37 hours in transit!) from my first trip to Uganda. It was my second trip to Africa, with the first being to South Africa six years ago. The main purpose of the trip was to plan, with my colleague Lauren Chapman, some new studies on adaptation by fishes to extreme (low oxygen) environments. However, my first trip to any new location also becomes an adventure in natural history and photography. During these adventures, I was motivated to write a post based on a series of natural history anecdotes, which I will also seek to tie into a book I happened to reading at the same time.

Following in Lauren's foot steps.
My current bed time (and plane time) reading is The Monkey’s Voyage by Alan de Queiroz. The subtitle of the book is How Improbable Journeys Shaped the History of Life. The goal of the book is to contrast old and new views of biogeography, the study of where species are found and why. The old view is that the distribution of organisms and faunas across the world is almost entirely shaped by vicariance events that sunder formerly contiguous landmasses. These events including land masses splitting through continental drift, mountain ranges rising, large rivers forming, and so on. Under this view, the species found in New Zealand, for example, are remnants of an early Gondwanaland biota that persisted (and diversified) following the isolation of New Zealand from a larger land mass that included Australia. By contrast, the new view is that the distribution of terrestrial organisms is shaped to a larger extent by rare long distance dispersal across even large ocean distances. Under this alternative view, New Zealand’s fauna is mainly shaped by over-water dispersal from Australia long after the two islands split apart. (Interestingly, the new view is actually an even older view. Darwin spent considerable time studying mechanisms of long distance dispersal, although perhaps he wouldn’t have if continental drift had been known.) De Queiroz clearly favors the new view, marshalling extensive evidence that biogeography is strongly shaped by long distance dispersal.

Reading biogeography in books is interesting but experiencing it in person is transformative. For the last 14 years, most of my field work has taken place in South America, including Trinidad, Galapagos, Panama, and Chile, alongside shorter trips to Brazil, Barbados, Roatan, and other locations. Now my recent trip to Uganda, combined with my earlier South African trip, has brought home in a personal sense the differences between “New World” and “Old World” biotas.

A South African lion showing off his dental array.

A South African hippo showing off his even more impressive dental array.
The most in-your-face contrast, of course, would be the classic African large-mammal spectacles: elephants, hippos, buffalo, giraffes, lions, leopards, wildebeest, zebras, cheetahs, camels, and so on – most of which I have now seen in the wild. Although the New World certainly does have large mammals (moose, bison, bears, capybaras, tapirs, jaguars), they are not nearly as striking, abundant, or dramatic a spectacle. However, this contrast is somewhat disingenuous given that the New World had many similar forms (mastodons, mammoths, lions, sabre-toothed cats, camels) until their extinction in the Pleistocene not that long ago. (And, of course, bison recently did, and caribou still do, present huge migratory spectacles.) So, but for vagaries of our particular point in time, the large-mammal faunas of the two continents might not have seemed quite so different.

A brown bear from my cabin in Northern BC, Canada.

Yes, moose are huge - this one in Lake Nerka, Wood River Lakes, Alaska.
A classic contemporary contrast is Old World monkeys (and apes) versus New World monkeys. The two groups differ in a number of ways, including various aspects of facial shape and – iconically – the prehensile tail of New World but not Old World monkeys. In Panama, I have been able to observe howler monkeys, white-faced monkeys, spider monkeys, Geoffroy’s tamarins, and others. In Kibale National Park in Uganda, I was able to observe olive baboons, grey-cheeked mangabeys, blue monkeys, redtail monkeys, red colobus, black-and-white colobus, L’Hoest’s monkey, galagos (bush babies), pottos, and – the most amazing of all – chimps. (Kibale is said to harbor the highest primate biomass in the world.) Later at Lake Nabugabo, I saw vervets (more about these later), which I had also – along with baboons – seen in South Africa. Excepting chimps and baboons, and despite some differences in appearance, the two sets of monkeys strike one as superficially similar. They all move with varying degrees of frenetic activity through forest canopy feeding on a diversity of insects, leaves, and fruits. Thus, we here have a similar ecological set of organisms in the two worlds, with the new world monkeys having radiated from a single common ancestor colonizing the new world, perhaps by long-distance dispersal of just a few individuals from Africa (as argued by de Queiroz and others). 

