September 5, 2008 | David F. Coppedge

How Not to Prove Positive Selection

Erase all that evidence for positive natural selection in the genes you’ve read about.  It’s all misleading confusion based on “certain poorly conceived statistical methods,” argues Austin L. Hughes, an evolutionary biologist at the University of South Carolina.  Writing a commentary in PNAS,1 he accused, “Thousands of papers are published every year claiming evidence of adaptive evolution on the basis of computational analyses alone, with no evidence whatsoever regarding the phenotypic effects of allegedly adaptive mutations.
    Why would Hughes make such a damning statement among colleagues of the National Academy of Sciences, who are overwhelmingly pro-evolution?  The reason: he wanted to praise a new study that does it right.  In the same issue of PNAS,2 a study by Yokoyama is “solidly grounded in biology,” Hughes said.  Instead of presenting “more of the same” evidence of positive selection without tying it to adaptation at the phenotypical level, this paper related changes in visual pigment genes to actual benefits for the organism.
    More on that later.  What’s interesting about evolutionary claims in perspective is that Hughes presented this paper as if it were a rarity, a first step in the right direction after decades of error.  “Sequences of DNA provide documentary evidence of the evolutionary past undreamed of by pioneers such as Darwin and Wallace, but their potential as sources of evolutionary information is still far from being realized,” he began.  “A major hindrance to progress has been confusion regarding the role of positive (Darwinian) selection, i.e., natural selection favoring adaptive mutations.”  That’s when he criticized the methods in “thousands of papers” that rely on “certain poorly conceived statistical methods” that fail to show how the genetic changes relate to adaptive benefits to the organism in its ecological niche.
    Hughes described how the typical paper uses an “unwarranted generalization” from one classic case in which relative frequencies of synonymous and nonsynonymous mutations3 seemed to be related to selectional pressure.  Since then, evolutionists have recklessly applied instances of dN > dS as evidence of positive selection.  This assumption is “demonstrably false,” Hughes argued, because due to the stochastic nature of mutations, such inequalities are just as likely to occur by chance, without any adaptive value.  “Yet, despite their shaky foundations, numerous publications have used these methods as the basis for claims of positive selection at the molecular level.”  In fact, using the Yokoyama et al paper to test the codon-based methods and Bayesian methods so often used in the literature, Hughes found them to be “100% off-target.”  The mutations putatively showing positive selection, in other words, bore no relation to the ones Yokoyama et al found to be adaptive.  “These results support the theoretical prediction that, because of the faulty logic in their underlying assumptions, codon-based focus mainly on statistical artifacts rather than true cases of positive selection.”  Has he just falsified “thousands of papers … published each year”?
    Hughes is not done with his bombshell barrage yet.  Next, he criticized Neo-Darwinism itself – at least some widely-held assumptions about its record in the genes:

Contrary to a widespread impression, natural selection does not leave any unambiguous “signature” on the genome, certainly not one that is still detectable after tens or hundreds of millions of years.  To biologists schooled in Neo-Darwinian thought processes, it is virtually axiomatic that any adaptive change must have been fixed as a result of natural selection.  But it is important to remember that reality can be more complicated than simplistic textbook scenarios.

Adaptive change can occur by simple genetic drift, for instance.  Hughes suggests that some of the genomic changes for visual pigments occurred by this method.  But then, how is an evolutionary biologist to find genetic evidence for positive selection at all?  Hughes is merciless in his conclusion:

In recent years the literature of evolutionary biology has been glutted with extravagant claims of positive selection on the basis of computational analyses alone, including both codon-based methods and other questionable methods such as the McDonald-Kreitman test.  This vast outpouring of pseudo-Darwinian hype has been genuinely harmful to the credibility of evolutionary biology as a science.  It is to be hoped that the work of Yokoyama et al. will help put an end to these distressing tendencies.  By incorporating experimental evidence regarding the phenotypic effects of reconstructed evolutionary changes, this study sets a new standard for studies of adaptive evolution at the molecular level.  In addition, by providing evidence that non-Darwinian and Darwinian processes are likely to be involved in the evolution of adaptive phenotypes, it points the way toward a new, more realistic appreciation of the evolutionary process.

