( 1 ) Barry Hall’s lac Bug
( 2 ) Lenski’s Long Term E. Coli Evolution Experiment
Lenski’s E. coli Evolve Ability to Metabolize Citrate under Aerobic Conditions
( 3 ) Bacteriophage Lambda Evolves a New Protein Binding Site Using Four Mutations
( 4 ) The Significance of These Complex Mutations
Plate Tectonics: An Example of Evidences
Diverse Evidence for Evolution
( 5 ) The Core Issue in Rejecting Evolution
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( 1 ) Barry Hall’s lac Bug
Lactose is a sugar that bacteria can use as a food. In order to do this, they first have to cut lactose in half, releasing two simple sugars (glucose and galactose) that the metabolism of the cell can use for energy. In E. coli, the ability to metabolize lactose depends on having (1) a gene that produces the protein (the enzyme beta-galactosidase) that cuts the lactose, (2) an appropriate regulatory region which turns on this gene when lactose is present but turns it off (to conserve metabolic energy) when lactose is absent, and (3) another protein, called a permease, which imports lactose through the otherwise-impermeable cell membrane. These three functions are normally co-located on a stretch of DNA called the lac operon.
In 1982, Professor Barry Hall at the University of Rochester initiated a set of experiments using some E. coli in which he deleted the gene for the beta-galactosidase from this lac operon. The loss of this gene knocked out the operation of the three-part system, which made it impossible for the bacteria to metabolize the sugar lactose. He then let the bacteria reproduce in a nutrient mixture which contained galactose. The bacteria would only thrive if they managed to evolve a new metabolic pathway to digest galactose. The bacteria needed to evolve both (1) a gene which would produce the enzyme beta-galactosidase, and also (2) the appropriate controlling region of DNA. The growth medium contained the chemical IPTG, which promoted the cellular production of permease from the original lac genetic region, since that function had been disabled by Hall’s gene knock-out. Having the permease present in this way allowed the experimenters to detect any new mutants which had evolved the ability to cleave galactose.
As it happened, in the course of many generations, the bacterium did exactly that. Two mutations in a distant gene converted it into an effective gene for making beta-galactosidase enzyme, and further mutation in the DNA which controls the expression of this new gene gave it the needed galactose sensitivity. Neither part was greatly effective without the other. So, this is an example of a new multi-part genetic system, with a function which was new to this hacked bacterial strain, which was produced by mutation coupled with natural selection.
In further series of mutation and natural selection experiments, Hall found a new set of bacteria which had a new enzyme which was able to switch on the production of the lac permease, in addition to having the new regulated beta-galactosidase gene.
Intelligent Design proponent Michael Behe has attempted to minimize the significance of these observations. His main objection seems to be that this evolution of a new function took place by various alterations to existing genetic material. But that is exactly how evolution normally proceeds! Evolutionary biologists do not claim that fully functional, whole new genes suddenly appear in the genome ex nihilo. That seems to be what Behe is demanding. Rather, evolution is typically a matter of step by step modifications in the DNA which is already there.
Ken Miller rebuts all of Behe’s objections, and concludes:
Does Barry Hall’s ebg system fit the definition of irreducible complexity? Absolutely. The three parts of the evolved system are:
(1) A lactose-sensitive ebg repressor protein that controls expression of the galactosidase enzyme
(2) The ebg galactosidase enzyme
(3) The enzyme reaction that induces the lac permease
Unless all three are in place, the system does not function, which is, of course, the key element of an irreducibly complex system.
( 2 ) Lenski’s Long Term E. Coli Evolution Experiment
Richard Lenski’s group at Michigan State University has been running a long-term evolution experiment on asexual E. coli bacteria since 1988. Six populations (Ara-1 through Ara-6) were started from a one strain, and six further populations (Ara+1 through Ara+6) from a nearly identical strain. Each population is kept in a flask at 37 C with 10 ml of a growth medium which contains only enough glucose to support about 5 x 10+8 cells per culture. Each day, 0.1 ml of the previous day’s culture is transferred into 9.9 ml of fresh growth medium, and the cells reproduce up to the limit of the nutrients. The result is about 6.64 generations per day, or 2400 generations per year. Samples of each population are cryo-preserved every 500 generations. These preserved cells can be revived and further studied as needed. Fitness is periodically assayed by measuring growth rates versus an ancestral strain. A photo of the twelve population flasks is shown below.
Over the first 10,000 generations, each of the twelve populations demonstrated significant increases in fitness. The figure below shows the increases in average cell size as each population accumulates mutations to make them better adapted to their environment. Since then, fitness has continued to improve, but at ever-slower rates.
Clearly, these bacteria are accumulating multiple, step-by-step beneficial mutations, which improve their fitness in their environment. This is evolution in action.
The plots below show the numbers of mutations retained in each population, through about 50,000 generations. Six of the populations have evolved hypermutator strains, where point mutations appear much faster than usual. These populations show in Plot (a) as having 800-2500 mutations after 50,000 generations. Plot (b) is rescaled to show more clearly the trajectories in the six non-hypermutable populations, which have accumulated 60-110 mutations apiece. Across all the cells and all generations in a given flask (population), over a billion mutations have occurred; it is likely that every possible point mutation in the bacterial genome has been sampled many times over. However, most of these mutations did not spread and become fixed in the population as a whole. See STAN3–From Micro-Evolution to Macro-Evolution: Beneficial Mutations, the Pace of Evolution, and Increasing Genome Complexity for more details on numbers and patterns of the mutations in this experiment.