Black-and-white colobus in Kibale National Park, Uganda.

Red colobus in Kibale National Park, Uganda.

Redtail monkey in Kibale National Park, Uganda.
Grey-cheeked mangabey expressing displeasure in Kibale National Park, Uganda.

Chimp mom with sleepy baby in Kibale National Park, Uganda.
For birds, the classic contrast is between hummingbirds and sunbirds. Hummingbirds, such a ubiquitous, striking, and engaging component of New World environments, are entirely absent from the Old World. Instead, the Old World has a large radiation of the nectar-feeding sunbirds. The first sunbird I ever saw was in Cape Town, South Africa. I was on Table Mountain composing a photograph of a flowering bush in the foreground with Cape Town in the background far below. In the midst of a sequence of photographs, a sunbird landed right in the middle of the flowers – almost as if I had planned for it. In Kibale and Queen Elizabeth Parks in Uganda, I saw more sunbirds. However, despite the similar ecologies and exuberant colouration of both groups, which are not closely related, no one would mistake one for the other. For instance, the hovering flight of hummingbirds – perhaps their most obvious feature – is relatively rare in sunbirds.

Bronze sunbird, Kibale National Park, Uganda.
Both continents have wonderful radiations of small colorful frogs. While traveling along the swampy edge of Lake Nabugabo, we had stopped so I could take pictures of birds when one of the field assistants pointed to a tiny yellow-and-black-patterned frog on a reed we were holding on to. Mediocre bird photos immediately forgotten, I switched to macro equipment and started taking endless photographs of the frog. Then, in the space of just a few minutes, he pointed out two other species of frog clinging to other reeds less than a meter away. All were colorful but in various shades and patterns of green. Moreover, they all froze in place and didn’t move no matter how close my hands got or how much I manipulated the reeds or stuck a macro lens in their faces. This appearance and behavior was a surprise after the poison-dart frogs that I had seen in Panama and elsewhere in the Americas. When I asked the field assistants if these frogs were poisonous, they did not think so. It seems this group has specialized on camouflage whereas the New World Dendrobatids have specialized on conspicuousness. (I am no frog expert – perhaps a radiation of poisonous and conspicuous frogs exists in Africa – and I am generalizing – some New World frogs are very cryptic.)

Lake Nabugabo frog #1, which I still haven't taken the time to identify to species.

Lake Nabugabo frog #2, which I still haven't taken the time to identify to species.

Lake Nabugabo frog #3, which I still haven't taken the time to identify to species.
We later visited another part of the swamp and the same field assistant found another small-and-green-themed frog of seemingly yet another species. At this point, I was starting to feel incompetent in my ability to find critters and decided that I would find my own damn frog. So I went walking slowly through the marsh scanning blades of grass and other vegetation. Half an hour later, having still had no luck, I was about to give up when I saw a bit of movement near the water. “YES” I yelled, “I finally found one of the buggers” and, then, looking closer, I saw it was actually a finger-length chameleon. Even better – my first ever chameleon; and I found it myself (considerable boasting followed). The next hour was spent taking 217 photographs of the chameleon plus additional shots of what appeared to be a fifth small-green frog species that another field assistant found. Chameleons are another major radiation in the Old World – especially Madagascar – that is completely absent from the New World, which instead has a radiation of Anolis lizards that are absent from the Old World.

My chameleon. Found at Lake Nabugabo, Uganda.

Getting a closer look at my chameleon. Found at Lake Nabugabo, Uganda.

Lake Nabugabo frog #4, which I still haven't taken the time to identify to species.

Lake Nabugabo frog #5, which I still haven't taken the time to identify to species.

