The power of one base: The under-explored Rock pigeon demonstrates the power of micro-evolution

by Andrew Farrer

I was surprised to learn that the domestication of the Rock Pigeon (Columbia livia) has resulted in over 350 breeds. In a stunning example of macro-evolution within a species this domestication has led to some striking phenotype differences. Researchers have now started to explore the genomic diversity, genetic structure and phylogenetic relationships present in these birds. Studying the (relatively) simplistic genetic differences between breeds gives researchers a base to explore the more complex and less distinct differences between other avian species.

A Rock Pigeon takes flight (Creative Commons – Author: A.D. Wilson)

A Rock Pigeon takes flight (Creative Commons – Author: A.D. Wilson)

In a study by Shapiro et al. 38 domestic birds from 36 breeds and 2 feral individuals were sequenced, the results supporting the assumption that these birds would make good models. The Rock Pigeon isn’t on any endangered lists; its effective population size is roughly 521,000 which, apart from a recent bottle neck, has been stable for 1.5 million generations. More importantly, though, with a low expected Linkage Disequilibrium between gene pairs, association mapping techniques are possible (Association mapping exploits the fact that new alleles for new traits will still be close to ancestral genetic sequences. As such you can use SNP chips to explore specific regions of the genome in many individuals and link these differences with differences in phenotype).

Breed relationships were drawn up based on this initial examination and simulations showed that all the breeds had originated from a single ancestral population (rooted using sister species; Columbia rupestris). From this base, further analyses were conducted to associate specific genes and alleles with some of the phenotypic differences. (In case you were wondering, the two feral individuals included in this study appear to be descended from homing pigeons. Whether their ancestors escaped or got lost isn’t clear).

A Red Jacobin Pigeon. The white head is just visible (Creative Commons – Author: J. Gifford)

A Red Jacobin Pigeon. The white head is just visible (Creative Commons – Author: J. Gifford)

Change in plumage colouration is the primary result of bird domestication. This is followed by plumage and structural (skeletal and soft tissue) change and finally changes in behaviour. The head crest is an example of plumage alteration. This feature results from a reversed angle of feather projection on the neck. Growing upwards the feathers form a ring around the head, giving the bird a crown, or crest, of feathers. The degree varies between breeds. Some have no crest at all (the apparent ancestral state) whilst some have quite extreme crests (the Jacobin (pictured) hopefully doesn’t require a wide field of vision!). Importantly the presence of the crest follows a Mendelian recessive pattern, suggesting a single gene is responsible.

Re-sequencing confirmed that a single locus controlled the presence/absence of the crest. It was the pigeon homolog of the Ephrin receptor B2 gene, part of the tissue patterning and morphogenesis pathway in chickens and presumed to be similar in pigeons.  Comparison between 8 crested birds showed that the same mutation caused the presence of the crest in differing breeds. The non-synonymous substitution of a Thymine (T) for a Cytosine (C) generates the recessive allele, the substitution of one base in the entire genome, causes the presence of the crest. As noted the trait is recessive so an individual requires both copies of the gene to be the T allele to have a crest. The presence of the C allele in one or both genes results in no crest.

The same mutation in all crest bearing breeds suggests a single occurrence in a common ancestor or repeated selection from a standing occurrence in the original, wild Rock Pigeon population. With crested breeds not necessarily being more closely related to each other than to non-crested breeds the standing occurrence hypothesis seems viable. Although, it seems to me that in such an artificially selected group it would be easy, whilst the allele was not fixed in the population to breed it in or out of sister lineages or to breed in other alleles in different genes that cause two closer related breeds to appear differentiated. For instance if you took a stock of birds and split them into three populations, you could breed the crest into two whilst not the third. However, if you also select strongly for other traits in the second of the crested populations you would cause the two crested populations to differ at more loci than the first crested population and the non-crested population.

English trumpeter(Creative Commons - Author: J.Gifford)

English trumpeter (Creative Commons – Author: J.Gifford)

Contrast the Red Jacobin and the English Trumpeter (pictured); Ephrin receptor B2 gene causes the presence of both birds’ crest but the differences are caused by the effects of other genes along the pathway. For one single base change, though, to be the trigger for such an apparent trait really does show how tiny genetic changes can lead to phenotypic differences; differences which could easily impact upon fitness. Such an example shows how micro-evolution could easily generate macro-evolutionary changes; changes that could lead to speciation events. It may even be possible for a single base to make all the difference – it does here.

