Alternative splicing: function vs noise

This post is about a recent review of alternative splicing published by my colleague Ben Blencowe in the Dept. of Medical Genetics at the University of Toronto (Toronto, Ontario, Canada). (The other author is Jermej Ule of The Francis Crick Institute in London (UK).) They are strong supporters of the idea that alternative splicing is a common feature of most human genes.

I am a strong supporter of the idea that most splice variants are due to splicing errors and only a few percent of human genes undergo true alternative spicing.

This is a disagreement about the definition of "function." Is the mere existence of multiple splice variants evidence that they are biologically relevant (functional) or should we demand evidence of function—such as conservation—before accepting such a claim?

The Function Wars Part VII: Function monism vs function pluralism

This post is mostly about a recent paper published in Studies in History and Philosophy of Biol & Biomed Sci where two philosophers present their view of the function wars. They argue that the best definition of function is a weak etiological account (monism) and pluralistic accounts that include causal role (CR) definitions are mostly invalid. Weak etiological monism is the idea that sequence conservation is the best indication of function but that doesn't necessarily imply that the trait arose by natural selection (adaptation); it could have arisen by neutral processes such as constructive neutral evolution.

The paper makes several dubious claims about ENCODE that I want to discuss but first we need a little background.

Background

The ENCODE publicity campaign created a lot of controversy in 2012 because ENCODE researchers claimed that 80% of the human genome is functional. That claim conflicted with all the evidence that had accumulated up to that point in time. Based on their definition of function, the leading ENCODE researchers announced the death of junk DNA and this position was adopted by leading science writers and leading journals such as Nature and Science.

Let's be very clear about one thing. This was a SCIENTIFIC conflict over how to interpret data and evidence. The ENCODE researchers simply ignored a ton of evidence demonstrating that most of our genome is junk. Instead, they focused on the well-known facts that much of the genome is transcribed and that the genome is full of transcription factor binding sites. Neither of these facts were new and both of them had simple explanations: (1) most of the transcripts are spurious transcripts that have nothing to do with function, and (2) random non-functional transcription factor binding sites are expected from our knowledge of DNA binding proteins. The ENCODE researchers ignored these explanations and attributed function to all transcripts and all transcription factor binding sites. That's why they announced that 80% of the genome is functional.

Happy Darwin Day! 2020

Charles Darwin, the greatest scientist who ever lived, was born on this day in 1809 [Darwin still spurs tributes, debates] [Happy Darwin Day!] [Darwin Day 2017]. Darwin is mostly famous for two things: (1) he described and documented the evidence for evolution and common descent and (2) he provided a plausible scientific explanation of evolution—the theory of natural selection. He put all this in a book, The Origin of Species by Means of Natural Selection published in 1859—a book that spurred a revolution in our understanding of the natural world. (You can still buy a first edition copy of the book but it will cost you several hundred thousand dollars.)

The Function Wars Part VI: The problem with selected effect function

The term "Function Wars" refers to the debate over the meaning of 'function,' especially in the context of junk DNA.¹ That debate intensified in 2012 after the ENCODE publicity campaign that tried to redefine function to mean anything they want as long as it refutes junk DNA. This is the sixth in a series of posts exploring the debate and why it's important, or not. Links to the other five posts can be found at the bottom or this post.

The world is not inhabited exclusively by fools and when a subject arouses intense interest and debate, as this one has, something other than semantics is usually at stake.
Stephen Jay Gould (1982)Much of the discussion seems like quibbling over semantics but I'm reminded of a similar debate over the mode of evolution: is it gradual or punctuated? As Gould pointed out in 1982, there's a serious issue underlying the debate—an issue that shouldn't get lost in bickering over the meaning of 'gradualistic.' The same warning applies here. It's important to determine how much of the human genome is junk and that requires an understanding of what we mean by junk DNA. However, it's easy to get distracted by focusing on the exact meaning of the word 'function' instead of looking at the big picture.

lncRNA nonsense from Los Alamos

A group of scientists at the Los Alamos National Laboratory (Los Alamos, NM, USA) and their collaborators in Vienna (Austria) and Lethbridge (Alberta, Canada) have worked out the structure of Braveheart lncRNA from mice.

