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What Is the Red Queen Hypothesis?

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• Natural Selection
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Evolution is the changing in species over time. However, with the way ecosystems work on Earth, many species have a close and important relationship with each other to ensure their survival. These symbiotic relationships, such as the predator-prey relationship, keep the biosphere running correctly and keep species from going extinct. This means as one species evolves, it will affect the other species in some way. This coevolution of the species is like an evolutionary arms race that insists that the other species in the relationship must also evolve to survive.

The “Red Queen” hypothesis in evolution is related to the coevolution of species. It states that species must continuously adapt and evolve to pass on genes to the next generation and also to keep from going extinct when other species within a symbiotic relationship are evolving. First proposed in 1973 by Leigh Van Valen, this part of the hypothesis is especially important in a predator-prey relationship or a parasitic relationship.

Predator and Prey

Food sources are arguably one of the most important types of relationships in regards to survival of a species. For instance, if a prey species evolves to become faster over a period of time, the predator needs to adapt and evolve to keep using the prey as a reliable food source. Otherwise, the now faster prey will escape, and the predator will lose a food source and potentially go extinct. However, if the predator becomes faster itself, or evolves in another way like becoming stealthier or a better hunter, then the relationship can continue, and the predators will survive. According to the Red Queen hypothesis, this back and forth coevolution of the species is a constant change with smaller adaptations accumulating over long periods of time.

Sexual Selection

Another part of the Red Queen hypothesis has to do with sexual selection. It relates to the first part of the hypothesis as a mechanism to speed up evolution with the desirable traits. Species that are capable of choosing a mate rather than undergoing asexual reproduction or not having the ability to select a partner can identify characteristics in that partner that are desirable and will produce the more fit offspring for the environment. Hopefully, this mixing of desirable traits will lead to the offspring being chosen through natural selection and the species will continue. This is a particularly helpful mechanism for one species in a symbiotic relationship if the other species cannot undergo sexual selection.

Host and Parasite

An example of this type of interaction would be a host and parasite relationship. Individuals wanting to mate in an area with an abundance of parasitic relationships may be on the lookout for a mate that seems to be immune to the parasite. Since most parasites are asexual or not able to undergo sexual selection, then the species that can choose an immune mate has an evolutionary advantage. The goal would be to produce offspring that have the trait that makes them immune to the parasite. This would make the offspring more fit for the environment and more likely to live long enough to reproduce themselves and pass down the genes.

This hypothesis does not mean that the parasite in this example would not be able to coevolve. There are more ways to accumulate adaptations than just sexual selection of partners. DNA mutations can also produce a change in the gene pool only by chance. All organisms regardless of their reproduction style can have mutations happen at any time. This allows all species, even parasites, to coevolve as the other species in their symbiotic relationships also evolve.

• What Is the Evolutionary Arms Race?
• What Is Coevolution? Definition and Examples
• Symbiogenesis
• External Hierarchy of Life
• An Introduction to Evolution
• Parasitism: Definition and Examples
• Mutualism: Symbiotic Relationships
• Descent with Modification
• 4 Types of Reproduction
• Only Populations Can Evolve
• What Happens When Viruses Evolve?
• What Is Mass Extinction?
• The Evolution of Eye Color
• Commensalism Definition, Examples, and Relationships
• 5 Types of Asexual Reproduction
• Introduction to Evolutionary Psychology

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41 Testing the Red Queen Hypothesis

The Red Queen hypothesis—that sex evolved to combat our coevolving pathogens—can be tested by analyzing a few key predictions of this hypothesis:

• Sex is most beneficial where there is a high risk of infection
• Pathogens are more likely to attack common phenotypes (for example, clones) in a population, as opposed to the less-common counterparts (such as those that resulted from sex)
• In sexually reproducing populations, individuals choose mates that maximize diversity in their offspring

Note that all of these predictions implicitly rely on the heritability of being healthy (in this case, the ability to combat pathogens); specifically, parents must be able to pass along to their offspring genes for avoiding pathogens. Testing these predictions has resulted in several lines of evidence supporting the Red Queen hypothesis.

Prediction 1: Sex is most beneficial where there is a high risk of infection

An excellent system for testing this prediction involves a flatworm parasite in the genus Microphallus, a duck, and a small mud snail ( Potamopyrgus antipodarum ; Figure 7.6). This species of snail is able to reproduce sexually or asexually. The extent of sexual reproduction in a population of snails can be quantified by counting the number of males—asexual snails are all female.

The flatworm’s life cycle begins inside of the snail, where the worm emerges from its egg. Infected snails are consumed by ducks. Once in the duck’s intestine, adult worms have sex and produce eggs. Flatworm eggs are released, with duck feces, into the water, where they are ingested by snails and the cycle continues (Figure 2). Snails are harmed by this flatworm, largely because a symptom of infection is sterilization (the flatworm’s scientific name, Microphallus , translates to “small penis”).

Observations of this system in two New Zealand lakes (Alexandrina and Kaniere) revealed that snails are more likely to be sexual (measured by frequency of males) in shallow waters, where ducks feed, than in deeper waters, where ducks do not feed (Figure 3).

These results suggest that coevolutionary pressure is greater on the snails in the shallows, presumably because the feeding ducks effectively “close the circle” on the worm’s life cycle. Finally, higher infection rates in the shallows indicate that, in support of Prediction 1, above, sex is most beneficial where there is a high risk of infection.

Prediction 2:  Pathogens are more likely to attack common phenotypes in a population, as opposed to the less-common counterparts

In the Mexican desert there are isolated pools inhabited by a species of minnow.  Within these pools, populations of asexually reproducing individuals exist alongside sexually reproducing individuals.  Fish in these ponds exhibit “black spot disease,” which is caused by a parasitic flatworm.  Investigators have observed the frequency of sexual and asexual fish and the number of black spots in each type of fish in these ponds.  Clonal fish are likely to have the most common phenotype in these ponds (as they are genetically identical to each other), while the sexually reproducing fish will have a wide variety of infrequent phenotypes.  As the Red Queen predicts, the common type of fish (usually one of the clonal species) had the highest number of parasitic spots.  In ponds where there was a genetically diverse, sexually reproducing population, the sexual fish had fewer spots.

Additional evidence comes from the evening primrose (Figure 4), a flowering plant that–like the minnows, snails, and water fleas discussed above–exists in sexual and asexual forms.  Evening primrose can be damaged by mildew from a pathogenic fungus. The plants produce an enzyme protein called chitinase to defend themselves against this fungus. A recent comparison indicated that the sexually reproducing primrose plants had greater variety in the gene that codes for chitinase than did the asexual plants. In addition, the overall amount of chitinase expressed was higher in the sexual plants than in the asexuals. Finally, the researchers found that the plants that were more resistant to mildew damage had higher fitness (they produced more fruit, and thus more offspring) in the presence of that pathogen.  In evening primrose, greater diversity in a key gene renders an individual less susceptible to a pathogen, supporting the prediction that parasites are more likely to attack the most common phenotype in a population, and providing additional evidence for The Red Queen.

Know Your Pathogens

A pathogen is something that infects and causes a fitness cost in another organism.  Pathogens come in a wide variety; some of them are not even considered living!

Prions – Prions are non-living infectious agents that are misfolded proteins.

