From the Library of Prof. William B. Provine

I just saw the sad news that Will Provine, historian of population genetics, died peacefully at his home at the age of 73. Others will no doubt write of Provine’s legacy as a scholar and orator of the highest calibre, a fervent proponent of atheism and evolution that only a preacher’s son could be. I’m moved by his death to recall my experience of having Provine as a lecturer during my undergrad days at Cornell 20 years ago, where his dramatic and entertaining style drew me fully into evolutionary biology, both as a philosophy and as a profession. I can’t say I knew Provine well, but I can say our interactions left a deep impression on me.  He was an incredibly kind and engaging, pulling you onto what he called the “slippery slope” where religious belief must yield to rationalism.

I vividly recall Provine giving me a hard cover copy of the compendium of Dobzhansky’s papers he co-edited on our first meeting after class (pulled from a half-full box at the ready near his desk), and discussing the then-recent death of Motoo Kimura, who he was researching for his as-yet-unpublished history of the Neutral Theory. We met and talked about population genetics and molecular evolution several times after that, and for reasons I can’t quite recall, Provine ended up offering me keys to his personal library in the basement of Corson Hall. I’ll never forget the first time he showed me his library, with bookshelves lining what would have been a lab space, filled with various version of classic works in Genetics, Evolution, Development and History of Science. The delight he had in showing me his shelf of various editions of the Origin of Species was only matched by the impish pleasure he had in showing me the error in chromosome segregation on the spine of the first edition of Dobzhansky’s Genetics and the Origin of Species, or how to decode the edits to text of Fisher’s Genetical Theory of Natural Selection (see figure below).

IMG_0405a

In my first tour of his library, Provine showed me how to decode the revisions to the 1958 second edition of Fisher’s Genetical Theory of Natural Selection. Notice how the font in paragraph 2 is smaller that in paragraphs 1 and 3. Text in this font was added to the original plates prior to the second printing. Provine then handed me one of the many copies of this book he had on his shelf for me to keep, which is one of my few prized possessions.

His reprint collection was equally impressive (inherited from Sewall Wright from what I understand), with many copies signed, with compliments of the author, by the founders of the Modern Synthesis. Provine’s reprint collection was surpassed in value only by the FlyBase reprint collection in the Dept of Genetics in Cambridge, in my experience. I used Provine’s library to study quite often in my last year or so at Cornell, interrupting work on Alex Kondrashov’s problem sets by browsing early 20th century biology texts. Being able to immerse myself in this trove of incredible books left a lasting effect on me, and I have no doubt was a major factor in deciding to pursue academic research in evolution and genetics. Sadly while no longer physically intact, I am very glad to know the 5,000+ items in Provine’s library have been contributed to the Cornell Library, possibly the best place for the spirit of an atheist and historian to live on.

Multi-sample SNP calling circa 1994

Last November, when news of Fred Sanger‘s death was making its way around scientific circles, so too were many images of Sanger DNA sequencing reactions visualized as autoradiograms. These images brought back memories of a style of Sanger sequencing gel that I first saw in an undergraduate class on population genetics taught by Charles (“Chip”) Aquadro at Cornell University in the autumn of 1994, which left a deep impression on me. My personal photograph 51, if you will.

At the time, I was on course to be a high-school biology teacher, a plan that was scuppered by being introduced to the then-emerging field of molecular population genetics covered in Aquadro’s class. I distinctly remember Aquadro putting up a transparency on the overhead showing an image of a Sanger gel where each of the four bases were run in sets that included each individual in the sample, allowing single nucleotide polymorphisms (“SNPs”) to be easily identified by eye. This image made an extremely strong impression on me, transforming the abstract A and a alleles typically discussed in population genetics into concrete molecular entities. Together with the rest of the material in Aquadro’s class, this image convinced me to pursue a career in evolutionary genetics.

I emailed Aquadro around that time last year to see if he had such an image digitized, and he said he’d try to dig one out. A few weeks ago he sent me the following image, which shows the state-of-the-art in multi-sample SNP calling circa 1994:

SangerSequencingGel-RosyDmel-Aquadro

Multi-sample Sanger sequencing gel of a fragment of the Drosophila melanogaster rosy (Xdh) gene (credit: Charles Aquadro). The first four lanes represent the four bases of the “reference” sequence, followed by four sets of lanes (one for each base) containing sequencing reactions for each individual in the sample. Notice how when a band is missing from a set for one individual, it is present in a different set for that same individual. This format allowed the position and identity of variable sites in a sample to be identified quickly, without having to read off the complete sequence for each individual.

For those of us who now perform multi-sample SNP calling at the whole-genome scale using something like a Illumina->BWA->SAMtools pipeline, it is sometimes hard to comprehend how far things have progressed technologically in the last 20 years.