Kim, D.N., Thiel, B.C., Mrozowich, T., Hennelly, S.P., Hofacker, I.L., Patel, T.R., Sanbonmatsu, K.Y. (2020) Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution. Nat. Commun. 11:148 [doi: 10.1038/s41467-019-13942-4]

The authors point out in their paper that lncRNAs are difficult to work with and the 3D structures of only a small number have been characterized. There's nothing in the paper about the problems associated with determining the functions of lncRNAs and nothing about the number of lncRNAs except for this brief opening statement: "Long non-coding RNAs (lncRNAs) constitute a significant fraction of the transcriptome ..."

The Three Domain Hypothesis: RIP

The Three Domain Hypothesis died about twenty years ago but most people didn't notice.

The original idea was promoted by Carl Woese and his colleagues in the early 1980s. It was based on the discovery of archaebacteria as a distinct clade that was different from other bacteria (eubacteria). It also became clear that some eukaryotic genes (e.g. ribosomal RNA) were more closely related to archaebacterial genes and the original data indicated that eukaryotes formed another distinct group separate from either the archaebacteria or eubacteria. This gave rise to the Three Domain Hypothesis where each of the groups, bacteria (Eubacteria), archaebacteria (Archaea), and eukaryotes (Eucarya, Eukaryota), formed a separate clade that contained multiple kingdoms. These clades were called Domains.

Are pseudogenes really pseudogenes?

There are many junk DNA skeptics who claim that most of our genome is functional. Some of them have even questioned whether pseudogenes are mostly junk. The latest challenge comes from a recent review in Nature Reviews: Genetics where the authors try to place the burden of proof on those who say that pseudogenes are broken, nonfunctional, genes (Cheetam et al., 2019). The authors of the review try to make the case that we should not label a DNA sequence as a pseudogene until we can prove that it is truly nonfunctional junk.

I'm about to refute this ridiculous stance but first we need a little background.

Remember MOOCs?

We learned back in 2012 that Massive Open Online Courses (MOOCs) were going to transform higher education. People all over the world, especially in underdeveloped nations, would be able to learn from the best university professors while sitting at home in front of their computers. Several companies entered the market with high expectations of earning enormous profits while altruistically educating students who couldn't afford to go to university.

Are introns mostly junk?

There are many reasons for thinking that introns are mostly junk DNA.

1. The size and sequence of introns in related species are not conserved and almost all of the sequences are evolving at the rate expected for neutral substitutions and fixation by drift.
2. Many species have lost introns or reduced their lengths drastically suggesting that the presence of large introns can be detrimental in some cases (probably large populations).
3. After decades of searching, there are very few cases where introns and/or parts of introns have been shown to be essential.
4. Researchers routinely construct intronless versions of eukaryotic genes and they function normally when re-inserted into the genome.
5. Intron sequences are often littered with transposon and viral sequences that have inserted into the intron and this is not consistent with the idea that intron sequences are important.
6. About 98% of the introns in modern yeast (Saccharomyces cerevisiae) have been eliminated during evolution form a common ancestor that probably had about 18,000 introns [Yeast loses its introns]. This suggests that there was no selective pressure to retain those introns over the past 100 million years.
7. About 245/295 of the remaining introns in yeast have been artificially removed by researchers who are constructing an artificial yeast genome suggesting that over 80% of the introns that survived evolutionary loss are also junk [Yeast loses its introns].

The evolution of citrate synthase

Citrate synthase [EC 2.3.3.1] is one of the key enzymes of the citric acid cycle. It catalyzes the joining of acetyl-CoA and oxaloacetate to produce citrate.

acetyl-CoA + H₂O + oxaloacetate → citrate + HS-CoA + H⁺

We usually think of this reaction in terms of energy production since acetyl-CoA is the end product of glycolysis and the citric acid cycle produces substrates that enter the electron transport system leading to production of ATP. However, it's important to keep in mind that the enzyme also catalyzes the reverse reaction.

The "standard" view of junk DNA is completely wrong

I was browsing the table of contents of the latest issue of Cell and I came across this ....

For decades, the miniscule protein-coding portion of the genome was the primary focus of medical research. The sequencing of the human genome showed that only ∼2% of our genes ultimately code for proteins, and many in the scientific community believed that the remaining 98% was simply non-functional “junk” (Mattick and Makunin, 2006; Slack, 2006). However, the ENCODE project revealed that the non-protein coding portion of the genome is copied into thousands of RNA molecules (Djebali et al., 2012; Gerstein et al., 2012) that not only regulate fundamental biological processes such as growth, development, and organ function, but also appear to play a critical role in the whole spectrum of human disease, notably cancer (for recent reviews, see Adams et al., 2017; Deveson et al., 2017; Rupaimoole and Slack, 2017).

