Editor’s note: This is Part 3 of a 6-part series on ENCODE that Casey Luskin has been publishing this year in Salvo Magazine. Parts 1, 2, 3, 4, 5, and 6 have already been published. The prelude can be found here.

The ENCODE project presented strong, experimentally derived evidence that the vast majority of our genome has important biochemical functions, but the specific functions of most of our six billion nucleotides have yet to be determined. Perhaps a hundred years from now molecular biologists will have these puzzles largely solved. But our present ignorance leaves enough ambiguity for some evolutionary biologists to hide out in the hope that the bulk of our genome may still turn out to be junk.

Writing in Salvo 32, I showed that ENCODE critics are wrong in their main counterargument that research hasn’t detected function throughout the genome. Continuing that discussion, we’ll now examine three additional arguments from those who claim our genomes are largely junk DNA — and see why their circular logic doesn’t hold up.

The C-Value Paradox
Opponents of ENCODE often cite the “C-value paradox” as evidence that most DNA is junk.¹ According to this argument, the amount of DNA in an organism’s nucleus (its “C-value”) does not necessarily correlate with its overall complexity. For example, onion cells contain five times as much DNA as human cells, and humans have eight times more DNA than pufferfish. “Surely an onion doesn’t need some 40 times more DNA than a fish,” cries the ENCODE critic. “Much of that DNA must be junk!”

Appealing to the “onion test,” junk defenders argue that if most onion DNA is useless, the same must be true for human DNA. As one proponent puts it:

The onion test is a simple reality check for anyone who thinks they have come up with a universal function for non-coding DNA. Whatever your proposed function, ask yourself this question: Can I explain why an onion needs about five times more non-coding DNA for this function than a human?²

However, a number of observations and arguments mitigate the import of the C-value paradox and the “onion test.”

First, we find a positive correlation between genome size and cell volume (and the size of a cell’s nucleus), hinting at structural reasons for all that DNA.³ Onions can have very large cells, and by this hypothesis, it’s unsurprising that their genomes are also gigantic. Other correlations — like a negative correlation between genome size and metabolic rates in various vertebrates — also hint at functional reasons for the C-value.⁴

A second response asks, Who are we to dictate how much DNA an organism needs? We are far from fully understanding the simplest bacterial genomes — much less our own. Evolutionists use this excuse to claim that most of our genome must be junk. But we might retort: “Why can’t it be possible that onions are using most of their DNA?” Indeed, some single-celled protozoans — supposedly “simple” organisms — use massive amounts of non-coding DNA during their reproductive processes.⁵

Finally large-genomed organisms like the onion seem to have undergone genome duplications. This phenomenon, called polyploidy, shows that genomes can occasionally multiply in size, especially in plants.⁶ Perhaps the onion genome was originally designed small, but ballooned through such natural duplications. That this may have happened in a few plant species that doesn’t therefore mean that most DNA in most species is junk.

Some might find it surprising, and thereby persuasive, to learn that the onion has a huge genome, but the “onion test” is touted mostly for rhetorical effect. Logically speaking, it does not demonstrate that giant genomes are mostly junk, nor does it say anything about whether our own genome is junk-laden. The C-value paradox is a weak rhetorical argument that ignores empirically derived evidence showing mass genomic function.

Very Little DNA Is “Conserved”
After raising the C-value paradox, ENCODE critics often follow with a logical argument. “Only about 10 percent of our DNA is ‘conserved,’ or has a similar sequence, compared to the genomes of other mammals,” they point out. “This means that only about 10 percent of our genome is under selection to preserve the DNA sequence.” They then reason: “Since natural selection is the only force that creates and preserves functional elements in our genome, it’s impossible that more than about 10 percent of our genome is functional.”

This argument was on display in a 2014 paper claiming that only 8.2 percent of human DNA is functional because only that percentage of our genome is “conserved” between humans and other mammals like mice and pandas.⁷ But there’s a glaring problem with this thinking: it assumes that all DNA sequences are the result of undirected mutation and selection to begin with, and that biological function only comes from natural selection. Throw out the assumption of an evolutionary origin of species and there’s no reason to believe that only conserved DNA can be functional. After all, an intelligent agent could independently design functional genetic elements with widely divergent DNA sequences in the genomes of different species–no “conservation” required.

Only if we assume that strictly unguided evolutionary mechanisms produced our genome can we infer that such a small fraction of our genome is functional. Under this logic, when evolutionists cite the preponderance of junk DNA as evidence for evolution, they engage in circular reasoning.

Junk proponents seem blind to these flaws. A co-author of the 8.2-percent paper boasted, “our approach is largely free from assumptions or hypotheses.”⁸ Apparently he was forgetting about assumptions and hypotheses like evolution.