Perhaps equally dramatic are the changes in the larger social and scientific value placed on the use of sequence analysis and the identification of variation in natural populations. At that time, the Aquadro lab was referred to in a friendly, if somewhat disparaging, way as the “Sequence and Think Lab” by others in the department (because “all they do in that lab is sequence and think”). As the identification of natural molecular variation in humans quickly becomes the basis for personalized medicine, and as next-generation sequencing is incorporated into more basic molecular biological techniques, it is impressive to see how quickly the “sequence and think” model has moved from a peripheral to a central role in modern biology.

 

On the 30th Anniversary of DNA Sequencing in Population Genetics

30 years ago today, the “struggle to measure genetic variation” in natural populations was finally won. In a paper entitled “Nucleotide polymorphism at the alcohol dehydrogenase locus of Drosophila melanogaster” (published on 4 Aug 1983), Martin Kreitman reported the first effort to use DNA sequencing to study genetic variation at the ultimate level of resolution possible. Kreitman (1983) was instantly recognized as a major advance and became a textbook example in population genetics by the end of the 1980s. John Gillespie refers to this paper as “a milestone in evolutionary genetics“. Jeff Powell in his brief history of molecular population genetics goes so far as to say “It would be difficult to overestimate the importance of this paper”.

Arguably, the importance of Kreitman (1983) is greater now than ever, in that it provides both the technical and conceptual foundations for the modern gold rush in population genomics, including important global initiatives such as the 1000 Genomes Project. However, I suspect this paper is less well know to the increasing number of researchers who have come to studying molecular variation from routes other than through a training in population genetics. For those not familiar with this landmark paper, it is worth taking the time to read it or Nathan Pearson‘s excellent summary over on Genomena.

As with other landmark scientific efforts, I am intrigued by how such projects and papers come together. Powell’s “brief history” describes how Kreitman arrived at using DNA to study variation in Adh, including some direct quotes from Kreitman (p. 145). However, this account leaves out an interesting story about the publication of this paper that I had heard bits and pieces of over time. Hard as it may be to imagine in today’s post-genomic sequence-everything world, using DNA sequencing to study genetic variation in natural populations was not immediately recognized as being of fundamental importance, at least by the editors of Nature where it was ultimately published.

To better understand the events of the publication of this work, I recently asked Richard Lewontin, Kreitman’s PhD supervisor, to provide his recollections on this project and the paper. Here is what he had to say by email (12 July 2013):

Dear Casey Bergman

I am delighted that you are commemorating Marty’s 1983 paper that changed the whole face of experimental population genetics. The story of the paper is as follows.

It was always the policy in our lab group that graduate students invented their own theses. My view was (and still is) that someone who cannot come up with an idea for a research program and a plan for carrying it out should not be a graduate student. Marty is a wonderful example of what a graduate student can do without being told what to do by his or her professor. Marty came to us from a zoology background and one day not very long after he became a member of the group he came to me and asked how I would feel about his investigating the genetic variation in Drosophila populations by looking at DNA sequence variation rather than the usual molecular method of looking at proteins which then occupied our lab. My sole contribution to Marty’s proposal was to say “It sounds like a great idea.”  I had never thought of the idea before but it became immediately obvious to me that it was a marvelous idea.  So Marty went over on his own initiative, to Wally Gilbert’s lab and learned all the methodology from George Church who was then in the Gilbert lab.

After Marty’s work was finished and he was to get his degree, he wrote a paper based on his thesis and, with my encouragement, sent the paper to Nature. He offered to make me a co-author, but I refused on long-standing principle. Since the idea and the work were entirely his, he was the sole author, a policy that was general in our group. I had no doubt that it was the most important work done in experimental population genetics  in many years and Nature was an obvious choice for this pathbreaking work.

The paper was soon returned by the Editor saying that they were not interested because they already had so many papers that gave the DNA sequence of various genes that they really did not want yet another one! Obviously they missed the point. My immediate reaction was to have Marty send the paper to a leading influential British Drosophila geneticist who would obviously understand its importance, asking him to retransmit the paper to Nature with his recommendation. He did so and the Editor of Nature then accepted it for publication. The rest is history.

Our own lab very quickly converted from protein electrophoresis to DNA sequencing, and I spent a lot of time using and updating the computer interface with the gel reading process, starting from  Marty’s original programs for reading gels and outputting sequences. We never went back to protein electrophoresis. While protein gel electrophoresis certainly revolutionized population genetics, Marty’s introduction of DNA sequencing as the method for evolutionary genetic investigation of population genetic issues was a much more powerful one and made possible the testing of a  variety of evolutionary questions for which protein gel electrophoresis was inadequate. Marty deserves to be considered as one of the major developers of evolutionary and population genetics studies.

Yours ,

Dick Lewontin

Some may argue that Kreitman (1983) did not reveal all forms of genetic variation at the molecular level (e.g. large-scale structural variants) and therefore does not truly represent the “end” of the struggle to measure variation. What is clear, however, is that Kreitman (1983) does indeed represent the beginning of the “struggle to interpret genetic variation” at the fundamental genetic level, a struggle that may ultimately take longer then measuring variation itself. According to Maynard Olson interpreting (human) genomic variation will be a multi-generational effort “like building the European cathedrals“. 30 years in, Olson’s assessment is proving to be remarkably accurate. Here’s to Kreitman (1983) for laying the first stone!

