BACKGROUND
the coexistence of asexual and sexual forms in nature has been reported mainly in plants and invertebrates.
concerning vertebrates, asexual reproduction in many forms has been found in fishes, amphibians and lizards.
the mechanisms promoting the coexistence of asexual fish living in sympatry with sexual ones have been investigated e.g.
phoxinus <cit> , poecilia <cit> , carasssius auratus <cit> .
sexual and asexual forms do not coexist easily.
sexual forms suffer from the twofold costs of sex  and, therefore, they should be outcompeted by asexual forms using the same resources and producing eggs at twice the rate  <cit> .
on the other hand, the long-term costs associated with the accumulation of deleterious mutation leading to fitness decline in asexual forms and their potential extinction within many generations have been predicted  <cit> .
moreover, asexual forms have a limited ability to adapt to environmental changes  <cit> , show reduced performance under stress  <cit> , and exhibit a higher susceptibility to parasites when compared to sexual forms ).
concerning the form of asexual reproduction termed gynogenesis, the offspring are formed parthenogenetically but egg development is induced by sperm of the same or closely-related sexual species.
gynogenetic females can only persist in sympatry with closely-related bisexual forms.
thus, this form of reproduction has some of the disadvantages of sexual and asexual reproduction  <cit> .
one of the most widely accepted hypotheses proposed to explain the maintenance and evolution of sex is the red queen hypothesis  <cit> .
this hypothesis postulates antagonistic coevolution between hosts and parasites leading to sustained oscillations in genotype frequencies, whereby hosts refine their defense strategies against parasites, and parasites, in turn, evolve counter-adaptations to these defense strategies.
the core idea is that parasites are generally under selection to infect the most common genotypes in the local host population.
parasite mediated frequency-dependent selection leads to genetic polymorphism in both the host and parasite  <cit> .
in species where asexual reproduction is possible , co-evolutionary interactions with parasites may select for sexual reproduction in hosts as a way of reducing the risk of infection in offspring  <cit> .
thus, the high level of parasite infection in common asexual clones could favor genetically diverse sexual individuals and promote the short-term coexistence of sexual and asexual populations.
in fish, several studies were conducted to compare the level of parasitism between sympatric asexual and sexual forms or species.
some of them support the red queen hypothesis and showed that asexual fish suffer from a higher parasite load than sexual fish .
however, no difference in parasitism between asexual and sexual forms of fish species was detected in several empirical studies .
the mhc is a family of highly polymorphic genes occurring in vertebrates that encode cell-surface glycoproteins responsible for the recognition of peptide fragments of self or non-self-origin and for presenting them to t cells, which can initiate an appropriate immune response resulting in the destruction of antigen-presenting cells or in antibody production  <cit> .
the high polymorphism is most pronounced in the peptide-binding region , which is at specific amino-acid sites directly in contact with the bound peptides.
mhc polymorphism is maintained by balancing selection driven by parasites or mating choice  <cit> .
for this reason, mhc is a suitable candidate for testing the mechanisms proposed for the coexistence of asexual and sexual species based especially on the red queen hypothesis.
the difference in the specific immunity, of which mhc is representative, may cause negative density-dependence mortality in the asexual form and thus maintain sexual reproduction in the populations.
the variability of mhc in asexual vertebrates has only been examined by a few studies  <cit> .
to our knowledge, parasite-mediated selection acting on mhc diversity has not yet been compared between sexual and asexual fish.
gibel carp  represents an interesting and unique species in which the coexistence of gynogenetic reproduction and gonochoristic reproduction is known.
the origin of this species is thought to be eastern russia and it colonized europe successively during the 20th century  <cit> .
in  <dig>  a non-native gibel carp permeated into the czech hydrologic system by migration from the danube river and, with human help and by its own invasive activities, gradually colonized most of the suitable biotopes of the three czech basins.
the former populations of c. gibelio were composed of triploid females with gynogenetic reproduction in which gynogenetic females used the sperm of males of other cyprinid fish species  for the activation of egg development  <cit> .
the solely female character of populations had been recorded up to  <dig>  when the first males were recorded in the populations.
a few years later, c. gibelio had formed stable mixed diploid-polyploid populations composed of sexually reproducing diploids, asexually reproducing dominant triploid females, and very rare triploid and tetraploid males  <cit> .