Slack, F.J. and Chinnaiyan, A.M. (2019) The Role of Non-coding RNAs in Oncology. Cell 179:1033-1055 [doi: 10.1016/j.cell.2019.10.017]

Cell is a high-impact, refereed journal so we can safely assume that this paper was reviewed by reputable scientists. This means that the view expressed in the paragraph above did not raise any alarm bells when the paper was reviewed. The authors clearly believe that what they are saying is true and so do many other reputable scientists. This seems to be the "standard" view of junk DNA among scientists who do not understand the facts or the debate surrounding junk DNA and pervasive transcription.

Here are some of the obvious errors in the statement.

1. The sequencing of the human genome did NOT show that only ~2% of our genome consisted of coding region. That fact was known almost 50 years ago and the human genome sequence merely confirmed it.
2. No knowledgeable scientist ever thought that the remaining 98% of the genome was junk—not in 1970 and not in any of the past fifty years.
3. The ENCODE project revealed that much of our genome is transcribed at some time or another but it is almost certainly true that the vast majority of these low-abundance, non-conserved, transcripts are junk RNA produced by accidental transcription.
4. The existence of noncoding RNAs such as ribosomal RNA and tRNA was known in the 1960s, long before ENCODE. The existence of snoRNAs, snRNAs, regulatory RNAs, and various catalytic RNAS were known in the 1980s, long before ENCODE. Other RNAs such as miRNAs, piRNAS, and siRNAs were well known in the 1990s, long before ENCODE.

How did this false view of our genome become so widespread? It's partially because of the now highly discredited ENCODE publicity campaign orchestrated by Nature and Science but that doesn't explain everything. The truth is out there in peer-reviewed scientific publications but scientists aren't reading those papers. They don't even realize that their standard view has been seriously challenged. Why?

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The evolution of de novo genes

De novo genes are new genes that arise spontaneously from junk DNA [De novo gene birth]. The frequency of de novo gene creation is important for an understanding of evolution. If it's a frequent event, then species with a large amount of junk DNA might have a selective advantage over species with less junk DNA, especially in a changing environment.

Last week I read a short Nature article on de novo genes [Levy, 2019] and I think the subject deserves more attention. Most new genes in a species appear to arise by gene duplication and subsequent divergence but de novo genes are genes that are unrelated to genes in any other clade so we can assume that they are created from junk DNA that accidentally becomes associated with a promoter causing the DNA to be transcribed. A new gene is formed if the RNA acquires a function. If the transcript contains an open reading frame then it may be translated to produce a polypeptide and if the polypeptide performs a new function then the resulting de novo gene is a new protein-coding gene.

The important question is whether the evolution of de novo genes is a common event or a rare event.

How many protein-coding genes in the human genome? (2)

It's difficult to know how many protein-coding genes there are in the human genome because there are several different ways of counting and the counts depend on what criteria are used to identify a gene. Last year I commented on a review by Abascal et al. (2018) that concluded there were somewhere between 19,000 and 20,000 protein-coding genes. Those authors discussed the problems with annotation and pointed out that the major databases don't agree on the number of gene [How many protein-coding genes in the human genome?].

Gerald Fink promotes a new definition of a gene

This is the 2019 Killian lecture at MIT, delivered in April 2019 by Gerald Fink. Fink is an eminent scientist who has done excellent work on the molecular biology of yeast. He was director of the prestigious Whitehead Institute at MIT from 1990-2001. With those credentials you would expect to watch a well-informed presentation of the latest discoveries in molecular genetics. Wouldn't you?

Contingency, selection, and the long-term evolution experiment

I'm a big fan of Richard Lenski's long-term evolution experiment (LTEE) and of Zachary Blount's work in particular. [Strolling around slopes and valleys in the adaptive landscape] [On the unpredictability of evolution and potentiation in Lenski's long-term evolution experiment] [Lenski's long-term evolution experiment: the evolution of bacteria that can use citrate as a carbon source]

The results of the LTEE raise some interesting questions about evolution. The Lenski experiment began with 12 (almost) identical cultures and these have now "evolved" for 31 years and more than 65,000 generations. All of the cultures have diverged to some extent and one of them (and only one) has developed the ability to use citrate as a carbon source. Many of the cultures exhibit identical, or very similar, mutations that have reached significant frequencies, or even fixation, in the cultures.

Several other laboratory evolution experiments have been completed or are underway in various labs around the world. The overall results are relevant to a discussion about the role of contingency and accident in the history of life [see Evolution by Accident]. Is it true that if you replay the tape of life the results will be quite different? [Replaying life's tape].