Viruses – Whether you consider viruses alive or not depends on your definition of life.  Viruses are protein-encased DNA or RNA entities that hijack a cell’s replication machinery to reproduce.  Viral infections include influenza, HIV, HPV, and herpes.

Fungal pathogens – Fungi are responsible for a variety of infections including mildew, thrush, athlete’s foot and smut.

Bacteria – Bacteria are prokaryotic organisms that occur everywhere. There are more bacteria in and on you than there are cells in your body. Fortunately, the vast majority of bacteria are benign. However, some bacteria cause problems such as urinary-tract infections, some kinds of pneumonia, ear infections, pertussis (whooping cough), chlamydia, gonorrhea, and syphilis.

Protists – Protists are single-celled eukaryotes that cause diseases such as malaria and amoebic dysentery.

Animals –Common animal pathogens include lice, many types of worms, and parasitic wasps.

Prediction 3: In sexually reproducing populations, individuals choose mates that maximize diversity in their offspring

If there is a fitness advantage to diversity, parents can best maximize their offspring’s potential (and have more grand-offspring) with careful mate choice. There are numerous examples of organisms preferring mates that increase offspring diversity, and shunning mates that might do the opposite. Even many hermaphrodites, with both male and female sex organs, seek other hermaphrodites for copulation…even if they are capable of self-fertilization.

An excellent model for studying mate choice is Atlantic Salmon, an important commercial fish that lives its life in the ocean and returns to freshwaters to mate (or spawn ). Sofia Consuegra and Carlos Garcia de Leaniz compared offspring diversity of salmon that were mated in a commercial fish hatchery (and unable to choose their mates) against that of salmon allowed to choose mates in the wild. The hatchery-spawned fish exhibited lower diversity than did the wild-spawned fish. Furthermore, hatchery-spawned fish displayed a greater number of roundworm parasites ( Anisakis ) then did their wild-spawned counterparts (Figure 5).  These results support the prediction that individuals will choose mates that maximize diversity in their offspring. Also, this work adds fuel to The Red Queen hypothesis by illustrating a potential benefit to Atlantic Salmon–namely, parasite avoidance.

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Red Queen hypothesis

The idea that, in order for a species to maintain a particular niche in an ecosystem and its fitness relative to other species, that species must be constantly undergoing adaptive evolution because the organisms with which it is  coevolving  are themselves undergoing adaptive evolution. When species evolve in accordance with the Red Queen hypothesis, it often results in an evolutionary  arms race .

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This year's Darwin Review: Revisiting the Red Queen

The red queen hypothesis states that in the battle for resources, species must continuously evolve just to keep up with their enemies, who themselves also evolve in response. this year, we revisit the seminal theory..

Proceedings B Darwin Reviews are special reviews published up to once a year. The aim of the Darwin Review is to showcase ideas and/or a field in biological science that is of very high interest to the whole diverse readership of Proceedings B, often being of particular relevance to strategic growth areas, importance to policy makers and/or having a bearing on the public that fund our science.

This year our Darwin review revisits a seminal theory in evolutionary research, Van Vaalen’s Red Queen Hypothesis. 40 years after its initial proposal the Red Queen is still informing research. Here the authors discuss their review and why now was the right time to highlight the Red Queen’s enduring legacy. You can read the full article here . Our previous Darwin review was on evolutionary medicine and can be found here .

Tell us about the Red Queen hypothesis.

The Red Queen hypothesis was proposed over 40 years ago by the late evolutionary biologist Leigh Van Valen. It advanced evolutionary thinking beyond the idea that organisms were merely matched to their physical environment by suggesting that interactions between species (such as between hosts and parasites, predators and prey) would also be important in driving evolutionary change. In essence, the Red Queen hypothesis states that in the battle for resources, species must continuously evolve just to keep up with their enemies, who themselves also evolve in response. The result is that species constantly change but, relative to their enemies, don’t actually get any fitter – like running on an evolutionary treadmill. The name for the theory came from Lewis Carroll’s ‘Through the Looking Glass’ (aka Alice in Wonderland). Alice finds herself in a race with the Red Queen, and despite running as fast as she can, Alice stays in the same place. The Red Queen hypothesis, doubtless partly due to this imaginative metaphor, has become one of the most influential ideas in evolution.

How has the theory influenced evolutionary biology research since its original proposal?

Van Valen was interested in macroevolution, that is speciation and extinction, and used the Red Queen hypothesis to explain the apparently constant rates of extinction observed in the fossil record. And although this pattern has subsequently been challenged, Red Queen thinking is still important in macroevolution in terms of understanding the biological, as opposed to environmental, causes of diversification and extinction. In the 1980s, the utility of the Red Queen concept was rediscovered as a way of explaining the ubiquity of sexual reproduction in nature. The idea starts by considering that hosts and parasites are evolving together. If the hosts are sexual females, they can produce offspring that are genetically diverse and therefore avoid parasite infection. However, if hosts are clonal females, their offspring lack the ability to be as genetically diverse and will be more susceptible to attack by parasites. Thus, coevolution with parasites prevents the clones from taking over and gives sexual species an advantage. Increasingly, biologists think of the Red Queen hypothesis, not only as an explanation for sex, but also as a means of explaining rapid evolutionary change in hosts and their parasites in general.

What prompted you to write this review?

The adoption of the Red Queen as an explanation for the evolution and widespread maintenance of sex led to a narrowing of the definition of the Red Queen hypothesis. This meant that the far richer perspective of Van Valen’s original conceptual leap was in danger of being lost. To some extent, we wrote the review because we believed it was necessary to reemphasize a broader importance of the Red Queen hypothesis because it offers a powerful way in which to understand natural communities, how they evolve, and how they work. It is an important and useful idea. We were also inspired by the data emerging from new approaches of studying species interactions, including genome sequencing and experimental evolution in the laboratory. These present a more complex and nuanced picture of the effect of species interactions on evolution that is entirely consistent with a Red Queen view of nature. Additionally, we were aware that much of theory developed to understand how conflicts between species evolve could be usefully applied to understand conflicts within species. A variety of conflicts have been defined in this context: intragenomic conflicts (e.g. over the representation of chromosomes in gametes during meiosis), sexual conflicts (e.g. between males and females over reproductive effort and timing) and parent / offspring conflicts (e.g. over the allocation of resources to progeny). However, the evolutionary dynamics of these conflicts has never formally been synthesized within the Red Queen concept, despite the clear ability for these conflicts to generate the type of ‘running to stay still’ evolutionary dynamics. One of our aims in this review was therefore also to attempt this synthesis.

What does the future hold the red queen hypothesis?

It will certainly be informative to see how useful and explanatory is the incorporation of conflicts within species into the Red Queen framework. Beyond this, we recognise that much of our appreciation of the Red Queen currently comes from studying binary relationships (one parasite in one host). Extensions of this to more realistic scenarios, by incorporating parasites that evolve with a range of hosts, and hosts that evolve with a range of parasites, are therefore needed. How these will affect the speed of the Red Queen needs to be determined. Finally, there has been increasing use of comparative genomics to examine evolutionary dynamics. However, these analyses are limited in that the function of many genes is not known, making it difficult to determine the contribution of the Red Queen. Commonly, for instance, expression patterns indicate that specific genes are involved in male-female interactions, and could therefore be subject to coevolution. However, at the moment, we cannot differentiate the subset of genes that are involved with the male-female interface that are likely to be subject to Red Queen forces from those involved in a variety of other sex-specific physiological functions. We also have incomplete ascertainment of genes at the host-pathogen boundary: we understand host genes that are generically involved in defence much better than those which alter microbial invasion into a cell. We expect the results of comparative genomic analysis of Red Queen type processes to be made sharper as our understanding of gene function improves.

