Lenski’s E-coli Citrate Experiment: New Genetic Information?

 

Lenski’s 15 year (E-coli generation = 31,500) E-coli experiment (along with nylonase) is perhaps one of the most famous argument used by evolutionists as an illustration of evolution creating irreducible-like systems or novelty in action. The Long Term Evolutionary Experiment (i.e. LTEE) involved a certain group of bacteria known as E-coli that were isolated under aerobic conditions and eventually had the new evolved the ability to metabolize citrate. Lenski’s E-coli experiment is a well known tactic used by evolutionists to refute creation/intelligent design arguments by implying evolution is capable of creating novel biological proteins for cellular functions that did not otherwise exist before. Is this position reasonable? A cross-examination between the arguments used by evolutionists and actual peer-reviewed literature revealed no based on numerous points:

Point # 1: Despite the evolutionary claim of E-coli’s ability to metabolize citrate as new genetic material, it is not at all completely new.

On first sight, it may seem that Lenski’s E-coli experiment does indeed seem to show evolution in action, considering the fact that E-coli orginally lacked the ability to utilize citrate. However, this is not at all completely true. Contrary to popular belief, the ability for E-coli to metabolize citrate is really nothing special.

As demonstrated in a July 1982 peer-reviewed paper published in the Journal of Bacteriology by Barry G. Hall:

“Chromosomal Mutation for Citrate Utilization by Escherichia coli K-12,”

“E. coli is not, however, totally inert toward citrate. It may utilize citrate anaerobically provided that a second substrate such as glucose or lactate is available to provide reducing power for the formation of succinate from oxaloacetate (13). The failure of E. coli to utilize citrate aerobically has been attributed to the absence of a citrate transport system (10), but the co-metabolism of citrate implies that E. coli does possess a citrate transport system that functions anaerobically (13).”- Barry G. Hall

Based on the paper, it seems that E-coli did indeed actually already had the ability to metabolize cirate in the first place although it was restricted. Originally, they are limited to utilizing citrate only under anaerobic conditions, meaning they were only capable of doing so under an environment that is absent of oxygen. So here’s a question to consider. Is citrate really something that is completely new? The true answer is no, as E-coli already possessed the genetic information to utilize citrate although restricted to the level where oxygen is absent.

Point # 2: E-coli’s ability to utilize citrate under oxic conditions is actually nothing more but a modification of an already existing system rather than a novel one.

By contrast, Lenski’s E-coli experiment involved isolation of the bacterial population with citrate under aeorboic conditions, i.e. environments with oxygen present. Due to the limitation stated above, they were unable to metabolize citrate. However, as Barry Hall has explained in his 1982 paper:

“(13). The failure of E. coli to utilize citrate aerobically has been attributed to the absence of a citrate transport system (10), but the co-metabolism of citrate implies that E. coli does possess a citrate transport system that functions anaerobically (13).”

To rephrase, the reason why E-coli lacked the ability to utilize citrate was due to the fact of E-coli lacking a ‘citrate transport system’ that although functional under the absence of oxygen, is non-functional under such conditions. Even Lenski himself has suggested this in his paper, “Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli,”

“A more likely possibility, in our view, is that an existing transporter has been coopted for citrate transport under oxic conditions. This transporter may previously have transported citrate under anoxic conditions (43) or, alternatively, it may have transported another substrate in the presence of oxygen. The evolved changes might involve gene regulation, protein structure, or both (61).” – Lenski

How did the citrare metabolism under aerobic conditions came about?

According to Barry G Hall in his 1982 paper, the ability for E-coli to metabolize citrate under oxygenated conditions was due to “genetic recombination” (the shuffling of pre-existing genetic information) that involved the activation of the so-called cryptic genes.

“The mutational activation of these genes suggests that they should be considered cryptic genes which have persisted in a silenced state since E. coli diverged from its Cit+ ancestor. That suggestion is supported by the observation that the kinetic parameters for citrate uptake in strain D2004 are of the same order as those for succinate uptake in E. coli (Km = 14 ,uM succi- nate; Vm,, = 20 ,umolImin per g [dry weight]) (11). It is unlikely that mutations conferring a specificity for citrate upon some already existing transport system would result in such a low Km for citrate. More detailed comparisons between the citrate transport system of strain D2004, the citrate transport systems specified by Cit+ plasmids in E. coli, and citrate transport systems of such citrate-utilizing organisms as Salmonella and Klebsiella should shed some light on the possibility that the citrate genes are cryptic in wild-type E. coli”

 Cryptic genes are silently phenotypic genes not normally expressed in the normal life cycle of organisms. They are silent genes and unexpressed, though giving the name cryptic. Interestingly though, cryptic genes are remotely activated and involved in helping organisms adapt to their environment whenever exposed to extreme environmental stress stimuli or selective pressure. They are activated by the cell rearranging its genome such as transposable genetic elements (genes that can copy-paste or cut-paste from one genome position to another or chromosome) Cryptic activation is believed to be a non-random and intelligent targeted response though this remains controversial.