Since Hughes put such a high value on the paper by Yokoyama et al,2 treating it as if it were the guiding light among thousands of papers lacking credibility, it bears taking a closer look.  The authors started immediately with assumptions based on evolution that they admitted are difficult to prove:

Vertebrate ancestors appeared in a uniform, shallow water environment, but modern species flourish in highly variable niches.  A striking array of phenotypes exhibited by contemporary animals is assumed to have evolved by accumulating a series of selectively advantageous mutations.  However, the experimental test of such adaptive events at the molecular level is remarkably difficult.

The authors referred to the evolution of visual pigments as “the deepest body of knowledge linking differences in specific genes to differences in ecology and to the evolution of species.”  This makes their subject matter the best case available for testing evolution with molecular methods.  They extracted rhodopsins from 5 deep-sea fish and compared them to 35 types of animals.  As Hughes had indicated, they showed that the standard codon-based, statistical inferences to positive selection are misleading.  This was a major emphasis in their paper.  In fact, four of their five major conclusions related to how traditional methods of assessing positive selection can be misleading.
    Then, using mutagenesis experiments, they purported to show that adaptive sensitivity to particular wavelengths of light in specific environments “evolved on at least 18 separate occasions.  These highly environment-specific adaptations seem to have occurred largely by amino acid replacements at 12 sites, and most of those at the remaining 191 (~94%) sites have undergone neutral evolution.”  In other words, evidence for genetic drift (neutral changes) swamped evidence for positive selection by 94%.  But even then, they started by assuming that the “ancestral” rhodopsin, which they “engineered” using evolutionary assumptions and mutagenesis, started with a maximal sensitivity to 500 nm light.
    Clearly, Cambrian-age ancestral rhodopsin is not available for study.  The ancestral rhodopsin on which their conclusions depend, therefore, was manufactured by them in the lab, based on their assumptions of evolutionary ancestry, millions of years, and the positions of animals in a phylogenetic tree, assuming the rhodopsins had diversified by natural selection.  The reasoning seems circular.  Even so, genetic drift was far more evident than positive selection.  And, to fit the data, they had to conclude that genotypes appeared and reappeared multiple times without any particular trend.  They said, “To complicate the matter further, evolutionary changes are not always unidirectional and ancestral phenotypes may reappear during evolution.
    Since no clear evolutionary pattern became evident without evolutionary assumptions,4 therefore, it is difficult to see how this paper could be judged any more objective than the thousands of papers Hughes criticized.


1.  Austin L. Hughes, “The origin of adaptive phenotypes,” (Commentary, Proceedings of the National Academy of Sciences, published September 3, 2008, doi:10.1073/pnas.0807440105.
2.  Yokoyama, Tada, Zhang and Britt, “Elucidation of phenotypic adaptations: Molecular analyses of dim-light vision proteins in vertebrates,” Proceedings of the National Academy of Sciences, published September 3, 2008, doi:10.1073/pnas.0802426105.
3.  Nonsynonymous mutations in a gene change the amino acid in the resulting protein.  Synonymous mutations do not, because some some of the 64 possible DNA codons have “synonyms” that code for the same amino acid (there are only 20 amino acids in most proteins).
4.  E.g, notice the evolutionary assumptions in this excerpt from the paper: “The ancestors of bony fish most likely used rhodopsins with [lambdamax-s (maximum sensitivity wavelength)] of ~500 nm (Fig. 1).  What types of light environment did these ancestors have?  The origin of many early vertebrate ancestors is controversial [i.e., the Cambrian explosion], but that of bony fish ancestors is clear [referring to a 1988 text on Vertebrate Paleontology and Evolution].  The fossil records from late Cambrian and early Ordovician, ~500 Mya, show that the ancestors of bony fish lived in shallow, near-shore marine environments (30�32).  Therefore, pigment a must have functioned as a surface rhodopsin and its lambda-max would be consistent with that roleInterpolating from the ancestral and contemporary rhodopsins, it is most likely that pigments b�d and f�h (lambdamax ~ 501�502 nm) were also surface rhodopsins, pigment i (496 nm) was an intermediate rhodopsin, and pigments e, j, and k (480�485 nm) were deep-sea rhodopsins (Fig. 1).  From their predicted lambdamax-s, it is also likely that pigments q, r, s, and v were intermediate rhodospins [sic] and pigment u was a deep-sea rhodopsin (Fig. 1)…. Based on the four types of dim-light vision, vertebrates show six different evolutionary paths (Fig. 1)….”  Later, they gave a Lamarckian description: “When moving into new dim-light environments, vertebrate ancestors adjusted their dim-light vision by modifying their rhodopsins.”

