Frozen Evolution. Or, that’s not the way it is, Mr. Darwin. A Farewell to Selfish Gene.

Box 31 What our British competitors discovered on

animals, and how we will soon beat them

Probably the most interesting discovery regarding the effect of latent toxoplasmosis on the behavior of animals was achieved by our British competitors in the laboratory of Joanne P. Webster. If you google “fatal attraction” and Toxoplasma, then the search engine won’t come up with the new release of the famous thriller, but many links to the research of this British team. In the year 2000 they were able to describe that brown rats with latent toxoplasmosis no longer avoid places rank with the smell of cat urine, but, to the contrary, seek them out (19). Neurophysiologists from Stanford University later showed that this is a highly specific phenomenon (20) (21). Infected brown rats (and mice) have an unimpaired sense of smell and avoid other dangerous stimuli; but the scent of cat truly attracts them. This study, which even I was initially skeptical of, has been repeated many times, in at least three different laboratories. So the described phenomenon is most likely real. It is, however, quite difficult to imagine how Toxoplasma can affect the nervous system of its host so specifically, that the switch from repulsive to attractive occurs only with the scent of cat. So how do we Prague scientists plan to beat them? It’s probably better that I keep it to myself. Otherwise, our students might realize the identity of these samples, whose odor they’ve been evaluating for our experiments for last two years.

Box 32 How and why a scientific publication is born

Officially, scientific publications ensue from research projects. A researcher writes a project, whose purpose is to answer a certain scientifically interesting question. If he succeeds with his project in the grant competition and obtains financial resources, he'll implement the planned studies and glean an answer to the questions he laid. He subsequently publishes this answer as a scientific article in a journal, so that the results of his study might be shared with as much of the scientific public as possible. I don't insist that my experiences are universally applicable, but, on the other hand, it doesn't seem to me likely that they would differ dramatically from the experiences of my colleagues. What I am sure about, however, is that my experiences and the official process of scientific publication are like night and day. Most of the results that we finally published were obtained as the by-product of other studies. Even when the results were directly related to the project from which they arose, they were most likely obtained long before the respective project was proposed for a grant. For only a completely irresponsible person submits to the grant competition a project that's not at least half-way done. A careful person, like I, generally enters a project at least 80% complete (and for the duration of his proposal's evaluation, crosses his fingers that the publications not come into the press before he is given the grant). But the institution giving money for an already completed project is not defrauded – the funds are used for another project, usually a follow-up, which becomes the subject of the next grant proposal. Publications don't usually come about so that other researchers in the field can use them, but in spite of the fact that other researchers can use them. Other researchers in the field are first and foremost competitors, and the ground must be cut from under their feet – i.e. they must be overtaken in solving questions. Yes, overtaking them usually means publishing a discovery before they do, but only a small percentage of articles actually publish a discovery – most of them show how it is and isn't possible to achieve that discovery in the future. Many publications come about so that a researcher can display a scientific feat to the grant agency and thus heighten his chances of later obtaining another grant. Many publications come about because a certain number of one's own publications are required of an applicant for a scientific or teaching degree or position (a PhD, associate professor or professor). But most publications arise from vanity. Like a gunfighter notches his pistol, a researcher collects his publications, delighting late at night in how his list is steadily growing. Yet today this proclaimed honorable competition, which in many cases resembles the manner of amateur sport until the 50s, is critically endangered by commercialization (but then again, what isn't?). The taxpayer began to care about what he's getting for the billions that go to science, and an educated taxpayer even begins to wonder whether he's getting the same number of publications for his taxes, as is a taxpayer in the U.S. or Great Britain (which he likely won’t get any time soon). So the respective government agencies don't waste time in motivating their researchers to the highest production of scientific articles in top journals. The number of articles is strictly observed, and based on their number and the ranking of the journals in which they were published, the respective scientific institutions and sometimes even teams are financed (see also Box 28 How to measure quality of science). In some countries this effort of the government bodies is carried out to an extreme. For example, in China, publishing an article in a prestigious journal brings the team and specifically the researcher such a great financial award, that researchers there are supposedly willing to pay foreign teams a more than decent sum for being added to the list of authors on their publication.