Meet the authors

Michael Brockhurst is Professor of Evolutionary Biology and a 50th Anniversary Chair at the University of York. He uses experimental evolution approaches to study the coevolution of species interactions with a particular focus on bacteria and their viral and genetic parasites. He is interested in the applied consequences of rapid microbial evolution in natural communities especially in clinically important pathogenic microbes.

Greg Hurst is Professor of Evolutionary Biology at the University of Liverpool. His main goal is to determine the ecological and evolutionary importance of heritable symbionts of insects (where symbiont is broadly defined and includes parasitism), and has an interest intragenomic and intra-specific conflicts more generally. His study animals include Drosophila, Nasonia wasps, butterflies and ladybirds, and the symbionts studied are largely microbial.

Kayla King is an Associate Professor at the University of Oxford. Her research explores the ecology and evolution of species interactions to ask fundamental questions about the maintenance of genetic and community-level diversity, sexual reproduction, and rapid evolutionary change. She focuses on interactions between hosts and their parasites as well as their microbiota.

Judith Mank is Professor of Evolutionary and Comparative Biology at University College London. She is interested in the constraints imposed on the genome by sexual conflict, and how selection navigates these restrictions of genome architecture to create intra-specific phenotypic diversity in the form of sexual dimorphism. She works on a range of study organisms, most recently birds, fish and flies.

Steve Paterson is Professor of Evolutionary Biology and a director of The Centre for Genomic Research at the University of Liverpool. He is primarily interested in understanding the forces that shape genetic diversity within host and parasite genomes. New genomic methods that allow us to address this question make it clear how much we owe to the Red Queen for our understanding of genome evolution and of the novel biology yet to be discovered from biotic interactions.

Tracey Chapman is Professor of Evolutionary Biology in the School of Biological Sciences at the University of East Anglia. She is interested in understanding the nature and the evolutionary potential of reproductive interactions between males and females. Such interactions are often subject to sexual conflict and can generate rapid evolutionary change.

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• Published: 09 December 2009

Phylogenies reveal new interpretation of speciation and the Red Queen

• Chris Venditti 1 ,
• Andrew Meade 1 &
• Mark Pagel 1 , 2  

Nature volume  463 ,  pages 349–352 ( 2010 ) Cite this article

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The Red Queen 1 describes a view of nature in which species continually evolve but do not become better adapted. It is one of the more distinctive metaphors of evolutionary biology, but no test of its claim that speciation occurs at a constant rate 2 has ever been made against competing models that can predict virtually identical outcomes, nor has any mechanism been proposed that could cause the constant-rate phenomenon. Here we use 101 phylogenies of animal, plant and fungal taxa to test the constant-rate claim against four competing models. Phylogenetic branch lengths record the amount of time or evolutionary change between successive events of speciation. The models predict the distribution of these lengths by specifying how factors combine to bring about speciation, or by describing how rates of speciation vary throughout a tree. We find that the hypotheses that speciation follows the accumulation of many small events that act either multiplicatively or additively found support in 8% and none of the trees, respectively. A further 8% of trees hinted that the probability of speciation changes according to the amount of divergence from the ancestral species, and 6% suggested speciation rates vary among taxa. By comparison, 78% of the trees fit the simplest model in which new species emerge from single events, each rare but individually sufficient to cause speciation. This model predicts a constant rate of speciation, and provides a new interpretation of the Red Queen: the metaphor of species losing a race against a deteriorating environment is replaced by a view linking speciation to rare stochastic events that cause reproductive isolation. Attempts to understand species-radiations 3 or why some groups have more or fewer species should look to the size of the catalogue of potential causes of speciation shared by a group of closely related organisms rather than to how those causes combine.

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Acknowledgements

We thank the Centre for Advanced Computing and Emerging Technologies (ACET) at the University of Reading for the use of the ThamesBlue supercomputer. We thank M. Turelli for calling our attention to Gillespie’s Poisson process model and for comments on earlier drafts of the paper. M. Steel pointed out the geometric distribution proof given in the Supplementary Information . This research was supported by grants to M.P. from the Natural Environment Research Council (NERC), UK, and the Leverhulme Trust.

Author Contributions C.V., A.M. and M.P. contributed to all aspects of this work.

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Venditti, C., Meade, A. & Pagel, M. Phylogenies reveal new interpretation of speciation and the Red Queen. Nature 463 , 349–352 (2010). https://doi.org/10.1038/nature08630

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7.4 The Red Queen

What does lewis carroll’s red queen have to do with sex [1].

In this scene from Through the Looking-Glass and What Alice Found There by Lewis Carroll, Alice and the Queen run with all their effort – yet make no progress.  Such is the claim that sex allows organisms to avoid extinction by keeping up in a very odd sort of race.

Alice never could quite make it out, in thinking it over afterwards, how it was that they began: all she remembers is that they were running hand in hand, and the Queen went so fast that it was all she could do to keep up with her: and still the Queen kept crying “Faster! Faster!”, but Alice felt she could not go faster, though she had not breath left to say so. 

The most curious part of the thing was, that the trees and the other things round them never changed their places at all: however fast they went, they never changed their places at all: however fast they went they never seemed to pass anything.  “I wonder if all the things move along with us?” thought poor puzzled Alice.  And the Queen seemed to guess her thoughts, for she cried “Faster! Don’t try to talk!”

Not that Alice had any idea of doing that.  She felt as if she would never be able to talk again, she was getting so much out of breath: and still the Queen cried “Faster! Faster!”, and dragged her along.  “Are we nearly there?” Alice managed to pant out at last.

“Nearly there!” the Queen repeated.  “Why, we passed it ten minutes ago! Faster!” And they ran on for a time in silence, with the wind whistling in Alice’s ears, and almost blowing her hair off her head, she fancied

“Now! Now!” cried the Queen. “Faster! Faster!” And they went so fast that at last they seemed to skim through the air, hardly touching the ground with their feet, till suddenly, just as Alice was getting quite exhausted, they stopped, and she found herself sitting on the ground, breathless and giddy. 

The Queen propped her up against a tree, and said kindly, “You may rest a little, now.”

Alice looked round her in great surprise.  “Why, I do believe we’ve been under this tree the whole time!  Everything’s just as it was!”

“Of course it is, “ said the Queen.  “What would you have it?”

“Well, in our country, “ said Alice, still panting a little, “you’d generally get to somewhere else – if you ran very fast for a long time as we’ve been doing.”

“A slow sort of country!” said the Queen.  “Now here, you see, it takes all the running you can do, to keep in the same place.  If you want to get somewhere else, you must run at least twice as fast as that!”

“I’d rather not try, please!” said Alice….

      Through the Looking-Glass and What Alice Found There by Lewis Carroll

Is sex part of an arms race?