It is worth noting that “beneficial” or “fitness” is always defined relative to a particular environment, typically the environment currently occupied by the organism. A strain of E. coli in “the wild” needs to cope with a variety of temperatures, acidities, availability of different nutrients, etc. A typical microbe therefore has versatile metabolic mechanisms to respond to variations in conditions. However, the bacteria in the Lenski experiment experience the same conditions, day after day, year after year, for thousands of generations. This particular environment is characterized by a limited amount of nutrient, primarily glucose. Thus, Lenski’s bugs have evolved to be greedy specialists in consuming glucose at a constant temperature of 37 C. To maintain other functions (e.g. the ability to metabolize the sugar ribose) in this situation is a waste of metabolic energy, and so it has been advantageous for these bacteria to accumulate mutations which disable some of these functions.
Lenski’s E. coli Evolve Ability to Metabolize Citrate under Aerobic Conditions
One of the most interesting features of this experiment is that after about 33,000 generations, one of the populations (“Ara-3”) evolved the ability to make use of the large amount of citrate which happened to be present in the growth medium. The photo below shows a close-up view of experiment flasks, where the middle (Ara-3) population is clearly more turbid (cloudy) than the others, due to the enormous bacterial cell mass in that flask.
The population designated Ara-3 (center) is more turbid because that population evolved to use the citrate present in the growth medium. Figure from Wikipedia article “E. coli long-term evolution experiment” ; original source: Brian Baer and Neerja Hajela – http://myxo.css.msu.edu/ecoli/citrateflasksphotos.html
Normally E. coli is unable to make use of citrate under the aerobic (presence of oxygen) conditions used in this experiment. The metabolic machinery for utilizing citrate exists in E. coli, and it can ferment citrate under anaerobic conditions in the presence of a reducing substrate, but it cannot transport citrate under oxic conditions.
In an effort to probe how this one population evolved this ability, the researchers resurrected (thawed) cells from previous generations in that population and let them evolve, to see whether these parallel lines would also develop the ability to metabolize citrate. A few of the lines restarted from generation 20,000 evolved the ability to utilize citrate, whereas none of the lines restarted from generation 15,000 could.
Apparently some key but essentially neutral mutation (which has not yet been identified) occurred between generation 15,000 and 20,000. This potentiating mutation (or complex of mutations) had no distinctive effect on citrate metabolizing, but under the conditions of this experiment it enabled some further mutation to produce a weakly effective Cit+ (citrate-metabolizing) variant by generation 31,500. This variant constituted about 0.5% of the cells in the population at generation 31,500, and rose to 15% and 19% at generations 32,000 and 32,500. At generation 33,000, the dominance of the Cit+ variant plummeted to 1.1%, presumably because the Cit- subpopulation produced a beneficial mutation which allowed it to out-compete the emerging Cit+ subpopulation. However, after a few hundred more generations the optical turbidity of the population took a step change to a higher level, indicating some further mutation at that point had increased the viability of the Cit+ subpopulation to a very high level. Thus, this set of mutations gave to the cells an important function (ability to digest citrate in the presence of air) which they did not have before. 
Young earth creationists and Intelligent Design proponents have tried to downplay the import of this evolutionary gain of function. Their usual rhetorical tactic is to characterize this as a minor tweak to the regulatory region of the existing citrate gene, as merely “turning on an existing switch in the genetic machinery” which did not involve evolution of any new information or added functionality. The graduate student chiefly responsible for this research, Zachary Blount, has stated that this description of the changes in the cells is simply “a lie”:
“….No, the ability to grow on citrate is not a matter of simply flipping a pre-existing regulatory switch. Continuing the electrical metaphor, the evolved Cit+ function is instead about rewiring. My dear little Cit+ cells gained their ability to partake of the previously forbidden citrate by a genetic duplication involving a gene, called citT, which encodes a transporter protein that is used during anaerobic growth on citrate.
“This duplication did something very special. You see, one of the major aspects of gene regulation is that genes have associated regulatory DNA sequences, including what are called promoters that control when genes are expressed. The citT gene is normally controlled by a promoter that tells the cell to turn it on only when there is no oxygen present. As shown in the Figure below, the gene duplication put one copy of citT next to, and under the control of, a promoter that normally controls another gene called rnk. The rnk gene is normally turned on when oxygen is present. The new association between citT and the rnk promoter – what we call the rnk-citT regulatory module – turns citT on when oxygen is present, and allows Cit+ cells to use citrate under the conditions of the LTEE. (To really feast on the citrate involved additional evolutionary changes, both before and after this rewiring, but I’ll leave that point aside for this post.)
“…True, the duplication responsible for Cit+ did rearrange components that were already there, but that rearrangement generated a new association between components that did not previously exist, and it produced a new function that also did not previously exist. To argue that rearrangements cannot produce innovation is akin to arguing that a novelist has done nothing creative in writing her novels because she only used words that already existed.” [emphasis in the original]
Here is the figure to which Blount referred, showing schematically the tandem duplication in the population that evolved the new ability to grow on citrate. This duplication produced the new rnk-citT regulatory module by placing the second copy (“Tandem Copy 2”) of the citT gene adjacent to the rnk promoter region.