Related Posts:

Calvin Bridges, Automotive Pioneer

Calvin Bridges in 1935 (Photo Credit: Smithsonian Institution Collections SIA Acc. 90-105 [SIA2008-0022])

Calvin Bridges (1889-1938) is perhaps best known as one of the original Drosophila geneticists in world. As an original member of Thomas Hunt Morgan’s Fly Room at Columbia University, Bridges made fundamental contributions to classical genetics, notably contributing the first paper ever published in the journal Genetics. The historical record on Bridges is scant, since Morgan and Alfred Sturtevant destroyed Bridges’ papers after his death to preserve the name of their dear friend whose politics and attitudes to free love were radical in many ways. Morgan’s biographical memoir of Bridges presented to the National Academy of Sciences in 1940 contains very little detail on Bridges’ life, and this historical black hole has piqued my curiosity for some time.

Recently, I stumbled across a listing in the New York Times for an exhibit in Brooklyn recreating the original Columbia Fly Room, which will be used as a set in an upcoming film of the same name directed by Alexis Gambis. Gambis’ film approaches the Fly Room from the perspective of a visit to the lab by one of Bridges children, Betsy Bridges. I recommend other Drosophila enthusiasts to check out The Fly Room website and follow @theflyroom and @alexisgambis on Twitter for updates about the project.

In digging around more about this project, I found a link to the Kickstarter page that was used to raise funds for the film. This page includes an amazing story about Bridges that I had never heard about previously. Apparently, after Morgan and his group moved to Caltech in 1928, Bridges built from scratch a futuristic car of his own design called “The Lightening Bug”. This initially came a big surprise to me, but on reflection it is in keeping with Bridge’s role as the main technical innovator for the original Drosophila group. For example, Bridges introduced the binocular dissecting scope, the etherizer, the controlled temperature incubator, and agar-based fly food into the Drosophilist’s toolkit.

Here is a clipping from Modern Mechanix from Aug 1936 describing the Lightening Bug:

Coverage of Calvin Bridge’s Lightning Bug in Modern Mechanix (Aug 1936).

Bridge’s Lightening Bug was notable enough to be written up in Time Magazine in May 1936, which described his car as follows:

It is almost perfectly streamlined, even the license plates and tail-lamp being recessed into the body and covered with Pyralin windows flush with the streamlining. There are no door handles; the doors must be opened with special keys. Dr. Bridges pronounced the Lightning Bug crash-proof and carbon-monoxide-proof. “My whole aim,” said he, “was to show what could be done to attain safety, economy and readability in a small car.”

Newshawks discovered that for months, when he got tired of looking at fruit flies, the geneticist had retired to a garage, put on a greasy jumper and worked on his car far into the night, hammering, welding, machining parts on a lathe. Now & then, the foreman reported, Dr. Bridges hit his thumb with a hammer. Once he had to visit a hospital to have removed some tiny bits of steel which flew into his eyes. It was Calvin Bridges’ splendid eyesight which first attracted Dr. Morgan’s interest in him when Bridges was a shaggy, enthusiastic student at Columbia.

Calvin Bridges next to the Lightening Bug (Time Magazine, 4 May 1936).

Gambis has also posted a video of the Lightening Bug being driven by Bridges taken by Pathé News. Gambis estimates this clip was from around 1938, but it is probably from 1936/7 since Bridges died in Dec 1938 and by the time Ed Novitski started graduate school at CalTech in the autumn of 1938 Bridges was terminally ill, but appears fit in this clip.  This clip clearly shows the design of Bridges’ Lightening Bug was years ahead of its time in comparison to the other cars in the background. I also would wager this is the only video footage in existence of Calvin Bridges.

The only other information I could find on the web about the Lightening Bug was a small news clipping that was making the rounds in local new April/May 1936:

LighteningBug

Interestingly, the only mention I can find of this story in historical accounts of the Drosophila group is one parenthetical note by Shine and Wrobel in their 1976 biography of Morgan that had previously escaped my notice. On page 120, they discuss how Morgan handled the receipt of his 1933 Nobel Prize in Physiology or Medicine (emphasis mine):

…Morgan was very modest about the honor. He frequently pointed out that it was a tribute to experimental biology than to any one man….As Morgan acknowledged the joint nature of the work, he divided the tax-free $40,000 award equally among his own children and those of Bridges and Sturtevant (but not of Muller’s). He gave no reason; in the letter to Sturtevant for example, he said merely I’m enclosing some money for your children. (Bridges, however, is said to have used his to build a new car.)

So there you have it: Calvin Bridges, Drosophila geneticist, was also an unsung automotive pioneer whose foray into designing futuristic cars was likely funded in part by the proceeds of the 1933 Nobel Prize!