 

References

Shapiro, M.D. et al (2013) Genomic diversity and evolution of the head crest in the rock pigeon. Science 339: 1063-1067

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“The single greatest experiment in the history of biology”

“The single greatest experiment in the history of biology.”  Quite an accolade.  Yet this is how Richard Lenski (no stranger to seminal investigations himself) described work carried out in 1943 by Salvador Luria and Max Delbrück.

Picture yourself in 1943.  Evolution has now been established as the explanation for the seemingly-designed nature of life, but many questions still remain.  One of the greatest in both its simplicity and importance is this: does genetic variation occur because of the action of selection, or is it present before selection acts?  This is so fundamental to our understanding of evolution as to seem obvious; however, before 1943 the issue was still very much up in the air.

The structure of DNA was not to be discovered for another 10 years, somewhat precluding the analysis of protein structure or DNA sequencing.  Luria and Delbrück therefore used a far more elegant method to infer whether bacteria had mutated before or after selection. They first calculated the expected theoretical variability in growth when multiple colonies were exposed to viruses under either hypothesis (mutation-endowed resistance or acquired resistance).  They then experimentally measured growth of multiple cultures of E. coli when exposed to viral challenge, and compared their experimental findings to the theoretical expectations under each hypothesis.  If the experimental measures matched the expectations of one of these hypotheses, the question would be answered.

To carry out their experiment they established hundreds of cultures from a single bacterial cell each, and grew each culture up for a set time.  They then assessed their viral resistance by plating them onto virally-infected agar, and observing how many colonies formed (and therefore how many bacteria from the original culture were resistant).

Their theoretical calculations threw up some interesting findings (trust me here).  They modelled the frequency distribution (where a category is plotted on the x axis, and the frequency of individuals in that category is on the y) of the number of resistant bacteria in multiple individual cultures expected under each hypothesis.  They found that under the mutation hypothesis, there will be a high variance between different cultures, with some cultures with high numbers of resistant bacteria, causing this distribution to have a long tail at the right hand end.  Both of these expectations stem from the many generations in which resistant mutants can arise: if a mutant arises during the first bacterial generation, half of the bacteria in that culture will be resistant to viral infection; if a mutant arises during the last generation, there will be only one resistant bacterium.  In contrast, under the acquired hypothesis, each bacterium has the same probability of becoming resistant to the virus upon being exposed to it, and each becomes immune or not at the same moment (when they are plated onto the viral agar); therefore the frequency distribution under acquired immunity will have very short tails, and low variance between cultures.

So they knew their expectations.  All that remained now was to expose E. coli cultures to viruses, plot the frequency distribution of resistant bacteria, and measure the variance between cultures.

Their findings emphatically supported the mutation hypothesis.  The frequency distributions of the different cultures formed a curve with a long right-hand tail, and revealed a large number of cultures with over nine resistant bacteria, as predicted by a resistant mutant arising early in the bacterial pedigree of a culture.  The variance between different cultures was also massively higher than their theoretical predictions under the acquired immunity hypothesis, and even higher than they predicted under the mutation hypothesis.

These results are simply inexplicable under the acquired immune hypothesis; the probability of a culture having over nine resistant bacteria is astronomically low, and the repeated observation of such cultures is nigh-on impossible.  However, they are perfectly explained by the mutation hypothesis.

Luria and Delbrück’s work also helped to settle a more philosophical question: is evolution a guided process?  By demonstrating that evolutionary change depends upon mutations that are randomly generated, they banished any element of guidance or divine, benevolent intervention from the evolutionary process.  When selection pressure is applied, time is up: the genetic variation has to be there already.