following the different advantages and disadvantages related to gynogenesis and sexual reproduction in gibel carp, a few studies  <cit>  were performed to compare growth as an important life trait between diploids and triploids expecting that gynogenetic triploids should exhibit fast growth than sexually reproducing diploids.
however, no difference in growth patterns between two forms was found.
the analyses of the control region and cytochrome b gene showed that both sexually reproducing diploids and gynogenetic triploid females of morphologically identified c. gibelio in the czech republic have the same origin  <cit> .
gibel carp is parasitized by a wide range of metazoan parasites  <cit> ), many of them  are specific to c. gibelio and the phylogenetically closely related species c. carassius.
for host specific parasites, long-term host-parasite coevolution  is hypothesized and such parasites are primarily responsible for the evolution of mhc polymorphism  <cit> .
in addition, gill and skin monogeneans represent the species richest group; they are pathogenic to gibel carp and closely-related fish species, causing fish mortality especially in fish aquaculture .
this study was focused on the variability of mhc genes and parasitism in two forms of gibel carp with different modes of reproduction and ploidy level.
first, we compared mhc variability and the selection pressure acting on mhc genes in sexual diploids and asexual triploids.
we expected that differences between sexual and asexual forms would be due to the negative frequency dependent selection acting in asexually reproducing females  and also due to the sexual selection acting on mhc diversity in sexual diploids.
we addressed the following specific questions 1) do sexual gibel carp exhibit a higher diversity of mhc genotypes than asexual gibel carp?
2) do asexual gibel carp  suffer from higher parasite loads than sexual gibel carp?
RESULTS
the mixed population of gibel carp in the locality studied was composed of 60% triploid individuals and 40% diploid individuals  with no variation between two consecutive years.
approximately the same number of triploid and diploid individuals , all of them with a similar total body size, were analyzed .
variability of dab genes
different genotype diversity was found for gynogenetic triploids and sexual diploids.
diploids expressed  <dig> genotypes whilst triploids expressed only  <dig> genotypes .
two genotypes were common in triploids, i.e.
53% of triploid females expressed the genotype including cagi-dab3* <dig> and cagi-dab3* <dig> alleles  and 26% of triploid females expressed the genotype including cagi-dab1* <dig>  cagi-dab3* <dig> and cagi-dab3* <dig> alleles .
other triploids expressed rare genotypes.
all genotypes of diploids were rare, i.e.
expressed by only one or two individuals except for the genotype including one allele cagi-dab1* <dig> found in  <dig> diploids.
the individuals of both sexual diploids and gynogenetic triploids expressed genotypes including the alleles of a single lineage  or the alleles of both lineages dab1-like and dab3-like.
due to the expression of the common genotype “a” of the dab3-like lineage in gynogenetic triploids, 62% of triploid individuals expressed the genotypes belonging to a single dab3-like lineage.
meanwhile, 32% of triploid individuals expressed the combined genotypes, whilst only 6% expressed the genotypes of the dab1-like lineage.
a different ratio of genotypes was found in diploids, where 45% of diploid individuals expressed the genotypes of the dab1-like lineage, 25% of diploid individuals expressed dab3-like genotypes, and 30% expressed the combined genotypes .
at population level, the total number of alleles expressed by sexual diploids and gynogenetic triploids was different .
a total of  <dig> different alleles were recorded in the population of gibel carp investigated, but only  <dig> alleles were shared between the two forms .
the majority of alleles were specific to either diploid or triploid genotypes.
three alleles shared by sexual diploids and gynogenetic triploids were expressed in common genotypes of triploids, and one shared allele was the most frequent allele found in diploid genotypes.
even if the diploids expressed a wide range of different genotypes, three alleles were more frequent than others i.e.
cagi-dab1* <dig>  cagi-dab1* <dig> and cagi-dab3* <dig> .
no significant influence of year of collection on the number of alleles expressed by individuals was found .
therefore, data from two years were pooled for the next analyses.
the number of alleles per individual  ranged from  <dig> to  <dig> dab alleles .
the number of alleles per individual expressed by triploids was significantly higher than the number of alleles expressed by diploids .
even the total number of different dab sequences was higher for sexual diploids than for gynogenetic triploids  at the population level; the mean overall nucleotide diversity was similar for both forms.
after translating the nucleotide sequences into amino-acid sequences, the alignment reflected the high level of non-synonymous substitutions in dab1-like and dab3-like genes in both forms.
the rate of nonsynonymous substitutions was significantly higher compared to synonymous ones .