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Directed Genome Sequencing: the Key to Deciphering the Fabric of Life in 1993

Seeing the #AAASmtg hashtag flowing on my twitter stream over the last few days reminded my that my former post-doc advisor Sue Celniker must be enjoying her well-deserved election to the American Association for the Advancement of Science (AAAS). Sue has made a number of major contributions to Drosophila genomics, and I personally owe her for the chance to spend my journeyman years with her and so many other talented people in the Berkeley Drosophila Genome Project. I even would go so far as to say that it was Sue’s 1995 paper with Ed Lewis on the “Complete sequence of the bithorax complex of Drosophila” that first got me interested in “genomics.” I remember being completely in awe of the Genbank accession from this paper which was over 300,000 bp long! Man, this had to be the future. (In fact the accession number for the BX-C region, U31961, is etched in my brain like some telephone numbers from my childhood.) By the time I arrived at BDGP in 2001, the sequencing of the BX-C was already ancient history, as was the directed sequencing strategy used for this project.  These rapid changes made discovery of a set of discarded propaganda posters collecting dust in Reed George’s office that were made at the time (circa 1993) extolling the virtues of “Directed Genome Sequencing” as the key to “Deciphering the Fabric of Life” all the more poignant. I dug a photo I took of one of these posters today to commemerate the recognition of this pioneering effort (below). Here’s to a bygone era, and hats off to pioneers like Sue who paved the road for the rest of us in (Drosophila) genomics!

Directed-genome-sequencing

From Electron to Retrotransposon: “-on” the Origin of a Common Suffix in Molecular Biology

Over the last year or so, I have become increasingly interested in understanding the origin of major concepts in genetics and molecular biology. This is driven by several motivating factors, primarily to cure my ignorance/satisfy my curiosity but also to be able to answer student queries more authoritatively and unearth unsolved questions in biology. One of the more interesting stories I have stumbled across relates to why so many terms in molecular biology (e.g. codon, replicon, exon, intron, transposon, etc.) end with the suffix “-on”? While nowhere as pervasive the “-ome” suffix that has contaminated biological parlance of late,  the suffix “-on” has clearly left its mark in some of the most frequently used terms the lexicon of molecular biology.

According to Brenner (1996) [1], the common use of the suffix “-on” in molecular biological terms can be traced to Seymour Benzer’s dissection of the fine structure of the rII locus in bacteriophage T4, which overturned the classical idea that a gene is an indivisible unit:

To mark this new view of the gene, Seymour invented new terms for the now different units of mutation, recombination and function. As he was a physicist, he modelled his terms on those of physics and just as electrons, protons and neutrons replaced the once indivisible atom, so genes came to be composed of mutons, recons and cistrons. The the unit of function, the cistron was based on the cis–trans complementation test, of which only the trans part is usually done…Of these terms, only cistron came to be widely used. It is conjectured that the other two, the muton and the recon, disappeared because Seymour failed to follow the first rule for inventing new words, which is to check what they may mean in other languages…Seymour’s pioneering invention of units was followed by a spate of other new names not all of which will survive. One that seems to have taken root is codon, which I invented in 1957; and the terms intron and exon, coined by Walter Gilbert, are certain to survive as well. Operon is moot; it is still frequently used in prokaryotic genetics but as the weight of research shifts to eukaryotes, which do not have such units of regulation, it may be lost. Replicon, invented by Francis Jacob and myself in 1962, seems also to have survived, despite the fact that we paid insufficient attention to how it sounded in other languages.

Thus, the fact that many molecular biological terms end in “-on” (initiated by Benzer) owes its origin to patterns of nomenclature in chemistry/nuclear physics (which itself began with Stoney’s proposal of the term electron in 1894) and the desire to identify “fundamental units” of biological structure and function.

While Brenner’s commentary provides a crucial first-hand account to understand the origin of these terms, it does not provide any primary references concerning the coining of these terms. So I’ve spent some time digging out the original usage for a number of more common molecular biology “-ons”, which I thought many be of use or interest to others.

The terms reconmuton and cistron were defined by Benzer (1957) [2] as follows:

  • Recon: “The unit of recombination will be defined as the smallest element in the one-dimensional array that is interchangeable (but not divisible) by genetic recombination. One such element will be referred to as a “recon.””
  • Muton: “The unit of mutation, the “muton” will be defined as the smallest element that, when altered, can give rise to a mutant form of the organism.”
  • Cistron: “A unit of function can be defined genetically, independent of biochemical information, by means of the elegant cistrans comparison devised by [Ed] Lewis…Such a map segment, corresponding to a function which is unitary as defined by the cistrans test applied to the heterocaryon, will be defined as a cistron.”

I have not been able to find a definitive first reference that defines the term codon the fundamental unit of the genetic code. According to Brenner (1996) [1] and US National Library of Medicine’s Profiles in Science webpage on Marshall Nirenberg [2], the term codon was introduced by Brenner in 1957 “to describe the fundamental units engaged in protein synthesis, even though the units had yet to be fully determined. Francis Crick popularized the term in 1959. After 1962, Nirenberg began to use “codon” to characterize the three-letter RNA code words” [3].