 

 

References

Luria, S.E. and Delbrück, M. (1943) Mutations of bacteria from virus sensitivity to virus resistance.  Genetics 28: 491-511

Lenski, R.E. (2011) Evolution in action: a 50,000-generation salute to Charles Darwin.  Microbe 6:30-33

The Salvador E. Luria Papers.  National Library of Medicine: Profiles in Science, accessed from

http://profiles.nlm.nih.gov/ps/retrieve/Collection/CID/QL

Luria-delbruck diagram.svg, Wikipedia, accessed from

http://en.wikipedia.org/wiki/File:Luria-delbruck_diagram.svg

Hurray for the parasite!

Could you ever feel sorry for a parasite?  Doubtful as it seems, a recent paper exploring the evolutionary conflict between the parasitoid wasp Leptopilina boulardi and the LbFV virus has left me not only supporting the parasite, but cheering at recent evidence that it seems to be putting up a good fight.

The lifecycle of L. boulardi seems particularly nasty: it lays a single egg in the larvae of Drosophila (fruit flies), which then hatches, consumes the larva from the inside, and eventually emerges from the now (thankfully) deceased host to repeat the cycle.  However, this wasp can itself become prey to a parasite: the imaginatively named Leptopilina boulardi filamentous virus (LbFV).  Ordinarily, an L. boulardi wasp would avoid larvae which have already been parasitized, to prevent her offspring having to compete with others.  However, wasps infected with LbFV will not discriminate between parasitized and non-parasitized larvae, and may lay eggs in a larva already hosting an egg (termed “superparasitism”).  This egg laying behaviour is a perfect example of an extended phenotype: “all effects of a gene upon the world” according to the idea’s originator Richard Dawkins (2008), but usually used in reference to those effects beyond the organism in whose genome the gene resides.

Superparasitism is great from the virus’s point of view: it gets an opportunity to infect the other eggs in the larva, as well as its usual route of infecting the offspring of the current host.  However, it is disadvantageous to the wasp, as it reduces the amount of food available to its offspring through competition with the other eggs.  Any wasp able to reduce or avoid superparasitism will therefore leave more offspring, and if this variation in superparasitism-avoidance is heritable, wasps will evolve resistance to superparasitism.

Researchers at the University of Lyon investigated this evolution in 30 wasp lines from five wild populations (with each population differing in its level of viral prevalence), aiming to answer three questions:

1. Is variation in superparasitism explained by the presence or absence of the virus?

2. Do viruses vary in the superparasitism phenotype they induce?

3. Does this variation correlate with variation in the viral titre (the amount of virus) in a wasp host?

They found that infection status of the wasp explains 77% of variation in superparasitism (measured as the number of eggs per parasitized larva): uninfected wasps had lower levels of superparasitism than infected wasps.  Thus, question number 1 was answered: a large part of the variation in superparasitism can be explained by viral presence.

The authors’ second finding was that lines differed in their degree of superparasitism – two (Sf12 and Av3) laid 8-10 eggs per larva and were therefore highly susceptible to viral manipulation; and two were less susceptible (Av8 and Go16), laying roughly half as many eggs per larva as the highly susceptible lines.  They also found a loose but significant correlation between the extent of superparasitism in one generation and the next.  From these results they concluded that not only is there variation in susceptibility to viral manipulation (leading to variation in the level of superparasitism), but that this variation is heritable, and therefore likely to have a genetic basis.

And finally, they found no difference between the viral titre of wasps with high and low levels of superparasitism.  The authors suggest that this may be due to a “tolerance mechanism”, giving a tantalizing hint as to the possible identity of this genetic difference in susceptibility.  One allele (form) of the same antiviral gene could endow some lines with high tolerance and hence low levels of superparasitism, whereas another allele produces lower tolerance and more superparasitism.  However, in the words of the authors, “we must be cautious about this conclusion, because only four lines were tested, which reduces the statistical power of the viral titre analysis.”

Puzzlingly, the authors found no geographical differentiation in the amount of viral resistance in wasp populations, even though the south’s higher viral prevalence leads to the expectation of higher resistance in the southern wasps.  The authors give three possible explanations: recent invasion of the virus into the wasp host; a high migration rate of wasps between north and south; and a high cost of tolerance.  While this last hypothesis is supported by their data (they found lower egg load in individuals of the Av12 line, which had higher tolerance), the authors admit that “this correlation needs to be tested over a wider range of parasitoid and virus genotypes.”