Even worse, ENCODE critic Dan Graur called it “‘idiotic’ to suggest that a part of the genome could be functional if it didn’t respond to pressure from natural selection.”⁹ He further charges that “what ENCODE researchers did not take into account . . . is that everything is shaped by evolution.”¹⁰ In Graur’s evolutionary world, the possibility that some important functional genetic element arose from a cause other than natural selection is simply inconceivable.

There’s another reason why the “sequence conservation” argument is unpersuasive. Whether or not one takes an evolutionary viewpoint, it’s apparent that many differences between species must be encoded somewhere. If species have unique physical or biochemical traits, they shouldalso have unique DNA sequences that encode those traits. As one paper correctly observes, non-conserved DNA “suggests taxon-related functions.”¹¹

In any case, ENCODE provides a nice empirical test of the evolutionary assumption that only conserved DNA can be functional: It finds evidence of mass functionality in “non-conserved” (i.e., unique) DNA sequences. As one lead ENCODE researcher explains: “Most elements defined by biochemical signatures lacked strong evolutionary conservation.”¹² Other ENCODE defenders argue that the research shows that “absence of conservation cannot be interpreted as evidence for the lack of function.”¹³

Mutational Load
The average person has between 70 and 150 mutations compared to his parents.¹⁴ Natural selection effectively “weeds out” extremely harmful mutations, but it doesn’t work efficiently enough to prevent slightly deleterious mutations from accumulating in the population. How can the human population tolerate so many mutations — such a high “mutational load” — without facing a disastrous crash?

Standard evolutionary thinking answers this question by inferring that we can tolerate all those mutations because our genome is mostly junk. If the vast majority of our genome isn’t doing anything, then most mutations will land in inconsequential locations and have a neutral (i.e., neither good nor bad) effect. Thus, humans can tolerate a high “mutational load” without facing major problems. One Scientific American article critiquing ENCODE makes this argument:

The third reason for accepting the reality of junk DNA is to simply think about mutational load. Our genomes, as of other organisms, have undergone lots of mutations during evolution. What would be the consequences if 90% of our genome were really functional and had undergone mutations? How would we have survived and flourished with such a high mutation rate? On the other hand, it’s much simpler to understand our survival if we assume that most mutations that happen in our genome happen in junk DNA.¹⁵

But this argument fails to recognize that not all functionally important DNA operates in the same way. Specifically, proponents of the “mutational load” argument assume that non-coding DNA responds to mutations similarly to protein-coding DNA.¹⁶ But different types of functional genetic elements may tolerate mutations in different ways. As two ENCODE-defending scientists point out, “protein-coding . . . sequences may have structure-function constraints and therefore mutational patterns different from those”¹⁷ in much non-coding DNA. They further observe:

Like words, [non-coding] regulatory sequences have more relaxed structure-function constraints than protein-coding sequences, which encode analog devices with strict chemical requirements. Indeed this is well supported by comparative analysis of gene promoters, which nobody disputes are functional, but where . . . function can be retained . . . in the absence of any recognizable primary sequence conservation.¹⁸

More importantly, like the other junk-DNA arguments we’ve examined here, this one doesn’t address ENCODE’s direct experimental results showing function for non-coding genomic regions. Rather, it infers junk based upon evolutionary considerations.

Like the “conservation” objection, the “mutational load” argument assumes that random mutation and natural selection are the only forces that shaped our genome. If our species was originally designed — but isn’t necessarily designed to live forever — then our genome could be largely functional and still experience a high “mutation load.”

Breaking the Circle
A few months after ENCODE’s results were published in 2012, junk-DNA advocate Sean Eddy published a paper promoting these objections:

ENCODE’s publicized interpretation would require that such nonconserved regulatory sequences account for 80-95% of the genome, far outnumbering evolutionarily conserved regulatory sequences. Given the C-value paradox, mutational load, and the massive impact of transposons, the data remain consistent with the view that the nonconserved 80-95% of the human genome is mostly composed of nonfunctional decaying transposons: “junk.” ¹⁹

Aside from the fact that ENCODE provides strong empirical evidence that most of our DNA is functional (see Salvo 32), we might pose the following questions to Eddy:

• What if the C-value has functional importance, and the “paradox” only pertains to the few species whose genomes have ballooned compared to their aboriginal design, but doesn’t mean their DNA isn’t functional, and doesn’t say anything about whether other genomes (including ours) are full of junk?
• What if species were intentionally designed with important and diverse functional genetic elements, but weren’t intended to live forever, such that seeking “conserved” sequences or studying the “mutational load” won’t reveal how much of the genome is functional?
• What if “transposons” — i.e., repetitive DNA — are actually important functional control elements in the genome (again, see Salvo 32), which we’ve mistakenly identified as selfish, junk DNA?

These arguments for junk only work if one assumes an evolutionary viewpoint. If one must assume an evolutionary view to conclude that the genome is full of junk, one cannot argue that junk demonstrates an evolutionary view.