To these Old versus New faunal contrasts that I already knew, the present trip added another. After having seen and photographed most of the diurnal primates, Lauren took me on a night walk to look for the nocturnal primates. Almost immediately, we saw a potto, which I am told is not common, and I was able to get some photographs with a long lens and flash. What immediately struck me about the potto was its slow, branch-hugging movement; kind of like a sloth. Hmmmm, what about sloths? Sure enough, Lauren confirmed that sloths are absent from the Old World just as potto-equivalent primates are essentially absent from the New World (although the latter does have night monkeys). 

Potto doing its sloth imitation in Kibale National Park, Uganda.
In this post, I have been a Natural History Tourist, giving my own superficial impressions of some differences between the two “worlds.” Although these impressions are based on relatively little experience in Africa, they made me ponder the biogeography debate I had been reading in de Queiroz’s The Monkey’s Voyage. Long distance over-water dispersal has certainly shaped the world’s fauna but vicariance ultimately seems more important through its role in limiting movement between land masses. On the one hand, long-distance dispersal does happen and is critical in shaping species distributions: without it we would not have any organisms on oceanic islands and many iconic organisms of large islands and continents would also be missing, including perhaps monkeys in the New World. On the other hand, faunas differ so much from place to place that effective long-distance dispersal must be very rare and vicariance provides the dominant factor assembling many communities.

I doubt de Queiroz would disagree with these points even though his book is very much focused on long-distance dispersal. Another way to explain the distinction is that long-distance dispersal is critical for explaining why species ARE in particular places whereas vicariance is critical for explaining why species ARE NOT in particular places.

All of this brings me back, believe it or not, to vervets, the first monkey I ever saw in the wild. This statement might seem surprising if you remember that vervets are Old World Monkeys whereas I had worked in South America for eight years before visiting Africa. In fact, my first experience with wild monkeys – the vervets – was in 2003 in Barbados. Yes: Old World Monkeys on New World Islands! It turns out that vervets were brought about 350 years ago to Barabados and some other Carribean islands by slavers. I don’t think other monkeys are naturally found on those islands, at least not on Barabados, so this isn’t a lesson in what happens when the two faunas collide. But if they did collide, who would win? Are New World Monkeys “better” than Old World monkeys? (They have that cool tail!) Would New World monkeys win in the New World and Old World monkeys win in the Old World (local adaptation!) – or vice versa (enemy release!)? Of course, countless such experiments are being undertaken with other organisms, as the field of invasion biology attests, but I don’t know any examples of recent human-mediated conflicts between ecologically-equivalent iconically-divergent faunas such as those described above. (Although placental dingos replaced, whether causally or not, the marsupial thylacine in Australia).

A Barbados vervet
How interesting it would be to bring hummingbirds to the Old World and sunbirds to the New, Anolis lizards to the Old and chameleons to the New, capybaras to the Old and hippos to the New, and so on. (Apparently Pablo Escobar, the drug lord, had a hippo herd that is now feral and expanding in Columbia.) Just think how much we would learn and how fun it would be to see the dynamics play out. Sadly, however, the likely ecological impact would exceed the value of the information gained thereby. I would much rather see Anolis in South American and chameleons in Africa than I would like to find out who would win in direct competition in nature. It is too bad we can’t have replicate worlds, some for conservation and some for experimentation.

Grey-crowned crane at Lake Nabugabo, Uganda.