It seems that E-coli rather obtained the ability to metabolize citrate via not by gene duplication but the mere activation of pre-existing cryptic genes. Despite what evolutionists claim that gene duplication created new genetic information of citrate in E-coli, it did not. Rather gene duplication was found to be more of an “amplifier” that helped them to metabolize citrate more effectively than they would without it. However, the modified pre-existing gene that gave them the capacity to utilize citrate under oxygen substrates had already existed long before gene duplication even took place.

How does this demonstrate that evolution is capable of creating brand new biological systems? Truth is it doesn’t. As  Hall has stated in his paper, the ability for E-coli to metabolize citrate was derived from an already existing complex biological system expressed from the activation of ‘cryptic genes.’ Rather than demonstrating the evolutionary origin of where biological systems actually come from, it only demonstrated a mere modification of an already existing system that adapted itself to function in oxygen conditions. Yet, no new component, no new novelty was actually generated in the evolution of citrate.

Point # 3: E-coli’s ability to use citrate is not new genetic information, not according to creationists but also to the 2016 peer-reviewed paper:

After doing a literature review, I came across an interesting 2016 peer-reviewed science paper entitled, “Rapid Evolution of Citrate Utilization by Escherichia coli by Direct Selection Requires citT and dctA,”

This paper, rather than supporting the neo-Darwinian synthesis of evolution, criticized the common evolutionary claim of Lenski’s experiment being great evidence for new genetic information. Despite such criticism over the evolutionary claim of new genetic information, somehow it managed to pass peer-review. As the researchers in the paper quote:

“We conclude that the rarity of the LTEE mutant was an artifact of the experimental conditions and not a unique evolutionary event. No new genetic information (novel gene function) evolved.”

No new genetic information? Well, there you have it folks. No new genetic information was actually generated in Lensi’s E-coli experiment.

Despite the fact that it took Lenski 15 years of experimentation for E-coli to metabolize citrate under aerobic conditions (i.e. 31,500 generations), the previously quoted study “Rapid Evolution of Citrate Utilization by Escherichia coli by Direct Selection Requires citT and dctA,” found E-coli already doing so in 12 to less than 100 generations.

and yet, contrary to the 2016 paper, the opposite was rather demonstrated:

“Importantly, potentiated and actualized E. coli Cit-strains were obtained in as few as 12 generations and refined phenotypes in fewer than 100 generations. Phenotypic and geneticanalyses of these E. coli Cit-strains provided insight into the mechanism for the adaptations and suggested why the LTEE took 33,000 generations to reach this phenotype. Also, this study provided a unique opportunity to compare the results of a direct selection with those of a long-term genetic screen. Finally, because this adaptation did not generate any new genetic information and required expanded expression of only two existing transporters (citT and dctA), generation of E. coli Cit-phenotypes in our estimation does not warrant consideration as a speciation event”

With just 12 to less than 100 generations, E-coli already had the ability to metabolize citrate under aerobic conditions. 12 to less than 100 is nothing compared to Lenksi’s 31,500 exact generations.

Also there was no significant difference between the E-coli in the 2016 paper to that of Lenski’s LTEE (Long Term Evolutionary Experiment) as they both had the same genetic material, making it a justifiable comparison. What was the reason why Lenski’s E-coli took more than 30,000 generations to develop citrate metabolism but only 12 to 100 in the 2016 paper? The reason was the difference in selection. The selection used in the 2016 paper was directive, as the E-coli were forced to try to metabolize citrate. Due to the extreme conditions, a certain population of E-coli was able to metabolize citrate quite rapidly in very few generations.