Box 33 How engaged women sniff out good genes

I don’t know, whether my colleague Jan Havlíček would agree, but I think that the most interesting result so far came from his project studying the differences in women's olfactory preferences for men's scent during the menstrual cycle (22). He was able to convince 48 men, students, to first complete a psychological questionnaire revealing dominance, and then to wear cotton tampons under their armpits for 24 hours, for the tampons to absorb their body odor. Then he placed the scent samples in brown glass containers and convinced 65 female students to evaluate how attractive they found each scent. It turned out that women in the fertile period of their menstrual cycle found more attractive the scents of men evaluated as dominant by the questionnaire. This result corresponds with the evolutionary psychological theory, which presumes that women choose their sexual partners according to the level of dominance. Dominance is hard to fake and a pretty expensive mark for its bearer, which honestly shows how superior his genes are and how many resources he has. A man can dissemble for some time, and try to look witty and intelligent, but if he’s trying to fake dominance and “doesn’t have it,” he’ll eventually be put in his place. . A more detailed analysis of our data showed something even more interesting. The effect pertained only to women with long-term partners; women without long-term partners preferred the scent of dominant men only minimally (Fig. 19). This goes to show that women who chose dominant men were seeking above all the man’s “good genes,” as opposed to his resources; they were interesting in only a short-term sexual partner. Women seeking a long-term partner, with long-term access to resources, did not put dominant men first. It seems evolution programmed women to follow a sort of mixed reproductive strategy. She preferred the dominant man (her sexual partner during the fertile period of her menstrual cycle) as the genetic father of her children, whereas her choice of social partner was subject to different criteria. It also works like this with other animals – for example, studies on swallows have show that attractive males are often poor social partners and are not as involved in feeding the young as are less attractive males. Females have it hard – they’d like to be faithful to their husbands, but nature ruins it for them. And those backwards vegetarians would like to stone them for it! Or was it the Shintoists or Lutherans? I’m not very good with religions, for in school the Communists only taught scientific atheism…

Fig. 19 The preference for the body odor of dominant men by engaged and not engaged women in different phases of their menstrual cycle. Preference for dominant men was expressed as the value of the correlation coefficient (r) between the dominance of the man based on a psychological questionnaire, and the average pleasantness of his body odor according to a given group of women. The graph demonstrates that all women preferred the odor of dominant men; however, the greatest preference was expressed by women in the fertile period of their menstrual cycle.

Footnote: By backwards I only mean that they haven’t yet read my book, don’t search for any other meaning. It was explained to me that I must be politically correct in the English version of my book and avoid joking about certain topics. It’s something I’m well-trained in; I had to practice auto-censorship for half my life.

Box 34 Is sexual reproduction related to defense against parasites?