Wednesday, June 3, 2015

Life history plasticity in lake-stream stickleback

When thinking about ecological speciation, we often assume that habitat-related local adaptation drives genetically based population divergence, and that the resulting phenotypic divergence promotes reproductive isolation. However, strong phenotypic divergence and associated reproductive isolation might also arise directly via phenotypic plasticity. In a paper that just went online, we start exploring the latter pathway to speciation in lake and stream stickleback populations in the Lake Constance basin (Central Europe). This system is cool because the stream populations consistently show the relatively small body size at reproduction typical of stream stickleback worldwide (Moser et a. 2012, PLoS One), whereas the lake fish reproduce at much larger body size (the photo below shows a representative reproductive male from Lake Constance and a tributary). Given that experimental work in many stickleback systems has shown that body size differences between populations represent a major sexual reproductive barrier, we wanted to understand how the body size differences among lake and stream fish arise. A combination of laboratory and field transplant work (see photos below) revealed that the divergence seen in the wild is largely plastic, most likely driven by differences in resource availability between the two habitat types: lake fish use a relatively poor limnetic food base, grow slowly, fail to reach reproductive size after one year, and hence reproduce only after two years at large size. Stream fish, by contrast, exploit a rich benthic food base, reach critical size after one year, reproduce directly at relatively small size, and die. In the laboratory and in field transplants, these life history differences disappear. We thus hypothesize that in this system, sexual reproductive barriers might have established immediately due to plastic divergence in life history. Evaluating this hypothesis experimentally is the next step. Also, it would be great to know if in other lake-stream stickleback systems, body size differences do have a stronger genetic component than in the Lake Constance basin.
If interested in the findings, see Moser et al. (2015), Evolutionary Biology: Lake-stream divergence in stickleback life history: a plastic response to trophic niche differentiation?

Tuesday, May 26, 2015

How to respond to reviewers

Previous posts in this "How to" series

4.     How to choose a journal (+ part 2)

Now its time for: How to respond to reviewers
Students starting in science might imagine that decisions on papers submitted to journals are rather straightforward outcomes such as “accept” or “reject” or seemingly "strong reject". The reality, however, is that most decisions are more on the order of “reconsider after revision” or “reject without prejudice,” or in their more modern forms, “reject with resubmission allowed” or “rejected in present form.” These more ambiguous decisions necessitate a careful revision of your paper in response to reviewer [more properly “referee”] comments, and a cover letter or “response to reviewers” that explains how you dealt with each comment. These revisions and responses are critical to the future of your paper, and the manner in which you implement them will make all the difference between whether your paper ultimately falls into the “accept” or “reject” category. In most of the above categories other than “reject,” your chance of acceptance is actually reasonably high provided you do a good job of the revisions and responses.

Having now published more than 150 papers, having reviewed more than 350 papers, and having been an Associated Editor (AE) for five journals, I have performed and encountered seemingly endless revisions and responses. Some work well and others don’t, and these alternatives typically translate into ultimate accept or reject decisions. This post is an attempt to distill those experiences down to a set of guidelines that can help you to optimize your revisions/responses so as to maximize your chances of acceptance and minimize the number of rounds of review. It is modified from a talk that Jonathan Davies and I give to grad students in Biology at McGill.

Image credit: Nick of

(By complete coincidence, a related post appeared today at Dynamic Ecology. And here is another recent one at Scientist Sees Squirrel.)

1. The response letter is critical

Most editors and reviewers will make their decision, or at least form a strong initial opinion, based entirely (or mostly) on the response letter, your “response to reviewer/editor comments”. This letter will be the first thing they read and – if they are satisfied with what you say – they might not even re-read the manuscript (MS) itself. Thus, you want to make sure that the editors and reviewers have all of the information optimally organized and explained in one place. Stated another way, the response letter is often just as important (maybe more so) than the changes you make to the MS itself. In preparing your letter, repeat all comments by the Associate Editor and the reviewers and make sure you respond to each immediately below. Repeat not only the negative comments you have to address but also all the positive comments. Repeating the latter is valuable because it can influence the other reviewers: “Hmmmm – the other reviewer quite likes this paper, maybe I am being a bit too harsh.”

As an aside, I often have my students write the response letter even before they modify the paper. This sequence helps to see in advance how their intended actions are likely to play out in the response letter, and doing so helps the students and coauthors to settle on the optimal set of changes. Of course, the letter will need to be modified as changes are made to the MS but it helps to settle the core elements first.