On the other hand, Lenski’s LTEE was not directive, as the E-coli were not forced only to citrate conditions. Rather the E-coli in Lenski’s experiment was free of selection, as they were exposed to other resources to obtain energy.

but regardless,  they were clearly criticizing Lenski’s finding:

In summary, E. coli can rapidly mutate to a Cit-phenotype in
a relatively short time if subjected to direct selection. This indi-
cates that the 33,000 generations to potentiate the evolutionary
resources for the Cit-phenotype do not reflect a direct requirement but merely experimental conditions. As such, Cit-mutants exemplify the adaptation capability of microorganisms but, as ofyet, the LTEE has not substantiated evolution in the broader senseby generation of new genetic information, i.e., a gene with a newfunction. Interestingly, our findings parallel the conclusions from bacterial starvation studies by Zinser and Kolter (44) in which E.coli adaptations were dominated by changes in the regulation ofpreexisting gene activities rather than by the generation of new gene activities, de novo. The LTEE isolation of Cit⁺ mutants has become a textbook example of the power of long-term evolution to generate new species. But, based on our results, E. coli arrives at the same solution to access citrate in days versus years, as originally shown by Hall (14). In either case, genes involved in the process maintain their same function but show expanded expression by deregulation. Because of this, we argue that this is not speciation any more than is the case with any other regulatory mutant of E. coli. A more accurate, albeit controversial, interpretation of the LTEE is that E. coli‘s capacity to evolve is more limited than currently assumed.

There you have it, no new genetic information was actually generated in the evolution of E-coli’s ability to metabolize citrate.

Problems With Dinosaur to Bird Evolution: The III Digits

One of the most widely held evolutionary claims in regards to the origin of species is the transition between Theropods and birds. Birds according to the evolutionary paradigm are considered to be the byproduct descendants from Theropod dinosaurs. The view that birds are the descendants from Theropods has been widely regarded as a scientific consensus. Just like how humans and their so-called closely related cousins the apes are asserted to have branch off from the last common ancestor, so too does the same concept apply for the transition between Theropods and early to modern day birds.

Proponents in favor of bird to dinosaur evolution have long relied in the fields of genetics, paleontology, comparative anatomy/embryology, to support this view. In addition, they hold these fields to be very strong evidence for their position in defense of dinosaur to bird evolution. In this article we are going to focus on one of the evidences that are claimed by these proponents to support this evolutionary view.

Evidence for Dinosaur to Bird Evolution: The III Fingered Digits

One of the evidence that is considered to be very strong evidence for dinosaur to bird evolution is the homology of the 3 fingered digits found between the hands of the advanced Triassic Theropods and in the forelimbs or wings that are found in the early and modern day birds. In the Triassic Theropod lineage, there were only 3 digits present in the theropod hands that were in the orders of Digits I, II, & III as demonstrated in the fossil record. Just like human hands, Digital I corresponded to the Thumb, Digit II for the index finger, and III for the middle finger. Because theropods in the Triassic period have lost Digits IV & V that were only present from their ancestors and birds are considered to have derived from the Theropod lineage that lost IV & V, it was predicted that the earlier to modern day birds should have the same evolutionary pathway of fingered digits. Theropods had Digits of I, II, & III and so the same homology should be true for birds if they truly derived from that lineage.

Studies in paleontology and comparative anatomy starting from the late 70s  investigating the developmental pathway of the fingers located in the bird wings/forelimbs have revealed that the fingered digits of the birds’ forelimbs do indeed follow the same pattern as dinosaurs. That is both the dinosaurs and birds follow Digits I, II, & III, exactly as predicted in the fossil record. Paleontologists have widely considered this as very strong evidence for dinosaur to bird evolution due to the strong homology present between them. This has been considered to be indisputable evidence that birds derived from the Triassic Theropod lineage. [1, 2, 3, 4]

The Conflict

Unfortunately, later studies in the field of embryology have challenged the common evolutionary view that the III digits present in birds provide very strong evidence for the dinosaur to bird transition. Contrary to the claims of paleontologists, embryologists have disputed the association of the I, II, & III digits homology as demonstrated in the fossil record and paleontological data. They claimed that the III digits in birds actually correspond to Digits II, III, & IV and not I, II, & III. The appearance of embryological data conflicting with that of paleontological have caused a great rage and controversy in regards to dinosaur to bird evolution. [5, 6, 7]