Two biochemists from down under, John Mattick and Marcel Dinger, agree that the case for junk DNA is based upon circular logic. They argue:

[T]he conclusion of lack of conservation of most of the human genome is largely based on a circular comparison with the rate of evolution of [repetitive DNA] . . . which are assumed to be largely non-functional and therefore evolving “neutrally.” . . . If the first assumption is incorrect, and increasing evidence suggests that it may be, the derived conclusion of nonfunctionality of the rest of the genome is also incorrect.²⁰

They conclude that ENCODE’s empirical evidence for functionality is the ultimate test: “differential expression (including extensive alternative splicing) of RNAs is a far more accurate guide to the functional content of the human genome than logically circular assessments of sequence conservation.”²¹ Bottom line: good evidence trumps bad theory.

A Great Divorce
Critics like Dan Graur charge that ENCODE is guilty of “divorcing genomic analysis from its evolutionary context”²² — and that’s exactly right. ENCODE’s empirically based finding that the vast majority of our genome is functional has withstood theoretical, evolution-based objections from critics. Maybe a divorce from evolutionary thinking is exactly what we need to liberate biology from bad evolutionary assumptions and explain what’s happening inside our cells.

References:
[1.] Alexander Palazzo and T. Ryan Gregory, “The Case for Junk DNA,” PLOS Genetics, 10:e1004351 (May 2014); W. Ford Doolittle, “Is junk DNA bunk? A critique of ENCODE,” Proceedings of the National Academy of Sciences, 110:5294-5300 (April 2, 2013); Dan Graur et al., “On the Immortality of Television Sets: ‘Function’ in the Human Genome According to the Evolution-Free Gospel of ENCODE,” Genome Biology and Evolution, 5:578-590 (2013); Sean Eddy, “The C-value paradox, junk DNA and ENCODE,” Current Biology, 22:R898-R899 (Nov. 6, 2012).
[2.] T. Ryan Gregory, “The onion test,” Evolver Zone Genomicron (April 25, 2007): genomicron.evolverzone.com/2007/04/onion-test. See also Nick Matzke, “ENCODE hype? From now on I’ll just reply: #oniontest,” Pandas Thumb (Sept. 5, 2012): pandasthumb.org/archives/2012/09/encode-hype-fro.html.
[3.] Thomas Cavalier-Smith, “Economy, Speed and Size Matter: Evolutionary Forces Driving Nuclear Genome Miniaturization and Expansion,” Annals of Botany, 95:147-175 (2005).
[4.] See Jonathan Wells, The Myth of Junk DNA (Discovery Institute Press, 2011), 85.
[5.] See Chen et al., “The Architecture of a Scrambled Genome Reveals Massive Levels of Genomic Rearrangement During Development,” Cell, 158:1187-1198 (Aug. 28, 2014).
[6.] John Mattick and Marcel Dinger, “The extent of functionality in the human genome,” The HUGO Journal, 7:2 (2013).
[7.] Rands et al., “8.2% of the Human Genome Is Constrained: Variation in Rates of Turnover Across Functional Element Classes in the Human Lineage,” PLoS Genetics, 10:e1004525 (July 2014).
[8.] “DNA mostly ‘junk’? Only 8.2 percent of human DNA is ‘functional,’ study finds,” ScienceDaily (July 24, 2014): sciencedaily.com/releases/2014/07/140724141608.htm.
[9.] Chris Woolston, “Furore over genome function,” Nature, 512:9 (Aug. 7, 2014).
[10.] Yudhit Bhattercharjee, “The Vigilante,” Science, 343:1306-1309 (March 21, 2014).
[11.] Richard Sternberg, “On the Roles of Repetitive DNA Elements in the Context of a Unified Genomic-Epigenetic System,” Annals of the New York Academy of Sciences, 981:154-188 (2002).
[12.] John Stamatoyannopoulos, “What does our genome encode?” Genome Research, 22:1602-1611 (2012).
[13.] Kellis et al., “Defining functional DNA elements in the human genome,” Proceedings of the National Academy of Sciences, 111:6131-6138 (April 29, 2014).
[14.] Palazzo and Gregory, ibid., note 1.
[15.] Ashutosh Jogalekar, “Three reasons why junk DNA makes evolutionary sense,” Scientific American (Sept. 13, 2012): blogs.scientificamerican.com/the-curious-wavefunction/2012/09/13/three-reasons-to-like-junk-dna.
[16.] Laurence Moran, “Genetic Load, Neutral Theory, and Junk DNA,” Sandwalk (Nov. 7, 2009): sandwalk.blogspot.com/2009/11/genetic-load-neutral-theory-and-junk.html.
[17.] Mattick and Dinger, ibid., note 6.
[18.] Ibid. (emphasis added).
[19.] Eddy, ibid., note 1.
[20.] Mattick and Dinger, ibid., note 6.
[21.] Ibid.
[22.] Graur et al., ibid., note 1.

Image: Ouroboros, by anonymous medieval illuminator; uploader Carlos Adanero [Public domain], via Wikimedia Commons.