When a species encounters a new environment on multiple occasions, but the same environment, how often does it re-use the same genes when adapting to the similar but novel habitats?  This is an unanswered question in evolutionary biology because such parallel adaptations can evolve by two genetic routes. First, the ancestral species might adapt simply by using genes that are already present (albeit at low frequencies) in the main body of the species—the so-called “standing genetic variation.” Every species, including ours, has a pool of genetic variants kicking around in its gene pool, many of them “neutral” or perhaps slightly deleterious but maintained by recurrent mutation. If the environment changes, these “standing” variants can be used as fuel for adaptation, so one might predict that the same variants might be used again and again when adapting to the same circumstance (so-called “parallel evolution”).

The other way to adapt is to use “new” mutations that happen to arise after one encounters a new habitat. In this case one might predict that multiple cases of adaptation to the same habitat might use different genetic variants, because many mutations are possible, they arise randomly, and selection will pick up only those that happen to arise.  (One exception here: if there are only one or a few possible mutations in the genome for adapting to the environment, then selection might have to wait for those to occur, and in the end you’ll also see the same mutations used again and again. This might explain why widely different species of insects adapt to organophosphate insecticides using mutations at the very same gene—an enzyme that detoxifies the insecticide—indeed, at the very same amino acid codon in that gene.) You can distinguish the two routes of adaptation by DNA sequencing: if the same standing variants are used repeatedly, they should show fundamental similarities in their DNA sequences, since they all derive from a common ancestral mutation.

A new paper in Nature by Felicity C. Jones et al. (there are a gazillion authors, though the last is the amiable David Kingsley, an evo-devo guy at Stanford who is the motive force behind most of this genetic work on sticklebacks), favors the former hypothesis: the re-use of standing genetic variation in multiple independent cases of adaptation.  The species is the threespine stickleback (Gasterosteus aculeatus).  The ancestral form of this fish was marine, but it has invaded freshwater—streams and lakes—multiple times, and each time it did so the fish evolved in similar directions.

So there are now two types, the marine (which does return to freshwater to breed) and the freshwater.  All the marine fish look pretty similar: they are larger, and deeper bodied, can survive better in salt water, and, most notably, are covered with bony armor and have pelvic spines. The freshwater forms are smaller and thinner, not tolerant of salt water, almost completely lack the bony armor of the marine forms, and many (but not all) such populations also lack the pelvis and the pelvic spine (hindfin).  The difference in armor plating and spiny-ness almost certainly reflects the relative lack of predators in the freshwater environment as opposed to the open ocean: it’s adaptive to not have to waste metabolic energy making protective structures you don’t need, and that’s supported by the fact that, when raised in a common environment, the marine form grows more slowly than the freshwater form.

Here’s a diagram showing the differences between the forms (top is freshwater, bottom marine; remember that these “forms” are classified not as different species, but as different subspecies of G. aculeatus). Note the divergence in armor plating, body size and shape, and the positions of the fins, the spines, and other “morphological landmarks” (diagram prepared by Dr. Felicity Jones of Stanford, first author of the paper, used with permission).

This more striking photo of the skeleton shows the big difference in bony armor and spines between the forms (remember that not all freshwater populations lack spines and pelvises, though all lack armor):

You can see a very nice lecture by David Kingsley on the evolution of sticklebacks here.  I recommend watching this one-hour video, produced by the Howard Hughes Medical Institute.

The Jones et al. paper used a clever technique designed to answer one question: since the freshwater form has evolved repeatedly throughout the world from the marine form (probably via some ancestors remaining in freshwater after breeding instead of returning to the sea), and has evolved parallel morphologies each time, how often is the genetic basis of this adaptive change similar? That is, how often has this parallel evolution used the same genetic variants?  The answer is “pretty often.”

The group used two clever ways to figure this out, which involved the tedious sequencing of 21 entire stickleback genomes: a reference genome and 10 pairs of fish from nearby localities, with one member of each pair being marine and one pair being freshwater. (The freshwater populations they studied lacked armor, but not spines or pelvises.) In essence, the method did a “family tree” of genes, and looked for those genes that all grouped together by habitat, i.e. were genetically similar among all freshwater forms, with the variants of those genes grouping in all saltwater forms. (Looking at most genes gives a “traditional” grouping, which groups fish by geographic region [i.e., Atlantic vs. Pacific] as opposed to habitat: that’s exactly what you’d expect if the evolutionary history of the forms was one of repeated colonization of freshwater habitats from saltwater.)

They found four nice results:

• The two methods each identified over 240 regions of the genome that responded in parallel when marine fish adapted to freshwater. That’s about 0.5% of the total genome.  Using only those genetic regions found in common with the two methods, the authors found 147 candidate regions for parallel adaptation (0.2% of the genome).  One of the regions that evolved in all freshwater fish contains a form of the autosmal Eda (hypohidrotic ectodermal dysplasia) allele, which is involved in formation of scales, plates, teeth, and other surface structures in fish and mammals.  This locus was implicated in previous work by the Kingsley lab, and the two forms that of the gene that make armor versus no armor differ in both coding and non-coding positions of the gene, so we’re not sure whether the difference in armor is due to a structural or regulatory change in the DNA.  The lesson, though, is that there has been substantial and large-scale use of the same genetic regions in parallel adaptation.  This is probably, as Kingsley has posited before,  because these variants (especially in Eda) are present in low frequency in all worldwide marine populations, and were simply present fortuitiously to serve as fuel for adaptation when climate change induced some marine populations to become permanent inhabitants of freshwater.