Although these two conflicting views were controversial, it was later confirmed by Alan in 2002 & Michael Richardson in 2003 using embryos that the development of bird forelimbs do indeed correspond to the digits of II, III, & IV unlike the Triassic Theropods of I, II, & III. By contrast, Digits I, II, & III that have been reported in the paleontology of birds were nothing more but the misleading result of convergent evolution. In other words, the III digits homology found between the dinosaurs and birds were due to different developmental pathways that converged independently to the same solution. Rather than those common characteristics being derived from common descent, birds acquired those digit similarities completely independent and distinct from the Triassic Theropod lineage. Thus, confirming the embryologists finding that the wing bird digits are II, III, & IV, not I, II, & III. [8, 9]

The Frameshift Mutation to the Rescue

Given the increasing evidence that the digit bird wings followed II, III, IV according to embryological papers and conflicted with the traditional I, II, III view predicted by the fossil record, the view that birds are dinosaurs was not dead yet. Some evolutionary biologists (despite others like Alan insisting that birds derived from an unknown lineage separate from Theropods) have insisted that such conflict between paleontology and embryology does not contradict the view of Theropods to birds transition. The reason, they claimed, is because of a frameshift that occurred during bird evolution. The Frameshift hypothesis predicts that the conflict of the birds possessing II, III, IV during embryogenesis can be best explained as the result of a frameshift mutation. This frameshift mutation is speculated to have occurred during bird evolution, where at that point the dinosaur digits of I, II, III have frameshifted into II, III, IV in the origin of bird evolution. Originally, on the onset action of bird evolution, the official digits were I, II, & III but since a frameshift has occurred, the birds digits shifted from I, II, III to II, III, & IV. A paper published in 1999 by J.P. & J.A. entitled, “A solution to the problem of the homology of the digits in the avian hand”.has been considered by some as a relief and savior to the conflicting view against dinosaur to bird evolution. [10]

Problems with the Frameshift Hypothesis

The frameshift hypothesis involves the introduction of complete homeosis, the transformation of one organ into another, arising from mutation in or misexpression of specific developmentally critical HOX genes. HOX genes are special protein-coding DNA segments that involve the protein synthesis of genetic switches. These proteins that act like on/off switches in the genome regulate the expression of other genes that play an important role in body-plan development by switching them on and off. HOX genes play a crucial in body-plan development, however, such body-plan structure is pre-determined by the cell long before the expression of HOX genes. Thus, any mutation in the HOX gene only affects the development of pre-determined body-plan structures. The Frameshift Hypothesis is considered as a resolution to the conflict between morphological and embryological data.

Molecular evidence in favor of the frameshift hypothesis by HOX gene expression is a 2004 paper published by Vargas & Fallon entitled, “Birds have dinosaur wings: the molecular evidence.” In this paper  Vargas & Fallon (2004) argue that the prospective digit 1 is characterized by the Hox d 13 expression alone while digits 2 to 5 have Hox d 13 and Hox d 12 expression. Since the anterior wing digit has only Hox d 13 gene expression, they argue it is digit 1 on the grounds of molecular homology, as quote unquote:

Our evidence contradicts the long standing argument that the development of the wing does not support the hypothesis that birds are living dinosaurs [11]

Unfortunately for these authors, their conclusion has been unconvincing as reported in the literature. A review paper published in the same year as Vargas & Fallon’s paper has criticized their claim that the solo presence of HOX D 13 expression only in the wing of birds has demonstrated unambiguously digits I, II & III. In their critique of Vargas & Fallon’s hypothesis Gallis (2005) argues convincingly that the molecular evidence cited is unconvincing as the mutants cited (eg talpid and Hox d deletion mutants) show only a weak correlation of Hox d 12/13 expression with digit identity. As quote unquote,

 

At the moment the data on mutant limbs does not present a challenge to the hypothesis that is based on developmental data; that is, the digits of bird wings are homologous to digits 2, 3, 4 in other amniotes. Other additional problems with the frameshift hypothesis are also highlighted in the same review paper as clearly demonstrated here.  [12,]

Although morphological data remain in conflict with that of embryological, not all embryological studies support the II, III, IV bird wing digit hypothesis. Fate mapping methodology based on Shh expression (Sonic Hedgehog Gene) and polarizing regions that relied on developmental/anatomical criteria have revealed that the digits of the birds are I, II, & III, exactly as predicted by the fossil record.