The origin of sexual reproduction is one of the greatest evolutionary mysteries. At first glance (as on second and The maybe even third), it would seem that when competing with an asexually reproducing species, which for one doesn’t throw out half of its reproductive potential in the production of useless males, the sexually reproducing species should quickly lose. Yet the opposite is true – the vast majority of known species reproduce sexually. Evolutionarily biologists have gradually suggested a whole range of possible explanations. One of these, which is reasonably supported by a number of studies, is called the Red Queen Hypothesis and presumes that sexuality evolved and is maintained in nature as one method of defense against parasites. The evolution of parasites is usually faster than that of their hosts; thus they are able to quickly adapt to the most frequently occurring form of the host species (i.e. the individuals with the most frequent genotype). If there are individuals with a combination of genes (genotype) most resistant against parasites, they’re asking for trouble in the next generation. They’ll leave behind the most offspring, and the new generation of parasites will adapt precisely to these offspring. Biological fitness in a world full of parasites, i.e. in our world, actually has a negative hereditability; individuals who have the most offspring in one generation, have the least offspring in the next. But this applies only to asexually reproducing species, where the gene combination of the parent passes to the offspring unaltered. With sexually reproducing species, in every generation each offspring has a random combination of genes, half from the father and half from the mother, so that the offspring differ genetically not only from their parents, but also from each other. Sexually reproducing species present a sort of moving target to parasites – as soon as the parasites adapt to gene combinations in one generation of hosts, these combinations disappear in the population, giving way to entirely new ones. An immensely important role in defense against parasites is played by MHC proteins. As described in Box 22 Antibodies and myths about them, these proteins transport parasite protein peptides from cell interior to cell surfaces, and so determine what signals (which peptides) the immune system will recognize to conclude the presence of a parasite. In a host species, each gene for an MHC protein occurs in many variants, meaning that it has many alleles; these variants can be differentiated by which peptides they bind and transport to the cell surface. For species, this is highly advantageous. If each MHC gene had but one variant, all the members of a species would recognize the presence of a parasite by the same peptides in its proteins. A parasite capable of rapid evolution, such as a virus, could remove these peptides from its proteins and become invisible to the host’s immune system. Because the individuals of a host species differ from each other in the variants of their MHC proteins, they also differ from each other in the peptide sets they use to recognize the presence of a given parasite. When the proteins of a parasite are cleaved, they form the same (enormous) set of various peptides; but the MHC proteins in each individual choose from this fundamental set a distinct subset of peptides they bring to T-cells to look over (see Box 22 Antibodies and myths about them). Thus parasites cannot adapt to a host species by removing from their own proteins the peptides the host uses to recognize their foreign nature – in each individual of the host species the peptides used are different. For a member of a host species, it is also advantageous to have a sufficient number of MHC protein variants; in that way it will be able to recognize a large spectrum of peptides, and thus also the foreign nature of a large number of parasitic species. This is apparently the reason why women, in choosing their sexual partner, prefer men with different variants of MHC genes than they themselves have (23). How women recognize these out of all the other men isn’t entirely clear, though it’s likely that they sniff them out – peptides have a relatively low molecular mass and after being loosened from MHC proteins may spread through the air. In a number of mammal species, a so-called vomeronasal organ is designated to detect them; but in humans this organ is not functional and it seems that the peptides are detected by a population of specialized cells in the olfactory epithelium. Signals from these cells go directly to the brain stem, as opposed to the cerebral cortex, so a woman’s conscious mind is never clued in – to her the given man just suddenly seems so inexplicably and irresistibly attractive...