In one human generation, HIV (the virus that causes AIDS) will reproduce over a million times. Given how natural selection works—via heritable variation and differential reproduction—human beings don’t stand a chance against this virus. How can we possibly adapt to such a fast-moving target? For that matter, how can any longer-lived organism compete with a quickly reproducing and quickly evolving enemy? Many of these enemies, or pathogens , such as viruses and bacteria, are also numerous and difficult to detect—invisible to the naked eye, they can enter a host’s body silently and reproduce with a fervor while their victims remain blissfully unaware. Given these challenges, how can any host organism defend itself against its would-be attackers? According to one hypothesis, outwitting pathogens is the whole point of sex.

The Red Queen

We are in the midst of an evolutionary arms race , in which host and parasitic pathogen must constantly adapt. Parasites must adapt to the host’s natural defenses, and host populations are under pressure to keep up with their ever-changing parasites.  This reciprocal evolution between two types of organisms (in this case, host and parasite) is a type of coevolution. According to the Red Queen Hypothesis , sex exists as a mechanism for keeping up with rapidly coevolving pathogens. By generating genetic diversity, sex makes host organisms a moving target.  Like Alice and the Red Queen in Lewis Carroll’s novel (Box 3), both host and parasite are running a race in which neither makes any observable progress. Yet, if the host organisms didn’t change dramatically with each new generation (if they didn’t have sex), they might go extinct.

Parasites adapt to exploit the most common type of host.  Therefore, a host that can produce offspring that have novel defenses against parasites would have an advantage over an organism producing clones–simply by making offspring that are different.

• Public Domain, https://commons.wikimedia.org/w/index.php?curid=2040219 ↵

The Evolution and Biology of Sex Copyright © 2020 by Sehoya Cotner and Deena Wassenberg is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License , except where otherwise noted.

Getting somewhere with the Red Queen: chasing a biologically modern definition of the hypothesis

Affiliations.

• 1 Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045, USA [email protected].
• 2 Biodiversity Institute, University of Kansas, Lawrence, KS 66045, USA.
• 3 Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045, USA.
• PMID: 29720444
• PMCID: PMC6012711
• DOI: 10.1098/rsbl.2017.0734

The Red Queen hypothesis (RQH) is both familiar and murky, with a scope and range that has broadened beyond its original focus. Although originally developed in the palaeontological arena, it now encompasses many evolutionary theories that champion biotic interactions as significant mechanisms for evolutionary change. As such it de-emphasizes the important role of abiotic drivers in evolution, even though such a role is frequently posited to be pivotal. Concomitant with this shift in focus, several studies challenged the validity of the RQH and downplayed its propriety. Herein, we examine in detail the assumptions that underpin the RQH in the hopes of furthering conceptual understanding and promoting appropriate application of the hypothesis. We identify issues and inconsistencies with the assumptions of the RQH, and propose a redefinition where the Red Queen's reign is restricted to certain types of biotic interactions and evolutionary patterns occurring at the population level.

Keywords: Red Queen hypothesis; competition; environment; extinction; macroevolution.

© 2018 The Author(s).

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• Research Support, U.S. Gov't, Non-P.H.S.
• Biological Evolution*
• Competitive Behavior
• Extinction, Biological*
• Models, Biological
• Paleontology
• Selection, Genetic

Red Queen Hypothesis, The

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• Nicholas Primavera 3  

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Red Queen’s Race ; The Red Queen Effect

The Red Queen hypothesis is an evolutionary hypothesis that states that all living beings must constantly adjust, evolve, and reproduce while attempting to survive ever-evolving predators.

Introduction

This hypothesis was first proposed by Leigh Van Valen in 1973. The term “Red Queen” is a reference to a statement made by the Red Queen to Alice, characters in the popular 1871 novel Through the Looking-Glass , written by Lewis Carol.

The Red Queen Hypothesis and it’s Relevance

The statement that sparked this hypothesis is “Now, here , you see, it takes all the running you can do, to keep in the same place” (Carroll 1871 ). Van Valen’s reference is essentially a metaphor for an evolutionary arms race. Predators that undergo a beneficial adaption may spark a change in selection pressure when it comes to a group of prey. This, in turn, would continue in a positive feedback loop, which gives rise to a form of antagonistic coevolution....

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Carroll, L. (1991[1871]). 2: The garden of live flowers. In Through the looking-glass (The millennium fulcrum Edition 1.7 ed.).

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Gat, A. (2009). So why do people fight? Evolutionary theory and the causes of war. European Journal of International Relations, 15 (4), 571–599.

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Vermeij, G. J. (1987). Evolution and escalation. An ecological history of life (pp. 369–370). Princeton: Princeton University Press.

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SUNY New Paltz, New Paltz, NY, USA

Nicholas Primavera

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Correspondence to Nicholas Primavera .

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Department of Psychology, Oakland University, Rochester, MI, USA

Todd K Shackelford

Viviana A Weekes-Shackelford

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University of Idaho, Moscow, ID, USA

Russell Jackson

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Primavera, N. (2021). Red Queen Hypothesis, The. In: Shackelford, T.K., Weekes-Shackelford, V.A. (eds) Encyclopedia of Evolutionary Psychological Science. Springer, Cham. https://doi.org/10.1007/978-3-319-19650-3_2663

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Red Queen revisited: Immune gene diversity and parasite load in the asexual Poecilia formosa versus its sexual host species P . mexicana

Fabian gösser.

1 Department of Animal Ecology, Evolution and Biodiversity, Ruhr-University Bochum, Bochum, Germany

Manfred Schartl

2 Department of Physiological Chemistry I, Wuerzburg University, Wuerzburg, Germany

3 Hagler Institute for Advanced Study and Department of Biology, Texas A&M University, College Station, Texas, United States of America

Francisco J. García-De León

4 Centro de Investigaciones Biológicas del Noroeste, S.C. (CIBNOR, S.C.), Instituto Politécnico Nacional No. 195, Col. Playa Palo de Santa Rita, La Paz, BCS, México

Ralph Tollrian

Kathrin p. lampert, associated data.

All relevant data are within the manuscript.

In accordance with the Red Queen hypothesis, the lower genotypic diversity in clonally reproducing species should make them easier targets for pathogen infection, especially when closely related sexually reproducing species occur in close proximity. We analyzed two populations of clonal P . formosa and their sexual parental species P . mexicana by correlating individual parasite infection with overall and immune genotype. Our study revealed lower levels of overall genotypic diversity and marginally fewer MHC class I alleles in P . formosa individuals compared to sexually reproducing P . mexicana . Parasite load, however, differed only between field sites but not between species. We hypothesize that this might be due to slightly higher genotypic diversity in P . formosa at the innate immune system (toll like receptor 8) which is likely due to the species’ hybrid origin. In consequence, it appears that clonal individuals do not necessarily suffer a disadvantage compared to sexual individuals when fighting parasite infection.

Introduction

Sexual reproduction is omnipresent in the majority of all animal groups [ 1 , 2 ] despite the notion that it includes very high costs [ 3 – 5 ]. This observation led to the assumption that the costs of sexual reproduction should be outweighed by its benefits, namely genetically diverse offspring due to allele recombination and the purging of deleterious mutations (Muller’s ratchet) [ 3 , 6 ]. Nevertheless the evolution and maintenance of sexual reproduction are still major questions in evolutionary biology [ 5 , 7 – 9 ]. One major generally accepted explanation for the maintenance of sexual reproduction is the Red Queen hypothesis [ 1 , 10 ]. It states that recombination results in a fitness advantage in biotic interactions. Recombination leads to fluctuating allele frequencies at loci that determine host fitness against parasite efficacy (Hamilton et al. 1990). Therefore, asexual organisms, particularly the ones co-occurring with closely related sexual species, should be an easier target for parasites and pathogens due to the lower genotypic diversity that compromises pathogen adaptation [ 11 ].