Evolution by Accident

Evolution by Accident
v1.43 ©2006 Laurence A. Moran
This essay has been transferred here from an old server that has been decommissioned.Modern concepts of evolutionary change are frequently attacked by those who find the notions of randomness, chance, and accident to be highly distasteful. Some of these critics are intelligent design creationists and their objections have been refuted elsewhere. In this essay I'm more concerned about my fellow evolutionists who go to great lengths to eliminate chance and accident from all discussions about the fundamental causes of evolution. This is my attempt to convince them that evolution is not as predictable as they claim. I was originally stimulated to put my ideas down on paper when I read essays by John Wilkins [Evolution and Chance] and Loren Haarsma [Chance from a Theistic Perspective] on the TalkOrigins Archive.

The privilege of living beings is the possession of a structure and of a mechanism which ensures two things: (i) reproduction true to type of the structure itself, and (ii) reproduction equally true to type, of any accident that occurs in the structure. Once you have that, you have evolution, because you have conservation of accidents. Accidents can then be recombined and offered to natural selection to find out if they are of any meaning or not.
Jacques Monod (1974) p.394The main conclusion of this essay is that a large part of ongoing evolution is determined by stochastic events that might as well be called "chance" or "random." Furthermore, a good deal of the past history of life on Earth was the product of chance events, or accidents, that could not have been predicted. When I say "evolution by accident" I'm referring to all these events. This phrase is intended solely to distinguish "accidental" evolution from that which is determined by non-random natural selection. I will argue that evolution is fundamentally a random process, although this should not be interpreted to mean that all of evolution is entirely due to chance or accident. The end result of evolution by accident is modern species that do not look designed.

First complete sequence of a human chromosome

A paper announcing the first complete sequence of a human chromosome has recently been posted on the bioRxiv server.

Miga, K. H., Koren, S., Rhie, A., Vollger, M. R., Gershman, A., Bzikadze, A., Brooks, S., Howe, E., Porubsky, D., Logsdon, G. A., et al. (2019) Telomere-to-telomere assembly of a complete human X chromosome. bioRxiv, 735928. doi: [doi: 10.1101/735928]

Abstract: After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome, we reconstructed the ∼2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.

How much of the human genome has been sequenced?

It's been more than seven years since I posted information on how much of the human genome has been sequenced [How Much of Our Genome Is Sequenced?]. At that time, the latest version of the human reference genome was GRCh37.p7 (Feb. 3, 2012) and 89.6% of the genome had been sequenced. It's time to update that information.

We have a pretty good idea of the size of the human genome based on quantitative Feulgen staining (1940-1980) and reassociation kinetic experiments from the 1970s (Morton, 1991). We can safely assume that the correct size of the human genome is close to 3,200,000,000 bp (3,200,000 kb, 3,200 Mb, 3.2 Gb) [How Big Is the Human Genome?]. That's the value cited most often in the literature. However, the actual values calculated by Morton (1991) were 3.227 Gb for the haploid female genome and less than that for the haploid male genome. The human reference genome contains all 22 autosomes plus one copy of the X chromosome and one copy of the Y chromosome. This gives a total of 3.286 Gb.

Reactionary fringe meets mutation-biased adaptation. 7. Going forward

This the last of a series of posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution. Arlin has established that the role of mutation in evolution is much more important than most people realize. He has also built a strong case for the influence of mutation bias. How should we incorporate these concepts into modern evolutionary theory?

Click on the links in the box (below) to see the other posts in the series.

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Reactionary fringe meets mutation-biased adaptation.
7. Going forward
by Arlin Stoltzfus
Haldane (1922) argued that, because mutation is a weak pressure easily overcome by selection, the potential for biases in variation to influence evolution depends on neutral evolution or high mutation rates. This theory, like the Modern Synthesis of 1959, depends on the assumption that evolution begins with pre-existing variation. By contrast, when evolution depends on the introduction of new variants, mutational and developmental biases in variation may impose biases on evolution, without requiring neutral evolution or high mutation rates.

Reactionary fringe meets mutation-biased adaptation. 5.5 Synthesis apologetics

This is part of a continuing series of posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution. In this post, Arlin explains how defenders of the Modern Synthesis react in the face of serious challenges to the theory that was formulated in the 1940s and 50s. Rather than reject the theory, they engage in various forms of "synthesis apologetics."

Click on the links in the box (below) to see the other posts in the series.