2. You need to convince the reviewers not the editor

Some people attempt to argue to the editor that the reviewer comments are not valid and should be ignored. The thought is that the editor will invalidate those comments and thereby let you proceed without addressing them. In the vast majority of cases, however, the editor will want to get the reviewers to agree to publish the paper and will be unlikely to overrule them (although it does happen). I therefore strongly suggest not trying to argue to the editor that the reviewer comments are invalid. Instead, you need to convince the reviewers, who will then help you convince the editor. In my experience, once you get a reviewer on your side, they will often then help to convince the other reviewer/editor too.

Further to the above, you should not say in your letter to the editor that the reviewer is unqualified or wrong or stupid or sloppy or anything like that. The editor usually selected the reviewers based on who they thought (or who you said) would be good reviewers – often people they know and respect and who could well be their friends. Thus saying the reviewers are unqualified is the same as insulting the judgement of the Editor. Moreover, your response letter – even if written only for the editor – will usually be provided to the reviewers. Thus, something you think you are saying in confidence to the Editor will often make it to the very person whom you are criticizing, which will only further bias them against your paper. Of course, there are exceptions when a reviewer really is personally insulting or overtly biased, in which case you should politely notify the Editor that the reviewer’s comments can be interpreted to be inappropriate. Of course, the other, more modern, strategy is to berate the journal on social media, which can lead them to issue a formal apology. However, I would not advocate this except in extreme cases, such as #addmaleauthorgate.

How editors select reviewers - Grod et al. (2010) Front. Ecol. Evol. 

3. (Re)define the problem

Reviews are often very extensive and different reviewers want different things – often many different things – some of which you can do and some of which you can’t. This can lead to very long and tedious response letters that serve to annoy and alienate the editor and reviewers. In such cases, it helps to define the critical problems at the outset of the response letter (sometimes the editor has helpfully defined them for you.) In essence, you write – just after the editor provides the key points – a short section that explains what you perceive to be the main criticisms, often rephrased in a way that best matches what you intend to do/argue about them. You then provide a short and focused explanation of how you have solved that problem or how, fortunately, the problem does not exist or isn’t too critical. The idea here is get the editor and reviewers focused on just a few key issues and show in a succinct and clear way how you have dealt with them. The implication (and truth) is then that the rest of the comments are really just minor things that didn’t influence the reviewer’s hesitation in accepting your MS. Thus, by showing right up front how you deal with the critical issues that you (or the Editor) think influence acceptance of the paper, you can set them to thinking right away that all will be well.

Types of reviewers - for more details see the awesome page:

4. Make actual changes to the MS whenever possible

You can respond to reviewer comments in two ways. First, you can change the MS to accommodate/ameliorate/fix the suggestions or criticisms. Second, you can try to argue your way out of making any changes. Take the first of these two options whenever possible. That is, whenever possible, DO something to the MS that helps to address each comment; and make sure to state that you have done so in your responses. However, it is likely that some reviewer suggestions are either impossible to implement or would actually make the MS worse or would totally change your intended meaning in a way you feel inappropriate. In such cases, you will need to argue your point. However, choosing to argue a specific point also means that you will want to have addressed as many of the other reviewer comments as possible with actual changes to the MS. Making these changes builds good credit with the reviewer and can give you some “free passes” on things you can’t (or don’t really want to) change. In essence, you should be careful to pick your battles, as the more you fight the more likely you are to lose the war. In some of the more difficult cases, you can meet the reviewer halfway by making some partial change (altering a graph, adding a new analysis in the supplementary materials, adding a qualifying statement), and I recommend you do this whenever possible.

As an aside , it is good to do these things even if your paper is rejected and then submitted elsewhere as it is reasonably common to get the same reviewers, who are annoyed at having to say the same thing they said previously and you seemingly ignored. And they really like it if you did what they suggested even if your paper was rejected. This happened to me once. I made many comments on a paper that got rejected from Journal A. I was then asked to review the same paper for Journal B. The author had - even though under no obligation to do so given the switch of journal - implemented essentially all of my suggestions, which were not trivial. I was very impressed and pleased and had many positive things to say about the MS, which as published in Journal B.