The term operon was introduced by Jacob et al. (1960) [4] and defined as follows (italics theirs):

  • Operon: “Celle-ci comprendrait des unités d’expression coordonée (opérons) constituées par un opérateur et le group de gènes de structure coodoneés par lui.”

The term replicon was introduced by Jacob and Brenner (1963) [4] and defined as follows (italics theirs):

  • Replicon: “Il est donc clair qu’un chromosome (de bactérie ou de phage) ou un épisome constitue une unité de réplication indépendante ou réplicon, dont la reproduction est régie par la présence et l’activité de certain déterminants qu’il porte. Les caractères des réplicons exigent qu’ils déterminent des systèmes spécifique gouvernant leur propre réplication.”

Near and dear to my heart is the term transposon, which was first introduced by Hedges and Jacob (1974) [7] (italics theirs):

  • Transposon: “We designate DNA sequences with transposition potential as transposons (units of transposition)”

The very commonly used terms intron and exon were defined by Gilbert (1978) [6] as follows:

  • Intron & Exon: “The notion of the cistron, the genetic unit of function that one thought to correspond to a polypeptide chain, must be replaced by that of a transcription unit containing regions which will be lost from the mature messenger – which I suggest we call introns (for intragenic regions) – alternating with regions which will be expressed – exons.”

And finally, Boeke et al. (1985) [8] defined the term retrotransposon in the following passage (italics theirs):

  • Retrotransposon: “These observations, together with the finding that introns are spliced out of the Ty upon transposition, suggest that reverse transcription is a step in the transposition of Ty elements…We therefore propose the term retrotransposon  for Ty and related elements.”

So there you have it, from electron to retrotransposon in just a few steps. I’ve left out some lesser used terms with this suffix for the moment (e.g. regulon, stimulon, modulon), so as not to let this post go -on and -on. If anyone has any major terms to add here or corrections to my reading of the tea leaves, please let me know in the comments below.

References:
[1] Brenner, S. (1995) “Loose end: Molecular biology by numbers… one.” Current Biology 5(8): 964.
[2] Benzer, S. (1957) “The Elementary Units of Heredity.” in Symposium on the Chemical Basis of Heredity p. 70–93.  Johns Hopkins University Press
[4] Jacob, F., et al. (1960) “L’opéron: groupe de gènes à expression coordonnée par un opérateur.” C.R. Acad. Sci. Paris 250: 1727-1729.
[5] Jacob, F., and S. Brenner. (1963) “Sur la regulation de la synthese du DNA chez les bacteries: l’hypothese du replicon.” C. R. Acad. Sci 246: 298-300.
[6] Gilbert, W. (1978) “Why genes in pieces?.” Nature 271(5645): 501.
[7] Hedges, R. W., and A. E. Jacob. (1974) “Transposition of ampicillin resistance from RP4 to other replicons.” Molecular and General Genetics MGG 132(1): 31-40.
[8] Boeke, J.D., et al. (1985) “Ty elements transpose through an RNA intermediate.” Cell 40(3): 491.
Credits:
Jim Shapiro (University of Chicago) gave very helpful pointers to possible places where the term “transposon” might have originally have been introduced.

On The Neutral Sequence Fallacy

ResearchBlogging.org

Beginning in the late 1960s, Motoo Kimura overturned over a century of “pan-selectionist” thinking in evolutionary biology by proposing what has come to be called The Neutral Theory of Molecular Evolution. The Neutral Theory in its basic form states that the dynamics of the majority of changes observed at the molecular level are governed by the force of Genetic Drift, rather than Darwinian (i.e. Positive) Natural Selection. As with all paradigm shifts in Science, there was much of controversy over the Neutral Theory in its early years, but nevertheless the Neutral Theory has firmly established itself as the null hypothesis for studies of evolution at the molecular level since the mid-1980s.

Despite its widespread adoption, over the last ten years or so there has been a worrying increase in abuse of terminology concerning the Neutral Theory, which I will collectively term here the “Neutral Sequence Fallacy” (inspired by T. Ryan Gregory’s Platypus Fallacy). The Neutral Sequence Fallacy arises when the distinct concepts of functional constraint and selective neutrality are conflated, leading to the mistaken description of functionally unconstrained sequences as being “Neutral”. The Fallacy, in short, is to assign the term Neutral to a particular biomolecular sequence.

The Neutral Sequence Fallacy now routinely causes problems in the fields of evolutionary and genome biology, both in terms of generating conceptual muddles as well as shifting the goalposts needed to reject the null model of sequence evolution. I have intended to write about this problem for years in order to put a halt to this growing abuse of Neutral terminology, but unfortunately never found the time. However, this issue has unfortunately reared its head more strongly in the last few days with new forms of the Neutral Sequence Fallacy arising in the context of discussions about the ENCODE project, motivating a rough version of this critique to finally see the light of day. Here I will try to sketch out the origins of the Neutral Sequence Fallacy, in its original pre-genomic form that was debunked by Kimura while he was alive, and in its modern post-genomic form that has proliferated unchecked since the early comparative genomic era.