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Reactionary fringe meets mutation-biased adaptation. 5.6 Synthesis apologetics
by Arlin Stoltzfus

Reactionary fringe meets mutation-biased adaptation. 5.4. Taking neo-Darwinism seriously

This is part of a continuing series of posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution. In this post Arlin discusses his view of neo-Darwinism and why it is inconsistent with macromutations and lateral gene transfer. He equates neo-Darwinism with the Modern Synthesis (1959 version), a comparison that might be challenged. Click on the links in the box (below) to see the other posts in the series.

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Reactionary fringe meets mutation-biased adaptation. 5.4. Taking neo-Darwinism seriously
by Arlin Stoltzfus
The Modern Synthesis is often described as the result of combining Darwinism and genetics. This description, in my opinion, is concise and historically accurate: the Modern Synthesis of 1959 is a sophisticated attempt to arrange the pieces of population genetics to justify a neo-Darwinian dichotomy in which variation merely supplies raw materials, and selection is the source of initiative, creativity and direction.

Religion vs science (junk DNA): a blast from the past

I was checking out the science books in our local bookstore the other day and I came across Evolution 2.0 by Perry Marshall. It was published in 2015 but I don't recall seeing it before.

The author is an engineer (The Salem Conjecture) who's a big fan of Intelligent Design. The book is an attempt to prove that evolution is a fraud.

I checked to see if junk DNA was mentioned and came across the following passages on pages 273-275. It's interesting to read them in light of what's happened in the past four years. I think that the view represented in this book is still the standard view in the ID community in spite of the fact that it is factually incorrect and scientifically indefensible.

Reactionary fringe meets mutation-biased adaptation. 6. What "limits" adaptation?

This is part of a continuing series of posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution. In this post Arlin discusses the role of adaptation and what determines the pathway that it will take over time. Is it true that populations will always adapt quickly to any change in the environment? (Hint: no it isn't!) Click on the links in the box (below) to see the other posts in the series.

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Reactionary fringe meets mutation-biased adaptation.
6. What "limits" adaptation?
by Arlin Stoltzfus
According to the hatchet piece at TREE, theoretical considerations dictate that biases in variation are unlikely to influence adaptation, because this requires small population sizes and reciprocal sign epistasis.

Yet, we have established that mutation-biased adaptation is real (see The empirical case and Some objections addressed). If theoretical population genetics tells us that mutation-biased adaptation is impossible or unlikely, what is wrong with theoretical population genetics?

Adaptation, before Equilibrium Day

Reactionary fringe meets mutation-biased adaptation. 5.3. How history is distorted.

This is the ninth in a series of guest posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution. Click on the links in the box (below) to see the other post in the series.

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Reactionary fringe meets mutation-biased adaptation.
5.3. How history is distorted.
by Arlin Stoltzus
In his famous Materials for the Study of Variation, Bateson (1894) refers to natural selection as "obviously" a "true cause" (p. 5). Punnett (1905) explains that mutations are heritable while environmental fluctuations are not, concluding that "Evolution takes place through the action of selection on these mutations" (p. 53). De Vries begins his major 1905 English treatise by writing that ...

"Darwin discovered the great principle which rules the evolution of organisms. It is the principle of natural selection. It is the sifting out of all organisms of minor worth through the struggle for life. It is only a sieve, and not a force of nature" (p. 6)

Morgan (1916), in his closing summary, writes:

"Evolution has taken place by the incorporation into the race of those mutations that are beneficial to the life and reproduction of the organism" (p. 194)

Reactionary fringe meets mutation-biased adaptation. 5.2. The Modern Synthesis of 1959

This is the eighth in a series of guest posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution.

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Reactionary fringe meets mutation-biased adaptation. 5.2. The Modern Synthesis of 1959
by Arlin Stoltfus
As we learned in What makes it new?, the newness of the effect of biases in the introduction process results from a classical assumption that evolution can be understood as a process of shifting the frequencies of existing alleles. How did this position emerge? Was it a technical, mathematical issue?

Reactionary fringe meets mutation-biased adaptation. 5.1. Thinking about theories

This is the seventh in a series of guest posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution.

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Reactionary fringe meets mutation-biased adaptation. 5.1. Thinking about theories
by Arlin Stoltzfus
A wikipedia page disambiguating "Modern Synthesis" defines neo-Darwinism as

"the state-of-the-art in evolutionary biology, as seen at any chosen time in history from the 1890s to the present day."