An example for an asexual species living in tight co-occurrence with a closely related species is Poecilia formosa . This fish originated as a hybrid of two sexual species Poecilia latipinna and Poecilia mexicana [ 12 – 14 ]. Even though it reproduced clonally, it still needs males of a closely related species, including the parental species, for reproduction (gynogenesis) [ 15 , 16 ]. Diploid and triploid clonal lineages can be found in the field [ 9 ]. Because of its reproductive mode, P . formosa lives in tight co-occurrence and forms mixed shoals with its host species [ 17 ]. High population densities in these shoals lead to regular infection with a range of parasites and pathogens [ 18 , 19 ]. A primary defense mechanism to resist pathogen infections is the immune system. Basically, pathogens or parasites are recognized, leading to signaling cascades that result in counteraction to infection [ 20 ]. The underlying immune genes play an important role in the response to pathogen infections and resistance to parasites, and consequently have been discussed as a genetic foundation on which selection can act [ 21 ].

Traditionally, innate and adaptive immunity are distinguished. The innate immune system reacts with an immediate non-specific response to pathogen infection [ 22 , 23 ]. In teleost fish the innate immune system is a fundamental defense strategy and a lot of immune defense parameters are more active and have a higher diversity than in mammals [ 24 ]. The signaling-cascades of the innate immune system are triggered by “Pattern Recognition Receptors” (PRRs), which react in binding “Pathogen Associated Molecular Patterns” (PAMPs). These PAMPs include lipopolysaccharide, peptidoglycans, bacterial DNA, viral RNA and other molecules [ 25 ]. Very important PRRs are the Toll-like receptors that recognize diverse PAMPs and can activate diverse signaling-cascades to protect against pathogens and parasites (Akira et al. 2006, Lemaitre et al. 1996). The first piscine TLR gene was reported in Carassius auratus (Stafford et al. 2003), followed by TLR genes in Fugu rubripes [ 26 ] and Danio rerio [ 27 ]. So far 20 TLR types (TLR1, 2, 3, 4, 5M, 5S, 7, 8, 9, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, 26) have been found in a variety of teleost species [ 28 ].

In addition to the innate immune response, the adaptive immune system, can be activated. The recognition of pathogens is improved as the immune system adapts its response during an infection. The response is then memorized even after the elimination of the pathogen or parasite. This immunological memory helps the adaptive immune system to launch a more rapid and intense response the next time this same pathogen or parasite is encountered [ 22 ]. Major histocompatibility complex (MHC) genes play an important role in the recognition of pathogens [ 22 , 29 ]. MHC genes have a great allelic diversity and the genotypic diversity can be extremely high at the population level. This diversity allows for the recognition of a wide range of pathogen derived protein fragments [ 30 ]. The MHC is found in all jawed vertebrates; however, the number of copies for the MHC genes can differ greatly between species [ 31 – 33 ]. MHC allele numbers and infection rate correlate inversely; however, some studies have shown that intermediate numbers of MHC alleles render the most effective protection against pathogen infection [ 34 ].

Immune genes are the most promising candidates to link genotypic diversity and pathogen resistance and therefore to test the Red Queen hypothesis. P . formosa provides excellent preconditions for proving or disproving the Red Queen hypothesis: sexual and clonal species occur in the same environment (mixed shoals) and are very closely related. So far, however, comparisons of clonal versus sexual Poecilia either focused on the analyses of parasite infections [ 18 , 19 ]or the genetic diversity of immune genes [ 9 , 35 ]. A simultaneous analysis of immune genotype and pathogen infection has not been done. Therefore, in this study, we investigated differences in the immune system between P . formosa and its closely related, sexual reproducing species P . mexicana and correlated those differences to parasite load in both species. As an indicator of parasite susceptibility a digenean trematode, Uvulifer sp., was used. All species of Poecilinae are regularly infected by this trematode, which uses water-snails as primary host, fish as a secondary host and piscivorous birds as final host [ 36 ]. Infection can be easily identified by distinct black spots on the skin of the fishes. These black spots develop because of the cercaries of Uvulifer sp., which penetrate the fish skin and provoke the production of a cyst around it. This event is followed by the migration of melanocytes, which lead to the appearance of the black spots. This is why infection with this parasite is also referred to as black spot disease (BSD) [ 36 – 38 ]. An infection with Uvulifer sp. is not deadly for the fishes but it is assumed that it comes with decreased energy levels for the host; also the penetration of the skin causes mechanical damage which is assumed to be costly for the host [ 38 ]. We compared the parasitic load ( Uvulifer sp.) in P . formosa (clonal) and P . mexicana (sexual) from two different locations, the Río Purificación and the Río Guayalejo, and compared the results with the overall genotypic variability as well as the genotypic variability at two different immune gene loci: MHC class I and TLR 8. Following the Red Queen hypothesis, we expected the clonal P . formosa to have lower genotypic diversity and higher parasite loads than the sexual P . mexicana . In addition, we looked for evidence of local adaptation in the immune genes and correlations between overall and immune gene genotypes. We found that while the parasite load differed significantly between field sites, the clonal and sexual species showed similar infection rates. It seems that the hybrid origin of the clonal P . formosa conveys an immune advantage, that at the individual outweighs the disadvantages of clonal reproduction.

Material and methods

Ethics statement.

In the field (Mexico), fish were handled very briefly to check for signs of Uvulifer infection and a small piece of the dorsal fin was cut for genetic analyses. All fish were released immediately after handling. The experiments complied with all laws of the country and were approved by the National Commission of Aquaculture and Fisheries (CONAPESCA) of the Mexican government (permit numbers DGOPA/16986/191205/8101 and DGOPA/02232.230706–1079).

Origin of samples, parasite counts and DNA extraction

Samples of Poecilia formosa and Poecilia mexicana were collected at two field sites in Mexico in 2010; in the Río Purificación (P) (24°04.711'N, 99°07.410'W), and in the Río Guayalejo (G) (23°16.624'N, 98°56.315'W). Seine nets were used to capture individuals. Collected specimens were identified, black spots on the body were counted, and a small piece of the fast-regenerating dorsal fin was clipped from each individual and stored in ethanol (70%) until further analyses in the laboratory. At the Río Purificación site 190 individuals were sampled: 118 P . formosa and 72 P . mexicana . At the Río Guayalejo site 46 P . formosa and 30 P . mexicana were sampled. After parasite count and fin clip, the fishes were immediately released.

Molecular analyses

DNA extraction was carried out using a Chelex protocol [ 39 ]. Overall genotypic diversity of the samples was determined in another (unpublished) study that included ten variable microsatellite loci (Sat1, KonD15, PR39, mATG31, mATG38, mATG44, mATG61, mATG78, mCA16 and mCA20 [ 40 , 41 ]. PCR reactions contained 10 mMTris–HCl (pH 8.85), 50 mMKCl, 0.1%Triton X-100, 1.5 mMMgCl2, 0.2 mMof each dNTP, 10 pmol of each primer and 0.05 U Taq polymerase. Reactions were performed in a total volume of 10μL using the following conditions: 5 min of denaturing at 94°C, 40 cycles of 30s denaturing at 94°C 30s, 30s annealing at 52°C for KonD15, 58°C for Sat1 and 55°C for all other primers and 30s extension at 72°C, followed by a final extension of 5 min at 72°C. PCR product size was analyzed on a Licor 4300 DNA Analyzer (Licor Biosciences, NE, USA).