This tandem duplication is the key mutation which enabled uptake of citrate, but by itself it only gave only weak performance. A further mutation just after generation 33,000 in a different spot in the bacteria’s genome greatly enhanced the cell’s growth in the presence of citrate. This second mutation involves the regulation of the dctA gene, enabling the reuptake of succinate or other C4-dicarboxylates that are exported in exchange for citrate import by the CitT system, thus completing a cycle that permits sustained citrate transport and utilization without an unbalanced loss of intracellular C4-dicarboxylate substrates.  It is the cells with this second mutation that grow so fast that they show high turbidity.
To summarize, at least three separate mutations were involved here: (1) a potentiating mutation(s) around generation 15,000-20,000 which of itself had no noticeable effect on citrate uptake; (2) a duplication of a stretch of DNA around generation 31,500, which created a new gene function by pairing a copy of the citrate uptake gene with a different promoter; and (3) another mutation around generation 33,000 which balanced out the cell’s C4-dicarboxylates in the presence of citrate uptake. This multistep, complex modification of the genome provided the cell with a new function (the ability to robustly take up citrate under aerobic conditions), with no loss in other functions. So again, this is clearly a “gain-of-function” set of mutations. Opponents of evolution try to downplay the significance of this complex mutational change, but the facts speak for themselves. 
( 3 ) Bacteriophage Lambda Evolves a New Protein Binding Site Using Four Mutations
A bacteriophage is a virus that infects and replicates within bacteria cells. The Lenski lab published a study  in 2012 involving a bacteriophage (“ lambda phage”) which infects E. coli. More details on this experiment, and a specific rebuttal to ID claims, were recently given by Lenski .
The phage normally works by using its protein called “J” to bind to a protein on the surface of the E. coli called “LamB”. To test the ability of the phage to evolve the capability of binding to a different protein, the researchers cultured lambda phage with a population of E. coli which had mutated such that only a few of the bacteria had the LamB protein present. The phage population could subsist at a low level on this tiny minority of the bacteria, but would only thrive if they evolved the ability to infect the rest of the bacterial population which lacked the LamB protein on its surface.
In an initial experiment, such a phage was found to evolve in one out of six flasks. Analysis showed that the phage had developed the capability of binding to a different E. coli protein, OmpF, while retaining its original ability to bind to LamB. Despite the efforts of Intelligent Design advocates to spin it otherwise, this again is a “gain-of-function”, pure and simple.
Lenski’s team then repeated the experiment on a larger scale, using 96 more bacteria/phage communities. In 24 of these 96 communities, the phage developed the capability to thrive by binding to the OmpF protein. This protein has a similar 3-dimensional structure as the original LamB, but differs considerably in amino acid sequence.
The researchers did genome sequencing to identify the mutations responsible for the new function in the phage. Figure 3 of their paper illustrates their findings. A portion of that figure is reproduced below. The labels across the top denote the point mutations at specific nucleotide positions along the phage genome. The top 24 rows show some of mutations observed in the 24 phage strains whose expressed J protein was able to bind to the E. coli OmpF. For comparison, a few rows from the lower half of the figure are shown to illustrate the mutations at these genome positions for some of the phage populations which did not evolve that capability.
Taken from Figure 3 of Meyer, et al., Repeatability and Contingency in the Evolution of a Key Innovation in Phage Lambda. Science 27 Jan 2012: Vol. 335, Issue 6067, pp. 428-432.
Caption of original Figure: “Mutations affecting the J protein in phage isolates from 48 independent populations of the large-scale experiment. Isolates are shown in rows (with alternate labels offset for readability) and mutations in columns; gray fill indicates an isolate has the mutation. The top 24 rows show phage isolates that can target the new OmpF receptor; the bottom 24 rows show phage that remain dependent on LamB.” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306806/
All of the top 24 rows (i.e. the strains able to bind to the different OmpF protein) shared four changes: at least one mutation between positions 2969 and 2999, mutations from A-to-G at nucleotide position 3034, from G-to-A at 3319, and a mutation at either position 3320 or 3321 (affecting the same codon as the mutation at 3319). We have labeled these four changes “A” to “D” as they appear from left to right in the figure above.
It is likely that these four canonical mutations, alone or in various combinations, improve the binding to the original LamB protein target in the E. coli, and that this selectively drove their initial appearance in various strains of the phage. However, all four of these mutational changes together are needed in order for the phage to robustly bind to the different OmpF protein.
In The Edge of Evolution Michael Behe had claimed that the appearance of a new protein binding site which required more than two mutations was, for all practical purposes, impossible – – beyond the “edge of evolution”. As noted by Dennis Venema, the evolution of this new protein binding capability in Lenski’s phage, requiring four distinct mutations, accomplished what Behe said was impossible.