• The genetic regions implicated in parallel evolution contain (or are close to) contain genes involved in “cellular response to signals, behavioural interaction between organisms, amine and fatty acid metabolism, cell–cell junctions and WNT signalling,” as well as kidney function, as expected for fish that have to live in freshwater vs. saltwater.  And of course the Eda gene was involved, as was known previously.

• Many of the genes occurred close together on chromosomes, i.e., they were physically linked.  Through clever sequencing techniques, Jones et al. determined that this physical linkage (on chromosome 1, 11, and 21) was caused by the presence of chromosomal inversions: sections of rearranged chromosomes that contain the selected genes.  The marine forms have one form of chromosomal architecture; the freshwater forms have another.  The association of newly evolving genes with such chromosome rearrangements is adaptive, because the inverted regions keep adaptive gene complexes together (a cross-over, or recombination, between different inversions leads to the production of chromosomes with duplicated or deficient regions, which cause death in their carriers. Recombinants having mixtures “wrong” genes, then, like kidneys for freshwater and armor for saltwater, can’t survive). The association of inversions with adaptive genetic differences is a sign that the marine and freshwater forms hybridized during or after their differentiation, for the associations will not be favored unless the forms have a chance to mate with each other during their divergence.

• Finally, the authors divided the genetic regions involved in parallel divergence into three classes: “coding regions” (those genome regions that involve a difference in protein sequence), “regulatory regions” (those regions where there is parallel adaptation but which contain no structural genes, so that the adaptation probably results, as in Eda, from differences in gene regulation that affect the expression but not the sequence of the protein product), and third, “probably regulatory” regions (regions that contain coding and noncoding sequences but where the authors couldn’t pinpoint a structural protein difference that distinguished all marine from all freshwater forms).  Jones et al. looked at 64 regions identified by both of their methods as having the strongest signs of parallel evolution, and here’s the breakdown of these 64 genetic regions involved in parallel adaptation:

As you can see, only about 1 in 6 of the evolved regions involved a change in protein sequence, while the rest involved likely or potential changes in gene regulation.  In this case, then, parallel evolution seems to have occurred largely through changes in when and how genes are expressed, not in the sequences of the genes themselves.  This goes along with evo-devotee Sean Carroll’s suggestion that evolutionary changes in animal form will largely involve changes in gene regulation rather than sequence.

Three caveats here, though.  First, Carroll’s hypothesis applied to animal form and not physiology (though he never explained why), and yet the changes we see in this study involve both form and physiology.

Second, the changes that Jones et al. studied were those involved in parallel adaptation: similar changes in multiple environments.  The chart above doesn’t tell us anything about the spectrum of genetic change involved in unique adaptations: those that were specific to one or some but not all instances of freshwater invasion. We know there are such genes, for Jones et al. describe some.

Third, other studies don’t show such a high concentration of regulatory regions vs. structural regions in the evolving genome. As Jones et al. note:

Mutations causing structural changes in proteins are the most abundant variants recovered in laboratory Escherichia coli and yeast evolution experiments. They make up 90% of 40 published examples of adaptive changes between closely related taxa, and 63–77% of the known molecular basis of phenotypic traits in domesticated or wild species. The larger fraction of regulatory changes implicated during repeated stickleback evolution may reflect our use of whole-genome rather than candidate gene approaches, stronger selection against loss-of-function and pleiotropic protein-coding changes in natural populations than in laboratory or domesticated organisms, or an increasing prevalence of regulatory changes at interspecific compared to intraspecific levels, including emerging species such as marine and freshwater sticklebacks.

To my mind, the data are still out on the Carroll hypothesis.  Five years ago, Hopi Hoekstra and I published a paper in Evolution (reference below) questioning whether the “rush to judgment” about the importance of regulatory genes in evolution was really warranted by the scanty amount of supporting data. Our point was not to push for the important of structural-gene changes in evolution (though they surely must be important, for many new genes, like globins or immune-system genes or olfactory genes, arise by the duplication of previously-existing genes followed by evolutionary divergence, and those are surely structural changes), but to call for more data to resolve the issue.  We were misunderstood, I think, as saying that evolution largely proceeds by structural gene changes, and people like Sean Carroll got mad at us. But at the time there simply weren’t enough data to resolve the issue.  I still don’t think there are, but I’m perfectly happy to accept a predominance of regulatory-gene changes in adaptive evolution if that’s what the data wind up showing.

The data of Jones et al. do show this for parallel evolution, and we need more work along those lines to resolve the issue. (That work, though, should involve both parallel and unique cases of adaptation.) But what is clear from their paper is that standing genetic variation can be used over and over again if a species has to adapt independently to similar habitats.  And that tells us something about how evolution worked, at least in this species.

This is a superb paper: congrats to Felicity Jones and to David Kingsley and his group. I love seeing long-standing evolutionary questions answered by a combination of classical morphological data and high-tech genetic work.

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F. C. Jones et al. 2012.  The genomic basis of adaptive evolution in threespine sticklebacks. Nature 484:55-61

Hoekstra, H. E., and J. A. Coyne. 2007. The locus of evolution: Evo devo and the genetics of adaptation. Evolution 61:995-1016.