For the investigation of MHC class I gens and their variability within P . formosa and P . mexicana and between the two species, the exon 2 region of the MHC class I locus was examined. Exon 2 is the antigen-presenting and therefore most variable region of the MHC class I locus [ 42 – 44 ]. For amplification of this region primers Tu1372 (forward) and Tu1373 (reverse) were used [ 9 , 42 , 43 ]. The 5´ end of the forward primer Tu1372 has a GC-clamp [ 45 ], which enhances resolution and prevents a complete splitting up of the PCR product during DGGE. PCR was carried out starting with 5min at 95°C initial denaturation followed by 30 s at 95°C, 30 s at annealing temperature (45°C), 30 s 72°C for 40 cycles followed by 30 minutes final elongation to counter the presence of double bands on the DGGE-gels (see below) [ 46 ]. To test for successful amplification of the desired DNA fragments (approx. 300 bp) a horizontal gel electrophoresis was used with a 1.5% agarose gel concentration [ 47 ]. Samples where then processed using a denaturing gel gradient (DGGE) approach (DGene system, Bio-Rad). Optimal running conditions for the MHC I alleles were: 6.5% polyacrylamid solution (37.5:1 ratio acrylamid/bisacryamid), 30–60% urea gradient, run temperature 60° C, runtime 20 h and a current of 60 V. The gel was loaded with 47 μL PCR product and 8 μL loading buffer (200 μL HPLC-water, 800 μL glycerine (Roth), 0.001g bromphenole Blue-Na-salt (SERVA)). For every sample, the bands that could be observed on the gels were counted. Bands of the same height/migration distance in different samples were interpreted as having the same DNA sequence. Every band observed for an individual was counted as a gene copy and the observed band pattern for that individual was interpreted as its genotype. Samples showing the same distinct combination of bands (= banding pattern) were interpreted as having the same genotype. To adjust for small variances in running length of PCR products between different gels, the same four samples that showed a high variability of bands were chosen as a standard and run on every gel. Samples that could not be scored unambiguously were excluded from further analyses ( Table 1 ).

The Toll-like receptor 8 (TLR 8) shows high levels of variability in the exon 2 region. Thus primers TLR1601 5´-TGACAATGCCTTCCAGGAAC-3´ and TLR1602 5´-ACCTGCTATGTTGGACAACG-3´ that amplify this region were designed using Geneious R6 ( http://www.geneious.com [ 48 ]). A GC-clamp (Sheffield et al. 1989) was attached to the 5´ end of the forward primer (TLR1601). The PCR was carried out starting with 5 min at 95°C initial denaturation followed by 30 s at 95°C, 30 s at annealing temperature (45°C), 30s 72°C for 40 cycles followed by 30 minutes final elongation. To test for successful amplification of the desired DNA fragments (ca. 500bp) a horizontal gel electrophoresis was used with a 1.5% agarose gel concentration [ 47 ]. For screening of TLR 8 diversity a urea gradient of 20–50% was used, while the other DGGE parameters were the same as described above. Four individuals were chosen as standard and run on every gel. As in the MHC analyses bands of the same height were interpreted as the same alleles and identical combinations of bands as the same genotype. Samples that could not be scored unambiguously were excluded from further analyses ( Table 1 ). To validate our DGGE-approach and to know how many different alleles of TLR 8 could be identified, 10 samples, representing nine of the genotypes found, were sequenced, with a prior cloning step (pGem-T Easy Vector System—Promega Corp.) ensuring successful Sanger sequencing of single alleles. All sequences were edited using Geneious R6 [ 48 ]. Sequences were trimmed according to quality, and aligned using the ClustalW-algorithm [ 49 ] implemented in Geneious. The consensus sequences of all samples were then aligned and visually inspected. Obvious sequencing mistakes, gaps or inserts were corrected by hand. An allele was only considered valid if we found it at least three times in the sequencing data. The sequencing results corresponded well with the DGGE banding patterns: All unique sequences resulted in a distinguishable DGGE band and could always be scored correctly.

Statistical analyses

For the analysis of the parasite load the two sample locations as well as both fish species were compared. First, normality distribution of the parasite load data was tested using the Shapiro-Wilk test. Since the parasite loads differed significantly from normal distribution (p-values between 1.34E-4 and 1.42E-14) we used non-parametric tests for all further analyses. Since within field sites diploid and triploid P . formosa did not vary in parasite load (Mann-Whitney U test Rio Guayalejo U = 170 p = 0,8264, Rio Purificacion U = 279 p = 0,5859) we decided to pool them. To test differences in parasite load between species within and among field sites we used a Kruskal-Wallis test followed by a Dunn’s posthoc test. Bonferroni correction was used to compensate for multiple testing.

A similar analysis was performed to test for differences in MHC allele numbers in species, ploidy and location. The Shapiro-Wilk test revealed that MHC class I allele counts were closer to normal distribution, however, still three of the six groups differed significantly from normal distribution (p-values between 2.19E-2 and 1.14E-16). A Mann-Whitney U test revealed significant differences between diploid and triploid P . formosa in the Rio Purificacion field site (Mann Whitney U = 81.5 p = 0.00026). Therefore, all six groups (diploid and triploid P . formosa and P . mexicana from each field site) were analyzed separately in the Kruskal-Wallis analyses. Dunn’s posthoc test was done to find specific differences between the groups and Bonferroni correction was used to compensate for multiple testing. All analyses were done using the program PAST version 3 [ 50 ].

Additionally, the effective number of clones (ENC), clonal diversity (CD) and clonal evenness (CE) after Menken et al. (1995) [ 51 ] were calculated for MHC class I, TLR 8, as well as for the genotypes originating from microsatellites. The ENC describes the number of clones, which actually reproduce in the population; CD describes the diversity of the population and CE the distribution of genotypes, where 1 is evenly distributed and 0 describes an uneven distribution. All analyses are based on the frequency (π) of the clonal lineages in the population: ENC = 1/(∑π 2 ); CE = ENC/(Number of genotypes); CD = 1 − ∑π 2 .

In the overall genotypic diversity we expected P . mexicana to have individual genotypes and consequently CD and CE to equal 1. We expected the clonal P . formosa to show lower values. At the immune gene level we expected shared genotypes in the clonal species but potentially also in the sexual species depending on allelic diversity or potentially local selection. Furthermore, we averaged the number of MHC I alleles found for the two sampled species in the two locations to see if there was a correlation between number of MHC I alleles and parasitic load. We also investigated the distribution of immune alleles in the two field sites to potentially find evidence of local adaptation. Finally, it was tested if the genotypes found for MHC class I always occurred in combination with distinct TLR 8 genotypes. To visualize the co-occurrence of distinct MHC I genotypes with distinct TLR 8 genotypes, the R package “circlize” (Version: 0.4.3 [ 52 ]) was used.