The Significance of These Complex Mutations
Behe published a literature review in 2010  which sought to minimize the significance of these sorts of experiments on bacteria and viruses in flasks. He states that in these experiments, what is observed is the “breaking and blunting” of genetic functionality, rather than an increase in functionality. In response, Paul Braterman  noted that Behe had to engage in numerous rhetorical contortions to press his case, and Jerry Coyne  pointed out that Behe had limited his review to situations which where are the least likely to yield new functional mutations. For instance, bacteria under normal circumstances can undergo substantial beneficial genetic changes by importing sizeable chunks of DNA from other microbes. However, in many of the laboratory experiments discussed by Behe, there was only one species of bacteria in the flask, so there was no possibility of such DNA exchange.
The reality is that in all three cases discussed above, a new (new to the starting organism) functionality arose via multiple mutations, which gave a significant improvement to fitness in the environment currently occupied by the organism. Moreover, in each of these cases the gain of the new function did not come about by “breaking or blunting” other important functions. This is indeed “evolution before our eyes.” 
That said, these changes fall far short of producing a new organelle or a whole new species. Is this all merely “micro-evolution”, with no relevance to the changes involved in, say, the divergence of modern humans from other primates?
Plate Tectonics: An Example of Evidences
An analogy may help in putting these mutation results in perspective. As Ron Miksha pointed out in The Mountain Mystery , prior to the 1960’s geologists had no clear idea as to how most mountain ranges were formed. Today nearly everyone accepts the notion that giant crustal plates move around and crumple into mountain ranges in zones where these plates are forced together. But the notion of “continental drift” on such a gigantic scale seemed so bizarre when it was suggested by Alfred Wagener in 1912 that it was rejected by most scientists : “Without detailed evidence and a force sufficient to drive the movement, the theory was not generally accepted: the Earth might have a solid crust and mantle and a liquid core, but there seemed to be no way that portions of the crust could move around”.
Nowadays we can use GPS measurements to directly measure the movements of the continents. For instance, we find that Africa is moving away from South America at a rate of about an inch (2.5 cm) per year. That is about as fast as your fingernails grow. If that were the only evidence we had of continental drift, perhaps severe skepticism would be warranted. In your entire lifetime, only about 7 feet (~2 meters) of relative motion will occur. Can this slow creeping really account for the tearing apart of whole continents, and the formation of a 3000 mile wide ocean?
Well, of course it can, if this process continues on and on, for many millions of years. However, if plate tectonics is real, there should be some other, corroborating evidence. And there is. The coastlines (or more precisely, the continental shelves) of Africa and South America, and (less obviously) of North American and Europe can be fit together like puzzle pieces. Moreover, distinctive geological formations can be found in matching locations on the coasts on either side of the Atlantic Ocean.
Dramatic confirmation of the spreading of the seafloor came with measurements showing symmetric magnetic striping in the oceanic crust on either side of the mid-Atlantic Ridge. This is clearly explained by basalt magma welling up all along this ridge, taking on the prevailing direction of the earth’s magnetic field as it cools and hardens, and then moving away from the ridge in either direction. The earth’s magnetic field is known to reverse its orientation at irregular intervals during geologic history, typically several hundred thousand years apart. Other evidences supporting plate tectonics include the radioactive dates of the rocks of the seafloor, the geographical patterns of fossils of various ages, and the location of belts of volcanos and earthquakes at the edges of the crustal plates.
Diverse Evidence for Evolution
With evolution, there is an analogous array of diverse evidence. If mutational changes have indeed been going on and on and on for billions of years, with accompanying evolution of today’s biota from much simpler ancestors, there should be some sort of progression visible in the fossil record, and there should be signs of common ancestry in today’s genomes.
If there has been no large-scale evolution, and thus modern plants and animals have always existed, we would expect to find modern plants and animals at all levels of the fossil record, possibly commingled with other, now-extinct creatures. But that is not what we find. What we actually find is a clear progression of initial appearances of various life-forms from the lowest (oldest) rock layers to the younger layers and on to the present, which cannot be explained away as the result of the hydraulics of Noah’s Flood .
In the lowest rock layers we find single-celled bacteria; after many hundreds of million years, simple multi-celled organisms like sponges and jellyfish appear. Millions of years later, in yet higher rock layers, we start to find fossils of soft-bodied animals known as Ediacaran fauna. Then in the rock layers from the Cambrian period (542 to 485 million years before the present), there is a rapid progression from mainly worms and slugs to an array of fauna which represent many of phyla which have persisted to the present .
The actual specimens within these phyla present in the Cambrian fossils, however, are typically quite unlike any modern species – – no modern fish or reptiles or mammals or flowering plants appear in the Cambrian. Trilobites (early arthropods which are distant cousins to today’s insects and crustaceans) always appear in sedimentary layers below any rocks containing dinosaurs, never above. Fossils of modern mammals are always found in layers above those of dinosaurs, never below. Moreover, fossils of modern marine animals like clams can be found in rock layers above fossils of large, active land animals like dinosaurs, so these sequences cannot be attributed to a world-wide Flood first burying marine creatures whilst land dwellers were climbing to higher ground.