Box 35 How I refuted Darwin

Darwin’s theory of evolution presumes that advantageous traits evolve in species as a result of the effect of natural selection. If stronger individuals are more likely than weaker individuals to survive to maturity and reproduce, then the average strength of the member of a species should increase each generation. For with each individual there are always mutations, some increasing and others decreasing the fitness (viability and fertility) of their bearer. Individuals bearing mutations which raise their fitness are more likely to reproduce than individuals without such mutations, and even more so than individuals with mutations that lower their fitness (this is the majority of mutations). Because mutations are passed down from parent to offspring, the fraction of individuals with advantageous mutations should steadily grow, and the average fitness of the member of a population should thereby increase each generation. The vast majority of biologists sees Darwin’s theory of evolution as entirely satisfying and doesn’t believe it necessary to change anything about it. But in reality this theory can explain evolution only for asexually reproducing organisms (such as bacteria or plants reproducing vegetatively, through tubers or rhizomes). In sexually reproducing organisms mutations can also be passed down from the parents to the offspring, but because the genes of both parents are mixed to form the genotypes of the offspring, then in each generation the same mutation occurs in the presence of different genes, changing its effect on the traits of the organism. A mutation (or, rather, a gene variant – allele) which raised fitness in one parent may, with a combination of genes with the other parent, actually lower the fitness of the offspring (or raise the offspring’s viability, but lower its fertility). In asexually reproducing organisms, the parents’ gene combination passes down to the offspring unchanged (at most enriched by some new mutation), so the parents’ traits are inherited by the offspring of the next generation. Unlike this, in sexually reproducing species new combinations of alleles arise in each generation, mixed from alleles of both parents. As a result of this, the parents’ traits are not inherited and the classical Darwinian mechanism of evolution cannot work here. This corresponds with the findings of modern paleontology, a science studying fossils. Paleontologists have shown that species change very little over the span of their existence, and when their time has comes to die out, they often look entirely the same as they did when they were formed. Of course, here and there an old species branches off a new one, which from the start is distinct from the ancestor species. If Darwin’s theory were also applicable to sexually reproducing species, the paleontological picture would be very different – above all in that species would have the capacity to change at any time during their existence, not just in the beginning. To explain how evolution can work in sexually reproducing organisms (which, by the way, is the vast majority of known species present on Earth), and simultaneously to define the picture of the species evolution provided to us by modern paleontology, sometime in the later 90s I created the frozen plasticity theory (24) (10). According to this theory, sexually reproducing species really cannot evolve through the effects of natural selection, firstly because their traits are not inherited (see the above), and secondly because the advantage or disadvantage of individual gene variants often depends on their numeric representation in the population. So species don’t act plastically (as Darwin imagined), but elastically – at first they happily react to natural selection, but as the numeric representation of individual alleles grows further from average, the individuals respond less to natural selection, until they finally stop reacting entirely. When natural selection is interrupted, the numeric representation of individual alleles (and thus also the average traits of the member of that species).returns to previous levels. But under certain conditions the species can switch over from an elastic to a plastic state, and, through the effect of natural selection, began evolving just as Darwin imagined. This happens, for example, when a small number of species members colonize an island far from the continent, where their offspring survive many generations as a small colony. During this time, chance (biologists call this genetic drift) erases almost all the alleles – from each gene there ideally remains but one. When the population increases sufficiently, there arise ideal conditions for Darwinian evolution. The population is large, so the fate of an individual is determined by natural selection, instead of chance, like in small populations. But at the same time, the individuals are genetically similar; therefore the mutations in each generation will occur in the presence of the same alleles – despite the fact that each individual gets new gene combinations mixed from two parents. Because both parents have nearly identical gene combinations (aside from maybe a few mutations), their genes can combine in whichever way but always achieve the same result. Therefore a species re-establishes evolutionary plasticity and can begin evolving. Yet over time the new species accumulates more and more alleles, often because the benefit of these alleles decreases as they spread throughout the population. The fate of such variants is determined by the so-called negative frequency dependent selection. So long as alleles remain rare after they’re formed, they are advantageous and spread throughout the population through natural selection; but when they become abundant, their merit decreases. Due to their frequency dependence, they can never become prevalent nor entirely disappear from the population (see Box 70 Genetic polymorphism: what it is, where it comes from, and how it’s maintained in nature?). A new species loses its plasticity and becomes more and more elastic. Paleontological data shows that a species persists in a plastic state only about 20 thousand years (less than one percent of the usual length of a species’ existence), which is a fairly long time for it to substantially change from its ancestor species, but also a short enough period of time in paleontological record that the formation of the new species looks like a sudden jump. The frozen plasticity theory further presumes that over time different evolutionary branches accumulate characters, which could become plastic only with great difficulty even during the formation of a new species. The accumulation of these eternally frozen characters is thanks to the process of sorting on the basis of stability (what is unstable come and goes, and what is stable remains). This accumulation of eternally frozen characters may explain why the rate of evolution over the course of Earth’s history has been slowing, and why the greatest evolutionary innovations in animals were formed over a relatively short period of time (possibly about ten million years), half a billion years ago (in the Cambrium) – and why, since that time, no drastically different body plans have appeared (25).