Because "neo-Darwinism" and the "Synthesis" are conflated with whatever is widely accepted, they are now regularly attacked on grounds that are completely unrelated to genuine neo-Darwinism or the original Modern Synthesis, e.g., as when a network of life (rather than a tree) is invoked as a contradiction of Darwinism. The attack by Noble (2015) on the

"... conceptual framework of neo-Darwinism, including the concepts of "gene," "selfish," "code," "program," "blueprint," "book of life," "replicator" and ˜"vehicle."

is entirely a critique of late-20th-century reductionism à la Dawkins, and addresses neither neo-Darwinism (selection and variation as the potter and the clay), nor the original Modern Synthesis, which is simply not reductionistic, but positively invokes emergent phenomena (population-level forces, the gene pool as dynamic buffer) in the service of selection as a high-level governing principle.

"The state of the art" is a phrase that needs no modification. Nothing good can come from linking it to the name of a dead person.

Reactionary fringe meets mutation-biased adaptation. 5. Beyond the "Synthesis" debate

This is the sixth in a series of guest posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution.

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Reactionary fringe meets mutation-biased adaptation. 5. Beyond the "Synthesis" debate
by Arlin Stoltzfus
The authors of TREE's hatchet piece imply that the theory of Yampolsky and Stoltzfus (2001) is somehow not new, citing ancient work from Dobzhansky and Haldane. In Box 1, they argue that this theory is part of "standard evolutionary theory," showing a 4-step derivation ending in Eqn IV, which is Eqn 2 of Yampolsky and Stoltzfus (2001), and informing the reader that this is based on "classical" results from Fisher, Haldane and Kimura, who are named, while Yampolsky and Stoltzfus are not named.

Yet, Fisher, Haldane, and Kimura did not make the argument in Box 1, did not follow the 4 steps, and did not derive Eqn IV!

Reactionary fringe meets mutation-biased adaptation. 4. What makes this new?

This is the fifth in a series of guest posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution.

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Reactionary fringe meets mutation-biased adaptation. 4. What makes this new?
by Arlin Stoltzfus
Scientists value novelty because it signifies untapped potential: a new idea has not been interrogated, applied, and extended. The more novel an idea, the greater its potential to re-shape our discourse and advance our understanding beyond the well tried ideas of the past.

Reactionary fringe meets mutation-biased adaptation. 3. The causes and consequences of biases in the introduction process

This is the fourth in a series of guest posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution.

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Reactionary fringe meets mutation-biased adaptation. 3. The causes and consequences of biases in the introduction process
by Arlin Stoltzfus
As discussed previously, mutation-biased adaptation occurs in the laboratory and in nature. In the cases that have been examined, modest several-fold mutational biases have modest several-fold effects on the changes involved in adaptation.

Reactionary fringe meets mutation-biased adaptation
Introduction
1. The empirical case
2. Some objections addressed
3. The causes and consequences of biases in the introduction process
4. What makes this new?
5. Beyond the "Synthesis" debate
    -Thinking about theories
    -Modern Synthesis of 1959
    -How history is distorted
    -Taking neo-Darwinism
      seriously
    -Synthesis apologetics
6. What "limits" adaptation?
7. Going forwardHow can this happen? Classical thinking says that mutation is a weak pressure easily overcome by selection. This "opposing pressures" argument was invoked by Fisher (1930), Haldane (1933) and Wright (1931), as well as Huxley, Ford, Stebbins, Simpson and others. On this basis, it is assumed that the effects of mutation bias will be seen only in neutral evolution, where the opposing pressure of selection is absent, or with unusually high mutation rates.

Reactionary fringe meets mutation-biased adaptation. 2. Some objections addressed.

This is the third in a series of guest posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution.

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Reactionary fringe meets mutation-biased adaptation. 2. Some objections addressed.
by Arlin Stoltzfus
In the previous post Part 1, we reviewed evidence from 8 analyses suggesting that modest several-fold biases in mutation may impose modest several-fold biases on the spectrum of changes involved in adaptation, including some legendary cases of natural adaptation.

Reactionary fringe meets mutation-biased adaptation
Introduction
1. The empirical case
2. Some objections addressed
3. The causes and consequences of biases in the introduction process
4. What makes this new?
5. Beyond the "Synthesis" debate
    -Thinking about theories
    -Modern Synthesis of 1959
    -How history is distorted
    -Taking neo-Darwinism
      seriously
    -Synthesis apologetics
6. What "limits" adaptation?
7. Going forwardIs the evidence strong enough already to conclude in favor of a bold new idea? The authors of the hatchet piece at TREE believe that nothing has been shown, arguing that the proposed effect is theoretically unlikely and is probably due to selection.