In Towers’ 2011 paper entitled, “Insights into bird wing evolution add digit specification from polarizing region fate maps” which the paper leads to the support of digits I, II, III & axis shift hypothesis, the authors have concluded that the three digits found in the forelimbs of neornithines developed from positions I, II & III. [13]

However, this view has remained inconsistent with the accepted early condensation patterns for digit progenitors, which show that the three hand digits of modern adult birds developed from the three middle positions. [14, 7, 9]. Furthermore, the assumption that a single frameshift mutation is capable of shifting Digits I, II III into II, III, IV has been shown to be unreliable given that such mutation reveals no adaptive advantage. Rather numerous frame shifts are required to even create an adaptive advantage, as demonstrated by Alan and other researchers. [15]

Another embryology paper that claims to support the Digits I, II, III view is a 2011 paper entitled, “Embryological Evidence Identifies Wing Digits in Birds as Digits I, II, and III. The methodology used was similar to Towers’ 2011 paper, and although they arrived to the same conclusion of Digits 1, 2, 3, it did not support the axis shift hypothesis.

However, just like Vargas & Fallon’s paper, this paper has been reviewed and criticized by a 2011 Nature paper entitled, “Comment on Embryological evidence identifies wing digits in birds as digits 1, 2, and 3.” Here, the authors critiquing the paper found the authors’ conclusions to be flawed and unconvincing due to incorrect methodology as highlighted here.

In summary, the peer-reviewed science literature shows no clear evidence that birds derived from Theropod dinosaurs. Studies in palenotology/comparitive anatomy criteria often tend to create contradictory results when compared to the data of embryology and positional criteria. The fact that these two fields don’t generally agree with each other has become rather a great difficulty for establishing dinosaur to bird evolution. In either case, both morphological and embryological analyses lead to the support that the bird digits are either I, II, III or II, III, IV. However, given the problems for the frameshift hypothesis, it has no longer been generally supported. Rather a new alternative hypothesis, known as the lateral shift, has been more supported in recent literature. However, lateral shift hypothesis unlike frameshift no longer predicts that the digits of the bird wings are I, II, III as predicted in the fossil record. The lateral shift hypothesis proposes that the digits of the wings are II, III, IV, showing no homology at all between the digits of Theropods and birds.

Given the contrary results, evolutionists can no longer claim using fingered digits homology as a back up. Rather than the literature demonstrating evidence of dinosaur to bird evolution, it has rather demonstrated quite the opposite.

References:

[1]   Gauthier, J. 1986. Saurischian monophyly and the origin of birds. Memoirs of the California Academy of Sciences, 8: 1-55.

[2] Ostrom, J. 1977. Archaeopteryx and the origin of birds. Biological Journal of the Linnean Society, 8: 91-182.

[3] Romer, A. S. 1966. Vertebrate Paleontology, University of Chicago Press, Chicago.

[4] Sereno, P.C. 1993. Shoulder girdles and forelimb of Herrerasaurus. Journal of Vertebrate Paleontology, 13: 425-450.

[5] Shubin, N. & Alberch, P. 1986. A morphogenetic approach to the origin and basic organisation of the tetrapod limb. Evolutionary Biology, 20: 319-387

[6] Muller, G.B.& Alberch, P. 1990. Ontogeny of the limb skeleton in Alligator mississipiensis: developmental invariance and change in the evolution of archosaur limbs. Journal of Morphology, 203:151-164.

[7] Burke, A. & Feduccia, A. 1997. Developmental patterns and the identification of the homologies in the avian hand. Science, 278: 666-669.

[8] Welten,.C.M., Verbeek, F.J., Meijer, A.H. & Richardson, M.K. 2005. Gene expression and digit homology in the chicken embryo wing. Evolution and Development, 7,18-28

[9] Nowicki, J. & Feduccia, A. 2002. The hand of birds revealed by early ostrich embryos. Naturwissenschaften, 89: 391-393.

[10] Wagner, G.P. & Gauthier, J.A. 1999. 1,2,3=2,3,4: a solution to the problem of the homology of the digits in the avian hand. Proceedings of the National Academy of Science USA, 96: 5111-5116.

[11] Vargas, A.O. & Fallon, J. 2004. Birds have dinosaur wings: the molecular evidence. Journal of Experimental Zoology (Molecular Development and Evolution), 304B, 85-89.

[12] Galis, F., Kundrat, M. & Metz, J. A. J. 2005. Hox genes, digit identities and the theropod/bird transition. Journal of Experimental Zoology (Molecular Development and Evolution), 304B: 198-205.

[13] Towers M, Signolet J, Sherman A, Sang H, Tickle C: Insights into bird wing evolution ad digit specification from polarizing region fate maps.

[14] “Embryological Evidence Identifies Wing Digits in Birds as Digits I, II, and III