Box 36 On peptides, and why they should interest a parasitologist

Proteins are generally considered the most interesting and varied molecules in modern cells. (Time will probably show that RNA molecules are even more interesting and varied, but so far not much is known about them.) Proteins make up the definite majority of enzymes, regulatory and signaling molecules, molecular motor and pumps, as well as a large part of cells’ molecular building-blocks. They therefore are the main component of a modern organism’s “hardware.” A protein molecule is chemically formed by a linear chain, in which twenty kinds of amino acids alternate in an apparently random pattern. The order of the twenty amino acids (relatively small organic molecules, which have in common only the fact that on one end they have a carboxyl and on the other an amino group, allowing them to unite using peptide bonds into a linear chain) is determined by the respective genes. This order decides the shape the chain will fold into, and thus what function it will perform. Yet the same shape and thereby similar function can be attained with different, though generally similar, amino acid sequences. Proteins are the main molecules used by the immune system to recognize the presence of parasites, whether they be viruses, bacteria or even Toxoplasma. A certain amount of each protein synthesized in the cell (intracellular) or brought in from the outside (extracellular), is cleaved using special enzymes into small pieces about 7-14 amino acids long. These peptides are, by the means of MHC proteins (MHC antigens), transported to the cell surface and there presented. Peptides from intracellular proteins are bound to and presented on MHC class I proteins; peptides from extracellular proteins, on MHC class II proteins. All cell surfaces are continuously examined by a type of white blood cell called T-cells (or T-lymphocytes). Each T-cell has on its surface a receptor with which it is able to recognize a certain peptide. In the body there are many groups of T-cells clones, with different receptors and thus recognizing different peptides. Unless an organism suffers from an autoimmune disease, it has only T-cells that recognize “non-self” peptides, peptides not present in the proteins synthesized by its own body. The body does produce T-cells that recognize any type of peptide, but all “immature” T-cells, which recognize a “self” protein, commit cell-suicide (apoptosis) while passing through the thymus. There are many types of T-cells; at this moment we are interested in cytotoxic and helper T-cells. Cytotoxic T-cells, or “killer” T-cells, recognize only peptides presented on class I MHC-proteins, peptides which come from intracellular proteins. True to their name, cytotoxic T-cells kill the unhappy cell presenting the foreign peptide – since the presence of a peptide that a cytotoxic T-cell can recognize signifies that “non-self” proteins, which most likely come from a parasite like Toxoplasma or a virus, are being synthesized in the presenting cell. Helper T-cells recognize peptides presented on class II MHC-proteins, peptides from proteins brought into the cell. While class I MHC-proteins occur on almost all types of cells, class II MHC-proteins are present only on certain cell types defending against parasites, such as B-cells or macrophages. (Dear reader, I simplify greatly, but believe me when I say it is for your sake). If a helper T-cell recognizes the presence of “its” foreign peptide on a cell, then it’s proof to the helper T-cell that the cell presenting the peptide is capable of ingesting a foreign intruder or proteins from a foreign intruder. The helper T-cell provides such a cell with growth factor (see also Box 22 Antibodies and myths about them).

It would be particularly advantageous for a parasite to succeed in removing from its proteins (gradually by evolution, through mutations in the respective genes) all the peptides not present in the proteins of its host. If the parasite could adapt its own peptide repertoire to that of its host, then the host’s immune system could not detect it. Similarly, it would be advantageous for the parasite to cut down its peptide repertoire as much as possible, getting rid of all the peptides it can afford to. This would eliminate of a large part of the target the T-cells in the host body use to recognize an invader, so the parasite could target a wider range of host species. It would likewise be advantageous for the host organism to cut down on its own expendable peptides, for then it could recognize a greater number of peptides as foreign. But all this is only a theory – my theory, to be perfectly clear. Seemingly, it shouldn’t be difficult to prove it. Potential hosts like mammals and birds, with an immune system based on the presentation of peptides, should have a sparser peptide library than potential hosts without such an immune system – hosts such as plants and free-living protozoa. Parasites should have a sparser peptide repertoire than their non-parasitic relatives; a specialized parasite, targeting but one host species, should have a similar peptide repertoire (containing the same peptides) as its host. Hybrids of two host species should have a richer peptide repertoire than both the parent species and should also be more susceptible to parasites (this has actually been confirmed). But surprisingly enough, over time it became clear that proving or disproving the existence of these (and other) phenomena stemming from my theory was extremely difficult. I threw in the towel over 20 years ago, followed ten years ago by my undergraduate and later graduate student Pavel Cindr. Five years ago not even my colleague Martin Mokrejš got very far, and currently my graduate student Michaela Zemková continues to wrestle with the problem. It seems there’s nothing to do but cross our fingers and hope.