In the vertebrate lineage, worm-like swimmers with a primitive notochord (no spine, no jaws yet) first appear in the Cambrian, followed in higher (younger) rock layers by jawed fish (fossils first seen 400-450 million years ago) , then tetrapod amphibians, then the first reptiles around 300 Mya, dinosaurs in yet higher rock layers, and finally modern mammals make their appearance. (The earlier types of animals often persist in the presence of the newer arrivals – – sponges, jellyfish, fish, amphibians, and reptiles persist in the fossil record and into the present, but the specific species of fossilized fish, amphibians and reptiles change over geological time.)
It is known that the fossil record is not complete, and the basic arithmetic of population genetics shows that transitional species will mainly occur in small, isolated populations which are unlikely to leave a fossil trace. Nevertheless, a steady stream of discoveries since Darwin’s time has filled in many of the major transitions in the animal family. For instance, a number of fossils with part fish/part amphibian characteristics, and with part reptile/part mammal characteristics, are found at about the times expected for these transitions. (See Realistic Expectations for Transitional Fossils ). Donald Prothero states , “We now have abundant evidence for how all the major groups of animals are related, much of it in the form of excellent transitional fossils”, and he supplies examples which include dinosaurs and modern mammals.
With the sequencing of genomes in the past twenty years, a whole new realm of evidence supporting evolution has become available. For instance, the patterns of “endogenous retroviruses” (ERVs) in the human and chimpanzee genomes are clear evidence that humans and chimps descended from a common ancestor. There are many other lines of genetic evidence for evolution, which are summarized by Douglas Theobald in “29+ Evidences for Macroevolution” .
The physical changes that appear from species to species to the next species in the fossil record are largely the result of ongoing mutational changes. Opponents of evolution claim that there is a some limit to mutational changes, that would prevent evolutionary changes going beyond some threshold (such as one “kind” developing into a different “kind”), but they have no actual evidence of such a limit . Theobald explains:
If the general observation of geneticists was that of genomic stasis and recalcitrance to significant genetic change, it would be weighty evidence against the probability of macroevolution. For instance, it is possible that whenever we introduce mutations into an organism’s genome, the DNA could back-mutate to its former state. However, the opposite is the case—the genome is incredibly plastic, and genetic change is heritable and essentially irreversible…. Extremely extensive genetic change has been observed, both in the lab and in the wild. We have seen genomes irreversibly and heritably altered by numerous phenomena, including gene flow, random genetic drift, natural selection, and mutation. Observed mutations have occurred by mobile introns, gene duplications, recombination, transpositions, retroviral insertions (horizontal gene transfer), base substitutions, base deletions, base insertions, and chromosomal rearrangements. Chromosomal rearrangements include genome duplication (e.g. polyploidy), unequal crossing over, inversions, translocations, fissions, fusions, chromosome duplications and chromosome deletions.
It is thus reasonable to infer that the same sorts of mutations observable today have been going on and on and on for geological history, and that these accumulated rearrangements of DNA over millions and millions and millions of years have led to large changes in physical features, including new organs, etc.  Indeed, the rates of genetic mutation and physical changes inferred from the fossil record match reasonably well with currently observed mutational rates and physical changes.
However, the evidence from the fossil record indicates that evolutionary changes typically proceed very, very slowly. For instance, it took something like 50 million years to evolve today’s large horses from different and much smaller ancestors. It is estimated that there are seven successive genera in the branchy lineage from Hyracotherium or Eohippus (size of a small fox, teeth for nibbling tips of branches, 3-4 toes per foot) to the present-day Equus horse (one toe per foot, large teeth for grinding up grass) . This gives an average phyletic taxonomic rate of 0.13 genus per million years, or 7.5 million years per genus. Triassic and earlier ammonites evolved at a rate of 0.05 genus per million years. The last common ancestor of hominids and chimpanzees is considered to have lived around 6 million years ago. These transitions did not involve significant whole new organs, yet they still spread over millions of years.
The reptile to mammal transition was a more significant transition, including many changes to the jaws and the reproductive system and elsewhere, although these were both still tetrapods. In the fossil record, this transition is spread over some 100 million years. This all indicates that it is unrealistic for us to expect to see a major change in bodily function evolve in a hundred years or even a hundred thousand years.
We simply don’t have enough information to track each of the thousands of mutations that would be involved in the transformation of one species in the fossil record to another. We have many fossils of hard body parts, but have recovered little DNA from fossils older than a few million year. For instance, we have no clearly sequenceable DNA from any dinosaur fossils. We can tabulate all the differences between the human and the chimp genomes, but we don’t know the genome of our common ancestor some six million years back; and even if we did, it would not be possible to reconstruct the exact sequence of mutations that led from that common ancestor to the two current species.
There are some cases, however, where there is enough information to reconstruct the mutations to a particular gene which gave rise to some significant new function. Long et al.  describe the mechanisms of gene duplication and exon shuffling, and note 22 genes in various species whose history has been reconstructed in some level of detail, using mutational steps of the types which are routinely observed today. For instance, the Antarctic notothenoid fishes have an unusual “antifreeze” glycoprotein (AFGP) which inhibits the formation of ice crystals in their bodies in the sub-freezing (-1.9 C) Antarctic waters. The AFGP is a polymer of a Thr-Ala-Ala glycopeptide monomer. It appears that in an ancestral copy of a trypsinogen protease gene the Thr-Ala-Ala region was expanded through multiple internal duplications. The exons coding for the protease sequences were lost, to yield the present form of the AFGP gene. Thus, a gene with an entirely different function was evolved, by means of mutations which are observable today (duplications, deletions, etc.). See details here.
Young earth creationists commonly complain about what has not yet been discovered – – for instance, we don’t possess fossils of every transitional form, and we do not know all the step by step mutations that produced the genomes of today’s organisms. But if we look at the evidence that HAS been found, instead of complaining or speculating about what has not yet been found, that evidence is overwhelmingly supportive of common descent driven by evolution.
The slow accumulation of mutations and the modest production of new functions that are observable in controlled laboratory experiments over the course of a few years or a few decades is analogous to the slow creep of the continents. In both cases, slow processes operating over many millions of years can produce enormous changes.
For both plate tectonics and macroevolution, there is abundant circumstantial evidence to complement the modest changes that we are able to observe in our day. To dismiss the evidence for evolution because we cannot observe a whole new organ develop within the span of historical human observation is as senseless as rejecting plate tectonics because we cannot personally see a whole continent torn apart to form a new ocean before our eyes.
( 5 ) The Core Issue in Rejecting Evolution
Having spent this much time addressing the physical evidence, I’ll briefly mention a factor which is actually more important for many people. The folks (in North America, at least) who reject evolution come largely from a conservative religious background. They have been told that evolution contradicts the Bible, and that “Darwinism” leads to a debased view of humans. Thus, they are predisposed to believe the proponents of Intelligent Design and of young earth creationism who publish a steady stream of articles that allegedly disprove evolution. (See A Creationist Speaker Comes to Town for my experience with a young earth creationist coming to give a workshop in my area ).
In order to become open to really hearing the evidence in favor of genuine science, these people need to realize that evolution is not a threat to their core values. The sneering and jeering of atheist biologists like Richard Dawkins, Jerry Coyne, and P.Z. Myers towards people of faith is counterproductive in this regard. Rather, it should be made known that many devout Christians, from Billy Graham to the Pope , find that “evolutionary creation” is fully compatible with their faith. Christian geneticist Francis Collins, director of the National Institutes of Health, former head of the Human Genome Project and author of The Language of God, established the Biologos Foundation which works to share this message. This entails applying a non-literal interpretation to some creation passages in the Bible, just as the church eventually let go of a literal interpretation of the verses which speak of a stationary earth.
The core problem is that young earth creationists misrepresent what the Bible is really about. In II Timothy 3:15-17 Paul states the purpose and function of the Scriptures: they make us “wise for salvation through faith which is in Christ Jesus”, and are “profitable for doctrine, for reproof, for correction, for instruction in righteousness”, so that we are “equipped for every good work.” This is all theology and morals. There is nothing here about authoritatively teaching geology or biology.
Likewise, Jesus said that the function of the Old Testament was to testify about him and his saving work (John 5:40; Luke 24:44), and Peter (I Pet 1: 10-12) wrote that prophets spoke of the sufferings and glory of Christ. This is “special revelation” of divine purposes which could not be inferred from the “general revelation” of nature. This is a biblical view of the Bible’s intent, which differs from some evangelical statements about inerrancy which mistakenly over-extend the Bible’s sphere of authority into general science or history.
If there really were clear evidence (e.g. rock layers from a recent worldwide Flood) of miraculous intervention on a geologic scale, or clear evidence of the un-natural origin of the human species, that would constitute a widely-accessible supernatural “sign” for unbelievers. However, Jesus flatly declared that no such sign would be given. Young earth creationism is thus founded on premises which run counter to what Christ himself taught (see Jesus on Seeing God in Nature: No Signs, No Justice, No Fear ). For a more general treatment of Bible passages dealing with creation issues, see Evolution and Faith: My Story, Part 2.
 The story of this flask gets even more interesting after the evolution of the robust Cit+ strain. That strain mushroomed in numbers each generation as it fed on the abundant citrate in the growth medium. In the process of metabolizing the citrate, this strain released the dicarboxylic acids succinate, fumarate, and malate into the medium. A strain of Cit- cells then evolved to make use of those dicarboxylic acids produced by the Cit+ bacteria. These two strains co-existed in the overall flask population. “Thus, the evolution of citrate consumption led to a flask-based ecosystem that went from a single limiting resource, glucose, to one with five resources either shared or partitioned between two coexisting clades.” – – from “Evolution and coexistence in response to a key innovation in a long-term evolution experiment with Escherichia coli”, by Caroline B. Turner, Zachary D. Blount, Daniel H. Mitchell, Richard E. Lenski , http://www.biorxiv.org/content/early/2015/06/17/020958 . (The timeline in the Wikipedia article indicates that this Cit- strain later disappeared from this Ara-3 population).
 For details on this second mutation see: Quandt, et al, Recursive genomewide recombination and sequencing reveals a key refinement step in the evolution of a metabolic innovation in Escherichia coli, PNAS February 11, 2014, vol. 111 no. 6, 2217-2222 http://www.pnas.org/content/111/6/2217.abstract
 Justin R. Meyer, Devin T. Dobias, Joshua S. Weitz, Jeffrey E. Barrick, Ryan T. Quick, Richard E. Lenski, Repeatability and Contingency in the Evolution of a Key Innovation in Phage Lambda. Science 27 Jan 2012: Vol. 335, Issue 6067, pp. 428-432. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306806/
 Behe M.J., Quarterly Review of Biology 85(4), 2010, 419-415.
 Paul Braterman https://pandasthumb.org/archives/2010/12/behes-review-in.html pointed out the rhetorical maneuvers employed by Behe in his 2010 review:
Behe constructs an elaborate apparatus for classifying mutations as “gain”, “modification”, or “loss” of what he calls a Functional Coded Element (FCT). The definition is skewed to make “gain” as difficult to prove as possible. The process needs to be understood at the molecular level, rather than simply in terms of phenotype expression. This enables him to dismiss as of unproven relevance the Lenski group’s famous demonstration of E.Coli acquiring the ability to metabolise citrate under anaerobic conditions. Moreover, advantageous removal of inhibition is treated as “loss”, but advantageous disruption of a function by IS duplication and insertion is classified as “modification”, rather than “gain”. Using these restrictive and asymmetric criteria, Behe classifies most sufficiently well-understood mutations in laboratory-bred bacteria as loss or modification, although he does recognise a few gains…..
But there are numerous well-known counterexamples, many of them discussed in this review.
The next stage is rhetorical dismissal of such counterexamples. Here the strategies include limiting the search (ignoring the massive creative role of gene duplication and polyploidy in eukaryotes, and of horizontal transfer followed by selection in bacteria themselves), narrowing the criteria (new functions don’t count unless they can be demonstrated to arise from additions, rather than any other kinds of alterations, to the molecular machinery), and inventing additional constraints (creation of a new category, the FCT, classifying the process as a loss if either material or function is lost at any stage in the change being discussed, dismissing changes in function as mere transformations, rather than novelties). This stage switches the emphasis from what is possible in principle, to the demand that each case be demonstrated in practice, and fully analysed in detail, at the molecular level.
Finally, any counterexamples still surviving this moving of the goalposts and restricting and tilting of the playing field are dismissed as untypical, and therefore unimportant.
 Jerry Coyne [ https://whyevolutionistrue.wordpress.com/2010/12/12/behes-new-paper/ ] noted that Behe limited his survey to precisely the sorts of experiments which are heavily biased towards NOT observing a dramatic increase in functional complexity:
Behe has provided a useful survey of mutations that cause adaptation in short-term lab experiments on microbes (note that at least one of these—Rich Lenski’s study— extends over several decades). But his conclusions may be misleading when you extend them to bacterial or viral evolution in nature, and are certainly misleading if you extend them to eukaryotes (organisms with complex cells), for several reasons:
- In virtually none of the experiments summarized by Behe was there the possibility of adapting the way that many bacteria and viruses actually adapt in nature: by the uptake of DNA from other microbes. Lenski’s studies of E. coli, for instance, and Bull’s work on phage evolution, deliberately preclude the presence of other species that could serve as vectors of DNA, and thus of new FCTs (Functional Coded elemenTs). This is not an idle objection, since we know that adaptation in natural populations of microbes often arises by incorporating new FCTs from other species. Pathogenicity and antibiotic resistance in bacteria, for example, arise in this way…
- In relatively short-term lab experiments there has simply not been enough time to observe the accumulation of complex FCTs, which take time to build or acquire from a rare horizontal transmission event. Finding adaptation via point mutations or loss of function is much more likely…
- Finally, Behe does not mention—and I think he should have—the extensive and very strong evidence for adaptation via gain-of-FCT mutations in eukaryotes. While that group may occasionally acquire genes or genetic elements by horizontal transfer, we know that they acquire new genes by the mechanism of gene duplication and divergence: new genes arise by duplication of old ones, and then the functions of these once-identical genes diverge as they acquire new mutations. Or, new genes can arise by unequal crossing-over between different genes, so that new genes arise by mixing bits of old ones.
 Some readers may wonder why the “nylon-eating bacteria” was not included in these list of complex mutations which give rise to new functions. It was found in 1975 that this bacterium in a pond in Japan had developed the novel ability to digest the waste products from nylon manufacture. In 1984 Susumu Ohno proposed that a key gene for this ability had arisen as a result of a “frameshift” mutation. Most types of mutations keep most of the protein products of a gene intact, while making a change in some portion of it. Thus, the expressed protein after the mutation is largely similar to the protein before the mutation. A frameshift mutation, on the other hand, can alter a gene such that an entirely new sequence of amino acids is produced, with little similarity to the original protein. https://en.wikipedia.org/wiki/Frameshift_mutation
For some years, this “nylon bug” was cited as an example of how a more or less random recoding of an entire gene product could produce a completely new and beneficial enzyme. However, papers published by Seiji Negoro since 2006 indicate that in fact no frameshift mutation was involved in the evolution of the 6-aminohexanoic acid hydrolase. Rather, it seems that the new enzyme came about by changes to two amino acids from a pre-existing gene. This is still a nice example of a new function arising from multiple mutations, but it is not as dramatic as a total remake stemming from a frameshift. See https://en.wikipedia.org/wiki/Nylon-eating_bacteria .