This paper also highlights the element of serendipity that seems to feature in some research: a “severe dysfunction” of their incubators caused the loss of many of their lines halfway through the investigation, meaning they had to reconstruct some lines.  However, this eventually supported their original claim that variation in the wasp genome leads to variation in level of superparasitism: their reconstructed “less susceptible” line displayed equally low levels of superparasitism as the original, strengthening the case for heritable variation in the susceptibility of wasps to the virus.

So in the end I was cheering for the wasp, which seems to be fighting back against this insidious (if fascinating) virus.  However, I was also cheering for the authors: they presented what could have been sensationalised data in a moderate and responsible manner, as well as pointing out the flaws in their method and the way that these could actually further inform their conclusions.

 

 

References

Dawkins, R. (2008) The Extended Phenotype.  pp.  264.  Oxford University Press

Martinez, J., Fleury, F. and Varaldi, J. (2011) Heritable variation in an extended phenotype: the case of a parasitoid manipulated by a virus.  Journal of Evolutionary Biology, Early View

Ignorance of evidence doesn’t make you scientific either

About a month ago a recent anti-evolutionary post caught my eye.  It neatly demonstrates two major misconceptions about evolutionary biology, and gives an ideal opportunity to talk about some powerful proofs of evolution provided by molecular genetics.

The basic thrust seems to be that Rick Perry is by far the superior candidate for the GOP Presidential nomination, due to his “acknowledgement of the holes in evolution theory”.  However, Mr Casey also takes the opportunity to air his own feelings about “why evolution will never be considered a fact”.

Mr Casey makes two staggeringly erroneous points in this piece: that evolution cannot be considered a fact because it cannot be observed (the moral of his match-and-candle parable); and that “not even Richard Dawkins, a leading evolutionary biologist from Oxford University, could name a single mutation that has added beneficial information.”

Just one of many examples of observable evolution is provided by the Long Term Evolutionary Experiment (LTEE) by the Lenski lab, an investigation into the evolution of 12 separate colonies of the bacterium Escherichia coli over 45,000 generations.  Usually each colony would grow, use up the glucose in the growth medium, and begin to die out; however, one colony of bacteria evolved the ability to survive on citrate (also present in the medium), and could therefore grow more.  The genetic basis of this ability has been tracked to two mutational events, one for each step in breaking down citrate, each adding beneficial information.

The globin gene family provides an even more detailed example of a suite of abilities provided by genetic mutation.  The original gene duplicated approximately 1.1 billion years ago, producing two copies.  One copy became the ancestor of all vertebrate haemoglobins, which bind oxygen in red blood cells, providing oxygen to the vast majority of cells in the body.  The other became the ancestor of all vertebrate myoglobins, which bind and store oxygen in muscle cells, allowing aquatic mammals to dive for extended periods (20 minutes in the case of Weddell seal).  We know the exact DNA sequence of these genes, and how mutation has added beneficial information (for more information, see sequencing studies here, here and here, as well as this previous BAB post).  Professor Dawkins is presumably aware of the globins, as he detailed their evolution in his book The Blind Watchmaker (p. 175 of the 2006 edition).

Skepticism is healthy in science, but rejecting an explanation when you lack a basic understanding of its proofs is not skeptical, but pure ignorance.

References

Blount, Z.D., Borland, C.Z. and Lenski, R.E. (2008) Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli.  PNAS 105: 7899-7906

Campbell, N.A. and Reece, J.B. (2005) Biology (Seventh Edition). Pearson Benjamin Cummings

Dawkins, R. (2006) The Blind Watchmaker, p.175.  Penguin

Goodman, M., Pedwaydon, J., Czelusniak, J., Suzuki, T., Gotoh, T., Moens, L., Shishikura, F., Walz, D. and Vinogradov, S. (1988) An evolutionary tree for invertebrate globin sequences.  Journal of Molecular Evolution 27: 236-249

Hardison, R. (1998) Hemoglobins from bacteria to man: evolution of different patterns of gene expression.  Journal of Experimental Biology 201: 1099-1117

Hoffman, F.G., Opazo, J.C. and Storz, J.F. (2011) Whole-genome duplications spurred the functional diversification of the globin gene superfamily in vertebrates.  Molecular Biology and Evolution, Advance Access