For investigation of immune gene variability within P . formosa and P . mexicana and also between the two species a total of 266 samples could be analyzed successfully. Parasitic load was higher in the Río Purificación than in the Río Guayalejo. In the Río Guayalejo field site, 78.1% of fish showed no sign of infection while in in the Río Purificación only 57.8% individuals did not show any black spots. Also in the Río Guayalejo the highest number of black spots in one individual was one, while at the Rio Purificacion site several individuals with more than 10 spots were found. While the locations clearly differed in parasite load, the species did not ( Fig 1 , Table 2 ).

Kruskal-Wallis test for equal medians and following Dunn’s posthoc test. Dunn’s z values above the diagonal, Bonferroni corrected p-values below the diagonal. Significant p-values are marked in bold typeface.

Overall genotypic diversity could be determined for all samples using the microsatellite markers. In total 144 diploid P . formosa , 20 triploid P . formosa and 102 P . mexicana from both locations were genotyped. In the Rio Guayalejo 27 unique genotypes were found for diploid P . formosa (32 individuals), while the triploids (14) showed seven different genotypes. Each of the 30 P . mexicana individuals had its own genotype ( Table 1 ). For the Río Purificación we identified 62 genotypes for 112 diploid P . formosa , 2 different genotypes for 6 triploid P . formosa and 69 unique genotypes for P . mexicana ( Table 1 ).Genotypic diversity was high in P . mexicana and diploid P . formosa in both field sites. Triploid P . formosa were less diverse than diploids but showed higher levels of genotypic diversity in the Rio Guayalejo field site ( Table 1 ).

For the MHC analysis 248 individuals yielded band patterns that allowed for further analysis. A total number of 35 MHC class I alleles were found. All alleles were present in P . mexicana , P . formosa had 31 different alleles. The highest number of different alleles for an individual was 15 in P . formosa and up to 17 different MHC alleles in P . mexicana . The triploid P . formosa had a total number of 21 different alleles with one individual having up to 10 distinguishable alleles. The median number of MHC I alleles for P . formosa was three in the Río Purificación and five in the Río Guayalejo. The triploid P . formosa had 5.5 in the Río Purificación and five in the Río Guayalejo. For P . mexicana we found the highest median number of MHC I alleles with eight in the Río Purificación and six in the Río Guayalejo ( Fig 2 ). The Kruskal-Wallis test showed significant differences in the data set. The Dunn’s posthoc test, however, revealed that this was only due to the low number of MHC class I alleles in diploid P . formosa from the Rio Purificacion field site. There were no other significant differences between species, ploidy or location ( Table 3 ). While P . formosa and P . mexicana had MHC I alleles in common, MHC I genotypes (distinct allele combinations) were never shared between the species ( Fig 3A and 3B ). We found 12 different genotypes for both species, five genotypes for P . formosa and seven genotypes for P . mexicana . Six of these genotypes were shared between the two locations, three in P . formosa and three in P . mexicana . One of the genotypes of P . formosa was only found in the Río Purificación. P . mexicana showed also one genotype exclusively in the Río Purificación and three genotypes only in the Río Guayalejo. Triploid P . formosa also had a private MHC I genotype that was not found in any of the other groups ( Fig 3A and 3B ). For all species and ploidy levels MHC class I diversity was higher in the Rio Guayalejo. MHC class I genotypes were also more evenly distributed in the Rio Guayalejo than in the Río Purificación ( Table 1 ).

Box plots show median plus upper and lower quartile and minimum and maximum values.

A) MHC alleles in all locations, species and ploidy levels. B) MHC genotypes in all locations, species and ploidy levels. C) TLR 8 alleles in all locations, species and ploidy levels. D) TLR8 genotypes in all locations, species and ploidy levels. (Pmex–Poecilia mexicana, 2n –diploid, 3n –triploid).

Kruskal-Wallis test for equal medians and following Dunn’s posthoc test. Dunn’s z values above the diagonal, Bonferroni corrected p-values below the diagonal. Significant p-values are marked in bold typeface. Rio P–Rio Purificacion, Rio G–Rio Guayalejo, P form– Poecilia formosa , P mex– Poecilia mexicana .

For the analyses of innate immunity the toll like receptor 8 (TLR8) was analyzed. 257 individuals yielded bands on the DGGE-gel that could be analyzed. Individual samples showed between two and five bands, with a total of seven different bands. Eleven different genotypes could be distinguished. Seven genotypes were identified for diploid P . formosa , two genotypes were found for the triploid P . formosa and four genotypes for P . mexicana . To validate the DGGE approach ten samples from nine different genotypes were chosen for sequencing. Six different alleles could be distinguished, but more alleles are possible as certain sequences only appeared two times, therefore not reaching our threshold of three discoveries in the dataset. Two of the alleles were found only in P . formosa , while the remaining alleles were shared between the two species ( Fig 3C ). We found two different genotypes for P . mexicana at each location. In Rio Purificacion a large majority of individuals (65 of 69) had the same genotype, while in the Río Guayalejo the distribution was more even: 27 to 18 ( Table 1 ). P . formosa showed five TLR 8 genotypes in Rio Guayalejo and four in Rio Purificacion. As in P . mexicana one genotype was very dominant in the Rio Purificacion, while in Rio Guayalejo genotypes were more equally distributed. Triploid P . formosa had only one genotype for both locations (= no clonal diversity) ( Table 1 , Fig 3D ).

With the genotypes found for TLR8 and MHC class I, we investigated if genotypes of the immune genes always occurred in distinct combinations. This could be indeed observed to some extent. As could be expected for a clonal organism, the diploid P . formosa that had the same genotype in the microsatellite assay also showed the same combination of MHC class I and TLR 8 genotypes. All triploid P . formosa shared the same TLR 8 genotype, but differed in their MHC class I genotype. Furthermore, we observed that TLR 8 genotypes of diploid P . formosa and P . mexicana correlated in most cases with particular MHC class I alleles with single deviations from the most common combinations ( Fig 4 ).

To facilitate the differentiation of alleles within species different shades of color are used.

The aim of this study was an immunogenetic analysis of the clonal fish, Poecilia formosa and one of its parental species, the sexually reproducing P . mexicana . In addition, parasite load (black spots—digenean trematode Uvulifer sp.) of both species was correlated with genotypic diversity: overall genotypic diversity (microsatellite markers) adaptive (MHC class I) and innate (TLR 8) immunity.

In contrast to our expectations based on the Red Queen hypothesis, the sexual and asexual species did not differ in parasite load. P . formosa and P . mexicana seemed to be affected by Uvuliver to the same degree. Interestingly, a similar observation was described earlier for the parasitic load of P . formosa in comparison to P . latipinna , the other parental species of P . formosa alongside P . mexicana . Despite looking for different macro- and micro-parasites, no significant difference could be observed between the two species [ 18 , 19 ]. Studies performed on asexual geckos and closely related sexual species revealed an even lower parasitic load with mites and other parasites of the asexual geckos in comparison to the sexual species. It was proposed that asexual vertebrates may have a higher resistance to parasites because of their hybrid origin [ 53 ]. The resulting combination of genes from two different parental species could be advantageous, a phenomenon called hybrid vigor [ 54 , 55 ]. This hypothesis has been has been supported by studies in hybridogenetic frogs [ 56 ] and was recently promoted for P . formosa [ 35 ].