The Neutral Sequence Fallacy draws on several misconceptions about the Neutral Theory, and begins with the abbreviation of the theory’s name from its full form (The Neutral Mutation – Random Drift Hypothesis) to its colloquial form (The Neutral Theory). This abbreviation de-emphasizes that the concept of selective neutrality applies to mutations (i.e. variants, alleles), not biomolecular sequences (i.e. regions of the genome, proteins). Simply put, only variants of a sequence can be neutral or non-neutral, not sequences themselves.

The key misconception that permits the Neutral Sequence Fallacy to flourish is the incorrect notion that if a sequence is neutrally evolving, it implies a lack of functional constraint operating on that sequence, and vice versa. Other ways to state this misconception are: “a sequence is Neutral if it is under no selective constraint” or conversely “selective constraint rejects Neutrality”. This misconception arose originally in the 1970s, shortly after the proposal of The Neutral Theory when many researchers were first coming to terms with what the theory meant. This misconception became prevalent enough that it was the first to be addressed head-on by Kimura (1983) nearly 30 years ago in section 3.6 of his book The Neutral Theory of Molecular Evolution entitled “On some misunderstandings and criticisms” (emphasis is mine):

Since a number of criticisms and comments have been made regarding my neutral theory, often based on misunderstandings, I would like to take this opportunity to discuss some of them. The neutral theory by no means claims that the genes involved are functionless as mistakenly suggested by Zuckerkandl (1978). They may or may not be, but what the neutral theory assumes is that the mutant forms of each gene participating in molecular evolution are selectively nearly equivalent, that is, they can do the job equally well in terms of survival and reproduction of the individual. (p. 50)

As pointed out by Kimura and Ohta (1977), functional constraints are consistent with neutral substitutions within a class of mutants. For example, if a group of amino acids are constrained to be hydrophilic, there can be random changes within the codons producing such amino acids…There is, of course, negative selection against hydrophobic mutants in this region, but, as mentioned before, negative selection does not contradict the neutral theory.  (p. 53)

It is understandable how this misconception arises, because in the limit of zero functional constraint (e.g. in a non-functional pseudogene), all alleles become effectively equivalent to one another and are therefore selectively neutral. However, this does not mean that an unconstrained sequence is Neutral (unless we redefine the meaning of Neutrality, see below), because a sequence itself cannot be Neutral, only variants of a sequence can be Neutral with respect to each other.

It is crucial in this context to understand that the Neutral Theory accommodates all levels of selective constraint, and sequences under selective constraint can evolve Neutrally (see formal statement of this in Equation 5.1 of Kimura 1983). This point is often lost on many people. Until you get this, you don’t understand the Neutral Theory. A simple example shows how this is true. Consider a single codon in a protein coding region that codes for a degenerate amino acid. Deletion of the third codon position would creat a frameshift, and thus a third position “silent” site is indeed functional. However, alternative codons for this amino acid are functionally equivalent and evolve (close to) neutrally. The fact that these alternative alleles evolve neutrally has to do with their equivalence of function, not the degree of their functional constraint.

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To demonstrate the The Neutral Sequence Fallacy, I’d like to point out a few clear examples of this misconception in action.  The majority of transgressions in this area come from the genomics community where people may not have been formally trained in evolution, but I am sad to say that an increasing number of evolutionary biologists are also falling victim to The Neutral Sequence Fallacy these days. My reckoning is that the The Neutral Sequence Fallacy gained traction again in the post-genomic era around the time of the mouse genome paper by Waterston et al. (2002). In this widely-read paper, putatively unconstrained ancestral repeats were referred to (incorrectly) as “neutrally evolving DNA”, and used to estimate the fraction of the human genome under selective constraint. This analysis culminated with the following question: “How can we cleanly separate neutral and selected sequences?”. Under the Neutral Theory, this question makes no sense. First, sequences cannot be neutral; and second the framework used to detect functional constraints by comparative genomics assumes Neutral evolution of both classes of sites (unconstrained and constrained) – i.e. most changes between species are driven by Genetic Drift not Positive Selection. The proper formulation of this question should have been: “How can we cleanly separate unconstrained and constrained sequences?”.

Here is another clear example of the Neutral Sequence Fallacy in action from Lunter et al. (2006):

Figure 5 from Lunter et al. (2006). Notice how in the top panel, regions of the genome are contrasted as being “Neutral” vs. “Functional”. Here the term “Neutral” is being used incorrectly to mean selectively unconstrained. The bottom panel shows how indels are suppressed in Functional regions leading to intergap segments.

Here are a couple of more examples of the Neutral Sequence Fallacy in action, right in the title of fairly high-profile comparative genomics papers:

Title from Enlistki et al (2003). Notice that the functional class of “Regulatory DNA” is incorrectly contrasted as being the complement of nonfunctional “Neutral Sites”. In fact, both classes of sites are assumed to evolve neutrally in the authors’ model.

Title from Chin et al (2005). As above, notice how the concept of “Functionally conserved” is incorrectly stated to be the opposite of “Neutral sequence” and both classes of sites are assumed to evolve neutrally in the authors’ model.

I don’t mean to single these papers out, they just happen to represent very clear examples of the Neutral Sequence Fallacy in action. In fact, the Lunter et al. (2006) paper is one of my all time favorites, but it bugs the hell out of me when I have to unpick student’s misconceptions after they read it. Frustratingly, the list of papers repeating the Neutral Sequence Fallacy is long and growing. I have recently started to collect them as a citeulike library to provide examples for students to understand how not to make this common mistake. (If anyone else would like to contribute to this effort, please let me know — there is much work to be done to reverse this trend.)

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So what’s the big deal here?  Some would argue that these authors actually know what they are talking about, but they just happen to be using the wrong terminology. I wish that this were the case, but very often it is not. In many papers that I read or review that perpetrate the Neutral Sequence Fallacy, I usually find further examples of seriously flawed evolutionary reasoning, suggesting that they actually do not have a deep understanding of the issues at hand. In fact, evidence of the Neutral Sequence Fallacy is usually a clear hallmark in a paper that the authors are most likely practicing population genetics or molecular evolution without a license. This leads to a Neutral Sequence Fallacy of the 1st Kind: where authors do not understand the difference between the concepts functional constraint and selective neutrality. The problems for the Neutral Theory caused by violations of the 1st Kind are deep and clear. Because the Neutral Theory is not fully understood, it is possible to construct a straw-man version of the null hypothesis of Neutrality that can easily be “rejected” simply by finding evidence of selective constraint. Furthermore, because selectively unconstrained sequences are asserted (incorrectly) to be “Neutral” without actually evaluating their mode of evolution, this conceptual error undermines the entire value of the Neutral Theory as a null hypothesis testing framework.

But some authors really do know the difference between these ideas, and just happen to be using the term “Neutral” as shorthand for the term “Unconstrained.” Increasingly, I see some of my respected peers making this mistake in print who are card-carrying molecular evolutionists and do know their stuff. In these cases what is happening is a Neutral Sequence Fallacy of the 2nd Kind: understanding the difference between functional constraint and selective neutrality, but using lazy terminology that confuses these ideas in print. This is most often found in the context of studies on noncoding DNA where, in the absence of the genetic code to conveniently constrain terminology, people use terms like “neutral standard” or “neutral region” or “neutral sites” or “neutral proxy” in place of  “putatively unconstrained”. While the meaning of violations of the 2nd Kind can be overlooked and parsed correctly by experts in molecular evolution (I hope), this sloppy language causes substantial confusion about the Neutral Theory by students or non-evolutionary biologists who are new to the field, and leads to whole swathes of subsequent violations of the 1st Kind. Moreover, defining sequences as Neutral serves those with an Adaptationist agenda: since a control region is defined as being Neutral, all mutations that occur in that region must therefore be neutral as well, and thus any potential complications of the non-neutrality of mutations in one’s control region are conveniently swept under the carpet. Violations of the 2nd Kind are often quite insidious since they are generally perpetrated by people with some authority in evolutionary biology, often who are unaware of their misuse of terminology and who will vigorously deny that they are using terms which perpetuate a classical misconception laid to rest by Kimura 30 years ago.

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Which brings us to the most recent incarnation of the Neutral Sequence Fallacy in the context of the ENCODE project. In a companion post explaining the main findings of the ENCODE Project, Ewan Birney describes how the ENCODE Project reinforced recent findings that many biochemical events operate on the genome that are highly reproducible, but have no known function. In describing these event, Birney states:

I really hate the phrase “biological noise” in this context. I would argue that “biologically neutral” is the better term, expressing that there are totally reproducible, cell-type-specific biochemical events that natural selection does not care about. This is similar to the neutral theory of amino acid evolution, which suggests that most amino acid changes are not selected either for or against…Whichever term you use, we can agree that some of these events are “neutral” and are not relevant for evolution.

Under the standard view of the Neutral Theory, Birney misuses the term “Neutral” here to mean lack of functional constraint, repeating the classical form of the Neutral Sequence Fallacy. Because of this, I argue that Birney’s proposed terminology be rejected, since it will perpetuate a classic misconception in Biology. Instead, I propose the term “biologically inert”.

But wait a minute, you say, this is actually a transgression of the 2nd Kind. Really what is going on here is a matter of semantics. Birney knows the difference between functional constraint and selective neutrality. He is just formalizing the creeping misuse of the term Neutral to mean “Nonfunctional” that has been happening over the last decade.  If so, then I argue he is proposing to assign to the term Neutral the primary misconception of the Neutral Theory previously debunked by Kimura. This is a very dangerous proposal, since it will lead to further confusion in genomics arising from the “overloading” of the term Neutral (Kimura’s meaning: selectively equivalent; Birney’s meaning: no functional constraint). This muddle will subsequently prevent most scientists from properly understanding the Neutral Theory, and lead to many further examples of the Neutral Sequence Fallacy of both Kinds.