Wow.  The damage to evolutionist credibility from these two papers can hardly be overstated.  Hughes just wiped away stacks and stacks of papers that Ken Miller and Eugenie Scott might have piled up in a courtroom to demonstrate the overwhelming evidence for evolution, then he held up a very weak paper as the best example yet.  We looked into that paper and found nothing but evolutionary assumptions buttressing evolutionary assumptions.
    Consider how weak their best evidence is.  They were talking about animals that already had eyes, retinas, optic nerves, brains and all the other organs and functions that support vision.  The only parameter that they studied was the wavelengths of light to which particular rhodopsin molecules are maximally sensitive, between 482 and 505 billionths of a meter.  But as we know from many phenomena in biology, compensating mechanisms are often at work.  It would be impossible to prove that a fish with a rhodopsin most sensitive to 489 nm would be any better evolved than one with a rhodopsin most sensitive to 502 nm, because the ganglion cells or optic nerve might compensate for the slight shift in sensitivity.  All we observe is that living fishes today are marvelously adapted to their particular ecological niches.
    We’re only talking about virtually indistinguishable shades of green light, folks!  Are you impressed with the creative power of natural selection?  Are you impressed with scientists’ ability to demonstrate evolution at the molecular level?  During the hundreds of millions of years in which animals supposedly evolved from trilobites to philosophers, the best evolutionists can show are slight changes to sensitivity to green light in just 12 positions in one protein molecule out of the thousands of exquisitely-adapted enzymes essential for vision.  Even then, the “evolution” demonstrated is predominantly from mutational drift, with no particular functional trend, and the changes (we are told) appeared, disappeared, and reappeared 18 times.  At the most optimistic, the changes they’re talking about are microevolutionary.  Even staunch young-earth creationists would have no difficulty believing that changes this small might occur in a few thousand years.
    Nothing the evolutionists have produced as evidence for natural selection (after the purge of papers by Hughes) is sufficient to distinguish between creation vs evolution models.  Should evolution be the only view sanctified in the schools?  Hughes was right on when he said that the “vast outpouring of pseudo-Darwinian hype has been genuinely harmful to the credibility of evolutionary biology as a science.”  He did nothing to repair the damage, and by pointing to an insipid paper as the best example yet after decades of hype, he actually made it worse.
    This effectively undermines everything the evolutionists have told us about divining evolutionary history in the genes (e.g., 06/13/2003, 04/30/2005).  Where else could we see it?  In the fossil record?  Ha! (07/21/2003), 05/21/2004, 05/10/2008).
    Undoubtedly the Darwin Party will spin this situation in their favor, by making it an illustration of the progress of triumphalist materialist secularist science.  For 8 years now, CEH has been exposing the charades behind the curtain where Charles the Extravagant, the Wizard of Flaws, and his loyal munchkins dupe people into thinking that evidence for evolution is overwhelming.  This is a prime example.  Now you know.  Get the word out!  Expose the charlatan!  We need to get out of this mythical fantasyland (09/04/2008) and back to the real world!  The Kansas School Board is counting on you!

You’re off to shame the Wizard, the Blunderful Wizard of Flaws
You’ll find he is a Wimp of a Wiz if ever a Wiz there was
If ever, oh ever, a Wimp there was the Wizard of Flaws is one because
Because, because, because, because, because
Because of the blunderful spins he does
You’re off to shame the wizard, the Blunderful Wizard of Flaws.

Use your brain.  Take courage.  Have a heart.  And bark, Toto, bark!

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Categories: Genetics

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