 Some direct rebuttals of the notion that the order of fossils in the rocks can be explained by a global Flood are given here: https://rationalwiki.org/wiki/Fossil_sorting_by_the_global_flood
and here: http://debunkingcreationism.blogspot.com/2012/11/real-geology-vs-flood-geology-via.html
Index fossils provide further evidence that life-forms have evolved over millions of years. Fossils which have a wide (sometimes global) geographic distribution but a relatively short time of appearance in the rocks are called “index fossils”, since they are useful in determining the relative ages of the rocks in which they occur:
These index fossils were all sea creatures, so it is not that the Pecten gibbus scallops all got buried in higher rock layers because they ran faster to higher ground as the Flood-waters rose whilst all the sluggish Paradoxides trilobites were left behind. At all levels there are fossils of animals that are big and small, skinny and fat, so this sequence is not a result of hydrodynamic sorting during one big Flood. Rather, the order of their world-wide appearance in the rock layers reflects their temporal appearance, then disappearance, across the times of deposition in the sedimentary rocks in which they are found. There are sound reasons to believe that that these deposition times extended over millions of years. The reality of this world-wide “faunal succession” in the sedimentary rock layers was recognized by geologists well before Darwin proposed a mechanism for how new species could arise.
 Contrary to the claims of anti-evolutionists, this “Cambrian explosion” of new life-forms is readily explicable within conventional evolutionary science. See Cambrian Contention: Disputing “Darwin’s Doubt”
 Donald Prothero, What Missing Link? NewScientist, 1 March 2008, 35-41 http://www.donaldprothero.com/files/92313517.pdf
 Michael Behe argued for such a limit to evolution in The Edge of Evolution, but his arguments fail, as shown by the examples here. This is further discussed in STAN 4: Assessing Limits to Evolution and to Natural Selection: Reviews of Michael Behe’s “Edge of Evolution” and John Sanford’s “Genetic Entropy”
 Michael Behe claimed that some biological systems are “irreducibly complex”, such that all the parts must be in place for the system to work – and hence these systems cannot be built up from step by step mutations. However, some of the examples described here directly disprove this thesis, and it has been addressed multiple times by the scientific community. https://en.wikipedia.org/wiki/Irreducible_complexity
 Motoo Kimura, The Neutral Theory of Molecular Evolution, (Cambridge: Cambridge University, 1983 Press) p. 63. Equus itself is excluded from the count of genera because its span is incomplete.
 Manyuan Long, Esther Betrán, Kevin Thornton & Wen Wang, “The origin of new genes: glimpses from the young and old,” Nature Reviews Genetics 4, 865-875 (November 2003) http://faculty.washington.edu/wjs18/Newgenes.pdf
 R. E. Lenski, “Is the LTEE breaking bad?”, Feb 2019. https://telliamedrevisited.wordpress.com/2019/02/26/is-the-ltee-breaking-bad
“The LTEE was designed (intelligently, in my opinion!) to be extremely simple in order to address some basic questions about the dynamics and repeatability of evolution, while minimizing complications. It was not intended to mimic the complexities of nature, nor was it meant to be a test-bed for the evolution of new functions. The environment in which the bacteria grow is extremely simple. …Indeed, the LTEE environment is so extremely simple that one might reasonably expect the bacteria would evolve by breaking many existing functions. That is because the cells could, without consequence, lose their abilities to exploit resources not present in the flasks, lose their defenses against absent predators and competitors, and lose their capacities to withstand no-longer-relevant extreme temperatures, bile salts, antibiotics, and more. The bacteria might even gain some advantage by losing these functions, if doing so saved time, energy, or materials that the cells could better use to exploit the limited glucose supply…… if one assumes the ancestral state of a gene is perfect, then there’s no room for improvement in its function, and the only possible functional changes are degradative. In my post on the polar bear case, I explained why the assumption that a gene is perfect (or nearly so) makes sense in certain situations. However, that assumption breaks down when an organism encounters a new environment, where the optimal state of a protein might differ from what it was before. …”
“…To his credit, Behe does write about the lineage that evolved the ability to consume the citrate. However, he dismisses it as a “sideshow” (p. 365), because he refuses to call this new capability a gain of function. Instead, Behe writes (p. 362) that under his self-fulfilling scheme “the mutation would be counted as modification-of-function—because no new functional coded element was gained or lost, just copied.” In other words, Behe won’t count any newly evolved function as a gain of function unless some entirely new gene or control region “poofs” into existence.
But that’s not how evolution works—unless you believe, as Behe apparently does, that God or some other “intelligent agent” intervened to insert new genetic information into various lineages during the course of history. (Suffice it to say that I don’t regard this as a scientifically useful hypothesis, because I don’t think it can be tested.) Evolutionary biology doesn’t require that new genes poof into existence. Instead, old genes and their products are coopted, modified, and used in new ways—a process called exaptation. For example, crystallin proteins in the lenses of our eyes derive from proteins that performed other functions. At a larger physical scale, the wings of birds and bats derive from the forelimbs of their four-legged ancestors, which in turn derive from fins of fishes….”
 R. E. Lenski, “Evolution goes viral! (And how real science works) ” , March 6, 2019. https://telliamedrevisited.wordpress.com/2019/03/06/evolution-goes-viral-and-how-real-science-works/