5. “I have now made this more clear in the MS”

If the reviewer is clearly wrong about something or if they missed something or misinterpreted something, never say so in as many words. Doing so can seem insulting or condescending (the reviewer missed something that you say is obvious) or it can imply that you think the reviewer is not doing a good job (as indeed they might not be).  Moreover, if the reviewer misunderstood or misinterpreted things, then other readers likely will too, and so you should change it. In essence, a reviewer’s misinterpretation of your study is YOUR fault, not the reviewer’s fault; or at least you should view it that way. In such cases, I first explain the reality and apologize for my mistake (“Fortunately, we did actually do XXXX but it was not sufficiently clear in the MS.”) and then state something like “We have now made this more clear through revisions to the text.” Of course, this means that you do indeed have to make it more clear in the MS – even if you thought it was clear to begin with.

6. Stop whining and just do the new analyses

With ever-increasing statistical sophistication (many would say over-complication), reviewers are likely to recommend some new analysis – no matter how hard you thought out and optimized your analysis in the first place. These new analyses will very rarely change any of your conclusions, and yet the reviewer thought they were important so it is not wise not to ignore them or try to argue them away. By far the simpler solution is to just do the new analysis (or graph or table) and place it in supplementary materials (if you don’t want to change the MS itself). You can then refer to this supplementary analysis in the text with a single sentence referring to the alternative analysis. Although doing the new analysis even when you are confident it won’t change the outcome can seem time-consuming and wasteful, it is even more time-consuming and wasteful to have the reviewer insist again that you do them, thus necessitating another round of revision. Just do the analyses the first time they are requested, which also lets the reviewer know you aren’t trying to weasel your way out of things because you are too busy/lazy or because you have actually done the analyses and found they go against your preferred conclusion. 
Real reviews compiled from my colleagues.

7. Be polite and respectful but not sycophantically so

Sometimes reviewers are insulting (see the above). Sometimes they clearly didn’t see something you had already put in the MS. Sometimes they appear to be complete idiots. Sometimes they are just wrong. However, you should never say any of these things in your response as reviewers will interpret them as being personal critiques. (And, as noted above, the reviewers will likely see things even if you intend them only for the editor.) Thus, no matter how annoying or rude or clueless reviewers are, you can never even hint that such is the case.
Balancing the above point, you should also avoid the temptation to repeatedly say “This is a great comment” or “We thank the reviewer for their comment” and so on. If you say it for every comment, then it means nothing. Instead, save such thank yous for key places, especially where you added an entirely new analysis or, seemingly paradoxically, where you can’t make a change. In this case, you are acknowledging that it is a good point but that you are able to make the change for logistical (or other) reasons.

8. Other procedural points

(a) Don’t paste the exact revised text (or even list the line numbers*) from your paper in the response letter. First, this wastes time as multiple changes are often needed and so the revised text and line numbers keep changing as the revision proceeds. Second, it greatly expands the length of the letter because you still need to explain it. Third, cutting out a single sentence and pasting it into the response letter means that it is out of context (which leads to the next point). Instead, simply explain the change you made and refer to the general part of the paper where you made the change. (Your supervisor or coauthor might have a different opinion here and you should obviously follow theirs in such cases.) *[It has been pointed out to me since writing this that Editors do often like line numbers]

(b) A great temptation is to simply insert a sentence into the MS to address a reviewer comment. Sometimes this works but more often it is out-of-place and out-of-context and awkward – so much so that people reading the paper can clearly see sentences that were added after the fact in response to reviewer comments. Instead, you need to re-read the entire MS (or at least the changed parts) with great care to make sure that any changes you make are seamlessly and effectively integrated into the whole such that they appear to have been there from the beginning. 


Of course, the above won't always work and, indeed, I have had papers rejected on the second round of review – although rarely. However, I do think the above steps will help you out. In closing, I will leave you with the famous video of Hitler responding to a peer review of his manuscript (which is apparently a lot less funny if you can speak German.)

Other resources:

Improving the reviewing process in Ecology and Evolutionary Biology