While the clonal and the sexual species did not differ in parasite load, there was a profound difference between the locations: Both species showed a significantly higher parasitic load in the Río Purificación than in the Río Guayalejo. Grave differences in parasitic load among field sites have been reported in other studies [ 57 ]. There are two alternative explanations for this observation. First the locations could differ in parasite frequency. The differences in number of infected individuals could reflect the actual number of parasites present in the habitat. The parasite Uvulifer sp. has a complex life cycle and depends on the occurrence of all of its three hosts for its survival. Lemly and Esch (1984) [ 58 ] e.g. showed that the shedding of cercaria of Uvulifer sp. correlated with peak abundance of water snails in ponds. If one of the hosts is absent in the location or only present in low abundance the total abundance of the parasite should be impacted. As low presence of the fish host can be ruled out (personal observation on catching success [ 59 ]) the abundance of little water snails or piscivorous birds could differ greatly between the Río Purificación and the Río Guayalejo.

An alternative (second) explanation would be adaptation. Fishes in the Río Guayalejo could be better adapted to parasite infections than fishes in the Río Purificación. Several studies investigated a potential correlation of parasite load with genotypic diversity. Especially MHC genes of class I and II were in the focus so far and found evidence of local adaptation [ 21 , 57 , 60 , 61 ].

To analyze potential genetic differences between field sites and species we analyzed the genotypic diversity of both species. For the overall genotypic diversity a microsatellite assay was used. The markers showed a high resolution in differentiating individual genotypes for P . mexicana . We are therefore confident, that P . formosa individuals with identical microsatellite genotypes belonged to the same clonal lineage. This was confirmed by the immune gene markers: P . formosa individuals with the same microsatellite genotype also shared the same immune genotype (identical MHC class I and TLR 8 combinations). Overall genotypic diversity was high in both field sites, as was immune gene diversity.

A high diversity of MHC class I alleles in fishes had been reported earlier [ 62 ]. Later, however, it was shown that that an optimum rather than a maximum number of alleles maximizes parasite resistance [ 21 , 34 ]. In our study P . mexicana individuals had significantly more MHC class I alleles than P . formosa . The higher average count of MHC class I alleles in P . mexicana in comparison to P . formosa could possibly be attributed to mate-choice if females preferred males with higher MHC I allele numbers over males with lower MHC I allele numbers [ 21 , 63 ]. Despite the difference in MHC allele number we did not find a species specific difference in parasite resistance: Both species were infected at similar rates. In addition, we could not find an optimal allele number correlating with low parasite susceptibility, but we noticed that within field sites a maximum number of MHC class I alleles did not maximize pathogen resistance. Interestingly, the most common MHC I genotype of P . formosa , did not show the highest parasitic load as would have been expected from the Red Queen hypothesis, where common genotypes are expected to be easier targets for parasite/pathogen infections. Instead all genotypes found for the Río Purificación showed a similar parasitic load. Especially the triploids with their very low genotypic variability should be susceptible to pathogen infections [ 10 , 21 , 40 ]. While other studies found that diploid P . formosa have a higher fitness than their triploid counterparts [ 15 ], it seems that the triploid fitness disadvantage is not due to higher parasite load due to common genotypes. A similar picture as for P . formosa was seen for P . mexicana . The more abundant MHC genotypes did not show enhanced parasite susceptibility as expected by the Red Queen hypothesis: All MHC genotypes (common or rare) showed similar levels of parasite infection.

In addition to MHC class I as part of the adaptive immune system, we analyzed a component of the innate immune system. We chose Toll like receptor 8 because Toll-like receptors had been shown to play a role in parasite control [ 64 ]). Eleven different genotypes were found for the TLR 8. Interestingly, the number of bands observed on the DGGE-gel were more than expected for a single copy gene: A maximum of two bands/alleles for diploids and three bands/alleles for triploids. Instead, we observed between two and five bands, with the majority of samples showing more than 2 bands/alleles. This means that in P . formosa the TLR 8 gene is most likely present in at least two copies with several alleles. Copy number variation between and even within species is quite common in teleost fishes [ 65 , 66 ] and TLR 8 has been reported to exist in two different variants in zebrafish, Danio rerio [ 67 , 68 ].

P . formosa and P . mexicana shared four of the 11 TLR 8 alleles but no genotypes, which is not surprising, considering P . formosa ´s hybrid origin. The hybrid origin might also explain the slightly higher level of genotypic diversity (ENC and CD Table 1 ) at the TLR 8 locus in diploid P . formosa compared to sexual P . mexicana . The two alleles found only for P . formosa are likely derived from P . latipinna , the other parental species. We found two different genotypes for each location for P . mexicana . While we observed that the common genotype of P . mexicana in the Río Purificación is heavily infected and the rare genotype is infection free, which is in accordance to the Red Queen hypothesis [ 1 ], a similar pattern could not be observed for individuals in the Río Guayalejo. Also, neither the most frequent genotype of P . formosa in the Río Purificación nor Río Guayalejo showed the highest levels of parasite susceptibility.

These results are somehow unexpected from the viewpoint of the Red Queen hypothesis. It might, however, be the result of sampling time. If in the co-evolutionary dynamics of fish and parasites high frequency MHC variants have just come up, the parasites might still be lacking behind [ 69 ]. In addition, at the end of the dry season highly infected clones might already be declining and therefore might no longer be the most dominant genotypes [ 70 , 71 ].

While we could not correlate certain alleles or genotypes with parasitic load or location we observed that the pattern of parasite susceptibility between the two locations matched the pattern we saw in the immune genotype diversity of MHC class I and TLR 8. We found a higher immune gene diversity but lower parasite load for both species in the Río Guayalejo than in the Río Purificación. This pattern was not reflected in the microsatellite data, even though microsatellite genotypes in P . formosa correlated with immune genotype. This finding is in accordance with the Red Queen hypothesis where we would expect high genotypic diversity to be connected with lower parasitic load.

In contrast to the predictions from the Red Queen hypothesis sexually reproducing P . mexicana did not show lower levels of parasite infection than the clonally reproducing P . formosa . Instead parasite load seemed equal in both species and correlated to field site instead. Similar observations have been made before but could so far not been explained. Molecular analyses of the overall genotypic diversity showed, that while the clonal species was quite diverse it was still less diverse than the sexually reproducing species and therefore fulfilled the preconditions for the Red Queen hypothesis. More specific analyses of the immune genes of the major histocompatibility complex came to the same conclusion. Our study of a multicopy innate immune gene (TLR 8) revealed that the diploid individuals of the clonal species were even more diverse at these loci than individuals from the sexual species. This is most likely due to their hybrid origin and might balance advantages in the fight of pathogen infection.

Acknowledgments

We are grateful to the National Commission of Aquaculture and Fisheries (CONAPESCA) of the Mexican government for the collection permits, nos. DGOPA/16986/191205/8101 and DGOPA/02232.230706–1079. We thank Peter Fischer and Matthias Stöck for help in the field. Financial support for this study was granted by the DFG (SFB 567 ‘Mechanismen der interspezifischen Interaktion von Organismen’), the University of Würzburg and the University of Bochum. We acknowledge support by the DFG Open Access Publication Funds of the Ruhr-Universität Bochum.

Funding Statement

This work was supported by grants to MS (German Research Foundation DFG projects SFB 567 ‘Mechanismen der interspezifischen Interaktion von Organismen,’ Scha408/10-1 and Scha408/12-1), the University of Bochum, the University of Cologne and the University of Würzburg. We acknowledge support by the DFG Open Access Publication Funds of the Ruhr-Universität Bochum. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability