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.
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).
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!
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