The focus of this post is on alternative hypotheses (theoretical arguments will be addressed later). For the sake of brevity, I will address just 2 of the many spurious objections offered by these authors in their quest to exemplify the Dunning-Kruger effect. For instance, they write "we stress that parallel genetic change underlying phenotypic convergence is not sufficient evidence for mutation bias being important in causing such convergence."

This is an inversion of the argument, common in the parallelism literature (see Bailey, et al. 2015), that the recurrence of exactly the same change is by itself evidence of selection.

In fact, the case for mutation-biased adaptation does not depend on such weak inferences. In the 8 analyses we reviewed, no change is designated as adaptive solely based on a pattern of recurrence. Instead, each mutational path has either (1) a genetic association with fitness or resistance, or (2) an experimentally verified molecular effect consistent with the adaptive story. Once adaptive changes have been identified, statistical tests are applied to detect an excess of changes of the mutationally favored class.

As another example, TREE's hatchet piece refers to selection as an independent force of adaptation, then attacks the strawman theory of mutation bias as an independent force of adaptation. To ensure that the reader is deceived about mutation-biased adaptation, and ill disposed toward this line of research, this strawman is repeated 5 times on the first page (figure).

Both arguments illustrate how reactionary minds fail to grasp new ideas, and see only perversions or inversions of cherished old ideas.

Now, let us set aside strawman arguments, to focus on genuine alternatives.

For instance, the authors suggest that transitions could be favored "owing to selection on genomic base composition," citing work on GC content. This hypothesis can not work. If the effect of selection is to conserve GC content, this can not explain a bias toward transitions, because the universe of GC-conserving mutations has a transition:transversion ratio of 0. Likewise, if the effect of selection is to change GC content, this can not explain the observed degree of bias, because the universe of GC-changing amino acid replacement mutations has roughly a 1:1 transition:transversion ratio, not large enough to explain results of Payne, et al. (2019) or Stoltzfus and McCandlish (2017).

A more plausible alternative raised by the authors, following Stoltzfus and Norris (2016), is that the observed evolutionary bias could be caused by a bias in protein-level fitness effects that happens to align with the mutation bias, e.g., they suggest that "selectively beneficial transitions and selectively beneficial transversions could also have different distributions of fitness effects."

Let us consider, for the 8 analyses addressed previously, the hypothesis that observed evolutionary biases are not due to mutation bias at all, but to a cryptic fitness bias that happens to align with the mutation bias.

First, in the studies by MacLean, et al. (2010), Sackman, et al. (2017) and Liu, et al. (2019), the authors measure fitness (or resistance). The data from MacLean, et al. (2010) reveal no correlation of mutation rate with fitness (figure).

In their model of effects in drug-resistant tumors, Liu, et al. (2019) find that the mutational factor (estimated mutation rate) explains more variance than the fitness-related factor (measured drug resistance). Results of one-step adaptation from Sackman, et al. (2017) are shown in the figure (left: transitions are in light gray, transversions are in dark gray; upper scale is selection coefficient, lower scale is number of evolved lineages out of 20). Here the mean selection coefficients for transitions and transversions are 0.37 (CI 0.053) and 0.40 (CI 0.18), respectively, i.e., transversions are insignificantly better (data from their Table 1).

Next, consider the experimental study by Couce, et al. (2015) shown in the figure below (courtesy of Alex Couce). Among resistant mutants in PBP3, the resistant mutT isolates (blue) overwhelmingly have the kind of mutations favored by mutT (left box), and the resistant mutH isolates (red) overwhelmingly have the kind of mutations favored by mutH (center box; other types of mutations are in the right box, which includes most of the black isolates indicating a wild-type parent).

The only way to explain this as a fitness effect would be to argue that (1) the mutT and mutH genotypes have widespread, strong, and utterly distinct epistatic effects on the fitness landscape for PBP3, i.e., each mut genotype induces a distinct set of beneficial alleles, and (2) the corresponding mutations for those alleles just happen to be (overwhelmingly) the same type of mutation favored by the mutator.  This is wildly implausible because it implies that the blue-red segregation of columns in the figure above is accidental.

What about the meta-analyses of transition-transversion bias? Could there be a fitness advantage of transitions that explains this effect?