Box 37 Does a cell conduct isoelectric focusing

Most biologically significant molecules, including proteins, carry both positive and negative charges on their surface; we call them amphiphatic molecules. In an environment with a high pH (a basic environment) where there are few free protons, positively charged protons loosen from the amphiphatic molecules and the molecules’ net charge become more and more negative. But in an environment with a low pH (an acidic environment), where there are many free protons, there these positively charged particles bind to amphiphatic macromolecules, whose net charge thus becomes positive. In an electric field, positively charged molecules move towards a negative electrode, while negatively charged molecules towards a positive electrode – a process called electrophoresis. If the electrophoresis of proteins or other molecules carrying both a positive and negative charge occurs in an environment with an existing pH gradient (in which the pH gets higher approaching a positive electrode and lower approaching a negative electrode), then proteins move towards a positive or negative electrode, depending upon their surface charge. When molecules with a positive initial charge reach an area of high pH, they gradually loose protons, and thus also their net positive charge. For this reason, in a certain area of the pH gradient, their movement stops. The same is true for molecules with a negative initial charge. At first they move towards a positive electrode, approaching an area of low pH, gaining more and more protons, and so gradually losing their negative charge – until they lose it entirely and their movement in the electric field comes to a complete stop. Each amphiphatic molecule (and thus every protein) has a pH at which its net charge and movement in an electric field are zero. We call this pH the isoelectric point and abbreviate it as pI. If we form an electric field in an environment with a pH gradient, then the proteins concentrate (focus) in certain locations based on their pI, separating different proteins from each other. When we have many types of ampholytes, with different isoelectric points, in solution, we don’t have to create a pI gradient ahead of time – it will form in the solution on its own due to the voltage between the electrodes. Biochemists use isoelectric focusing to separate complicated mixtures of proteins occurring in the cell; as well as to isolate a protein they’re interested in, or at least to detect its presence. In the year 1990, I published in the journal Biosystems the hypothesis that isoelectric focusing is also carried out by a living cell (26). I calculated that only a fraction of the energy the cell has at its disposal is needed for the cell to focus its macromolecules into different areas of its interior, accelerating and streamlining its metabolic processes. In the year 2009 I published in the Journal of Molecular Evolution an article in which I showed that evolutionary „invention“ of intracellular focusing could have prompted the origin of the eukaryotic cell – it could have made possible the existence of cells with a volume ten thousand times greater than that of typical prokaryotic cells (the cells of bacteria and archea) (27). In a small prokaryotic cell molecular transport can by managed by diffusion; but molecular transport spanning a large cell would take dozens of minutes by diffusion, which would intolerably lower the rate of all metabolic processes in the cell. So that cells could get bigger, they had to invent alternative mechanisms to transport molecules, and one of these mechanisms could have been intracellular isoelectric focusing. Incidentally, isoelectric focusing can explain even the usual way intracellular processes are regulated. When a cell is to switch on or off a certain chemical process, such as a metabolic pathway (perhaps a sequence of reactions leading to the synthesis of carbohydrates), it often phosphorylates or dephosphorylates a key enzyme of the metabolic pathway (by binding a negatively charged phosphate group to the enzyme, or removing it). This necessarily changes the enzyme’s isoelectric point, and results in the enzyme moving to a different part of the cell, somewhere the metabolic process can (or can’t) be carried out. Additionally, enzymes of individual metabolic pathways can be concentrated through isoelectric focusing into certain parts of the cell, so that the respective metabolic processes can proceed simultaneously and yet independently.