In my view, semantic switches like this are dangerous in Science, since they massively hinder communication and, therefore, progress. Semantic switches also lead to a distortion of understanding about key concepts in science. A famous case in point is Watson’s semantic switch of Crick’s term “Central Dogma” that corrupted Crick’s beautifully crafted original concept into the watered down textbook misinterpretation that is most often repeated: “DNA makes RNA make protein” (See Larry Moran’s blog for more on this).  Some may say this is the great thing about language, the same word can mean different things to different people. This view is best characterized in the immortal words of Humpty-Dumpty in Lewis Carroll’s Through the Looking Glass:

Others, including myself, disagree and prefer to have fixed definitions for scientific terms.

In a second recent case of the Neutral Sequence Fallacy creeping into discussions in the context of ENCODE, Michael Eisen proposes that we develop a “A neutral theory of molecular function” to interpret the meaning of these reproducible biochemical events that have no known function. Inspired by the introduction of a new null hypothesis in evolutionary biology ushered in by the Neutral Theory, Eisen calls for a new “neutral null hypothesis” that requires the molecular functions to be proven, not assumed. I laud any attempt to promote the use of null models for hypothesis testing in molecular biology, and whole-heartedly agree with Eisen’s main message about the need for a null model for molecular function.

But I disagree with Eisen’s proposal for a “neutral null hypothesis”, which from my reading of his piece, directly couples the null hypothesis for function with the null hypothesis for sequence evolution. By synonymizing the Ho of the functional model with the Ho of the evolutionary model, regions of the genome that would fail to reject the null functional model (i.e. have no functional constraint) will then be conflated with “being Neutral” (incorrect) or evolving neutrally (potentially correct), whereas those regions that reject the null functional model will be immediately considered as evolving non-neutrally (which may not always be the case since functional regions can evolve neutrally). While I assume this is not what is intended by Eisen, this is almost inevitably the outcome of suggesting a “neutral null hypothesis” in the context of biomolecular sequences. A “neutral null hypothesis for molecular function” makes it all to easy to merge the concepts of functional constraint and selective neutrality, which will inevitably lead many to the Neutral Sequence Fallacy. As Kimura does, Eisen should formally decouple the concept of functional constraint on a sequence from the mode of evolution by which that sequence evolves. Eisen should instead be promoting a “A null model of molecular function” that cleanly separates the concepts of function and evolution (an example of such a null model is embodied in Sean Eddy’s Random Genome Project). If not, I fear this conflation of concepts, like Birney’s semantic switch, will lead to more examples of the Neutral Sequence Fallacy of both Kinds.

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The Neutral Sequence Fallacy shares many sociological similarities with the chronic misuse and misconceptions about the concept of Homology. As discussed by Marabotti and Facchiano in their article “When it comes to homology, bad habits die hard“, there was a peak of misuse of the term Homology in the mid-1980s, which lead to backlash of many publications demanding more rigorous use of the term Homology. Despite this backlash and the best efforts of many scientists to stem the tide of misuse of Homology, ~43% of abstracts surveyed in 2007 use Homology incorrectly, down from 51% in 1986 before the assault on its misuse began. As anyone teaching the concept knows, unpicking misconceptions about Homology vs. Similarity is crucial for getting students to understand evolutionary theory. I argue that the same is true for the distinction between Functional Constraint and Selective Neutrality. When it comes to Functional Constraints on biomolecular sequences, our choice of terminology should be anything but Neutral.

References:

Chin CS, Chuang JH, & Li H (2005). Genome-wide regulatory complexity in yeast promoters: separation of functionally conserved and neutral sequence. Genome research, 15 (2), 205-13 PMID: 15653830

Elnitski L, Hardison RC, Li J, Yang S, Kolbe D, Eswara P, O’Connor MJ, Schwartz S, Miller W, & Chiaromonte F (2003). Distinguishing regulatory DNA from neutral sites. Genome research, 13 (1), 64-72 PMID: 12529307

Lunter G, Ponting CP, & Hein J (2006). Genome-wide identification of human functional DNA using a neutral indel model. PLoS computational biology, 2 (1) PMID: 16410828

Marabotti A, & Facchiano A (2009). When it comes to homology, bad habits die hard. Trends in biochemical sciences, 34 (3), 98-9 PMID: 19181528

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Credits:

Thanks to Chip Aquadro for originally pointing out to me when I perpetrated the Neutral Sequence Fallacy (of the 1st Kind!) during a journal club as an undergraduate in his lab. I can distinctly recall hot, embarrassment of the moment while being schooled in this important issue by a master. Thanks also to Alan Moses, who was the first of many people I converted to the light on this issue, and who has encouraged me since to write this up for a wider audience. Thanks also to Douda Bensasson for putting up with me ranting about this issue for years, and helpful comments on this post.

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