Stoltzfus and Norris (2016) analyzed data on 544 transitions and 695 transversions with experimentally measured fitness effects. Comparing various binary predictors, they considered the chance that a nominally conservative mutation is more fit than a nominally radical one, aka the AUC, which ranges from 0 to 1, with a null expectation of 0.5. Transition-transversion class is a weak predictor (AUC = 0.53, figure), out-performed by most biochemical factors, all 200 of which are out-performed by a conservative-radical distinction based on Tang's U (AUC = 0.64), an empirical measure of relative fixation probability computed from a large set of sequence alignments. Yet, the conservative-radical distinction from Tang's U corresponds to a mere 2.7-fold fixation bias in evolution. Using this relationship, Stoltzfus and Norris (2016) estimate that the transition:transversion distinction corresponds to a 1.3-fold fixation bias, with a confidence interval from 1.0 (no effect) to 1.6.

But these results use the entire distribution of mutations, including the worst ones that (in nature) would be removed by selection. Therefore, Stoltzfus and Norris (2016) truncated the data to see if a stronger benefit would emerge among benign mutations. Instead of getting stronger, the effect disappeared (their Fig. 1).

Next, Stoltzfus and Norris (2016) set aside the above data, and looked at an independent set of data from 4 studies of laboratory adaptation implicating 111 beneficial mutations with measured fitness effects. In the table below, the AUC value in the penultimate column is the chance that a transition is ranked higher than a randomly chosen transversion: the values are all < 0.5. That is, beneficial transitions rank slightly lower than beneficial transversions. The later study by Sackman, et al. (2017) (above) represents a 5th independent case in which beneficial transitions rank slightly lower than beneficial transversions.

Thus, available data, reflecting multiple lines of evidence, indicate that transitions simply do not have a fitness advantage that could explain a several-fold effect on amino acid changes in evolution.

Finally, note that Payne, et al. (2019) report evolutionary biases that cannot be explained by protein-level selection, including transition bias in non-coding changes, and the excess of Met-to-Ile transitions over Met-to-Ile transversions (which are twice as likely without mutation bias).

To summarize, in our evaluation of the cryptic-fitness-difference hypothesis, we find that: in 3 cases, the fitness effects were measured, with results that do not support the hypothesis; in 3 cases (counting 2 meta-analyses in Stoltzfus and McCandlish, 2017), the evidence indicates that the mutationally favored class (transitions) does not have a sufficient fitness advantage; in 1 case, the hypothesis is wildly implausible (Couce, et al., 2015); and in 1 remaining case, Storz, et al. (2019) invoke a mutational effect without any clear justification for assuming an absence of differential fitness effects.

Concluding thoughts

In recent years, systematic data have begun to accumulate on molecular changes implicated in phenotypic adaptation. The pattern emerging from these data is that the molecular changes implicated in adaptation are enriched for the kinds of changes that are favored by mutation, and this enrichment cannot be explained by a cryptic fitness bias that happens to align with the mutation bias.

We could treat this merely as a pattern, as a new and useful empirical generalization.

But there is much more to the story. Mutation-biased adaptation was predicted under a theory that contrasts sharply with classical thinking, which holds that internal tendencies of variation cannot cause evolutionary trends or biases, because mutation rates are too small: in order for mutation biases to be important, mutation rates must be very large, or the opposing pressure of selection must be absent, i.e., effects of biases in ordinary mutations will be limited to neutral evolution.

Yampolsky and Stoltzfus (2001) argued that this view, which derives from the mutation-selection balance model of Fisher and Haldane, assumes that evolution can be treated as a short-term process of shifting the frequencies of pre-existing alleles, without considering the (potentially biased) introduction of new alleles. Using a simple model, they showed that the efficacy of biases in introduction does not require absolute constraints, neutral evolution, or high mutation rates. They argued that this conclusion applies to developmental biases as well as mutation biases.

Thus, it is time to understand this theory, what it implies, and why it differs from classical thinking-- the topic of the next post in the series.

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Payne J.L., Menardo F., Trauner A., Borrell S., Gygli S.M., Loiseau C., et al. (2019). Transition bias influences the evolution of antibiotic resistance in Mycobacterium tuberculosis. PLoS Biol 17(5): e3000265. [doi: 10.1371/journal.pbio.3000265]

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Storz J.F., Natarajan C., Signore A.V., Witt C.C., McCandlish D.M. and Stoltzfus A. (2019) The role of mutation bias in adaptive molecular evolution: insights from convergent changes in protein function. Phil. Trans. R. Soc. B [doi: 10.1098/rstb.2018.0238]