standard errors estimates shown in parentheses were obtained from  <dig> bootstrap replicates.
numbers following dab gene groups shown in parentheses indicate the number of different allelic variants in the alignment.
mean amino-acid variability calculated for the total data set at the population level was slightly higher for sexual diploids than for gynogenetic triploids .
a similar nucleotide and amino-acid diversity for the dab1-like gene and for the dab3-like gene was found when comparing both forms.
however, the means of nucleotide and amino-acid diversity were higher in the dab3-like data set than in the dab1-like data set .
at the individual level, gynogenetic triploids had a significantly higher amino-acid diversity than sexual diploids .
however, when corrected for the number of alleles  no significant difference in amino-acid diversity was found between gynogenetic triploids and sexual diploids .
nucleotide diversity was not significantly different between the two forms of gibel carp .
however, the variance in both amino-acid diversity and nucleotide diversity was significantly higher in sexual diploids compared to gynogenetic triploids without correction for the number of alleles  and also using correction for the number of alleles .
positive selection in dab sequences
maximum likelihood estimates of parameters under different codon models of variable ω across sites and the number of pss for both forms of gibel carp are included in table  <dig>  the lrt statistic comparing the two models indicates that the alternative models m2a, m <dig> and m <dig>  fit the data significantly better  than the simpler models m1a, m <dig> and m <dig> , which indicates the action of positive selection at specific sites in dab sequences.
bayes identification of sites under positive selection calculated using the m <dig> model is included in figure  <dig>
the number following the model code given in parentheses represents the number of free parameters for the ω ratios.
parameters indicating positive selection are presented in bold type.
parameters in parentheses are presented for clarity only but are not free parameters; for example, under m <dig> p1 = 1-p0; pss are the positive selected sites identified using the beb method at *: p > 95%; **: p > 99%; ω is the selection parameter; pn is the proportion of sites that fall into the ωn site classes; p and q are the shape parameters of the β function .
the estimates from the m <dig> model suggest a different pattern of selection in dab1-like and dab3-like genes between both forms, i.e.
a different number and different positions of pss in sexual diploids and gynogenetic triploids were found .
concerning sexual diploids, a similar number of pss sites were under positive selection  in the dab1-like and dab3-like sequences .
<dig> out of  <dig> pss identified in dab1-like sequences and  <dig> out of  <dig> pss identified in dab3-like sequences corresponded to human antigen binding sites  identified using crystallography by brown et al.
<cit>  .
for dab sequence variants of gynogenetic triploids,  <dig> sites were identified as targets of positive selection in dab1-like alignment ,  <dig> of them identical to human abs.
a higher number of pss were detected in dab3-like sequences of gynogenetic females compared to the pss identified in dab1-like sequences in this form or pss identified in dab1-like or dab3-like sequences of sexual diploids.
<dig> out of  <dig> pss identified in dab3-like sequences of gynogenetic triploids corresponded to human abs.
all pss found in dab3-like sequences in sexual diploids were also identified as pss in dab3-like sequences of gynogenetic triploids.
<dig> pss found in dab1-like sequences of gynogenetic triploids were identified in dab1-like sequences of diploids.
mhc and parasitism
no significant difference in parasite species richness was found between the fish investigated in  <dig> and  <dig> .
therefore, the parasitological data were pooled for all analyses.
a total of  <dig> metazoan parasite species were found in the gibel carp studied.
among them, monogenea was the species richest group .
the prevalences of five dactylogyrus species were high  and the prevalences of another three ectoparasite species d. vastator, g. sprostonae and glochidum were moderate .
the presence of other parasite species was rare .
means of parasite species richness, total parasite abundance, abundance of dactylogyrus, and parasite diversity are shown in table  <dig>  when comparing infection by parasite species between the two forms of gibel carp , the prevalences of the five dominant dactylogyrus species were similar.
the intensities of infection for four of the five dominant dactylogyrus species tended to be higher in triploids than in diploids.
moreover, triploids were occasionally parasitized by four rare endoparasite species that were absent in diploids .
sc – specific parasite i.e.
parasitizing two congeneric c. gibelio and c. carassius, sph – specific parasite i.e.
parasitizing phylogenetically related species c. gibelio, c. carassius and cyprinus carpio.
g – generalist  parasite species.
host specificity  was taken from moravec  <cit> .
first, when fish mhc was expressed as the number of dab alleles, nested anova revealed no significant effects of fish mhc and reproductive strategy on parasite load measured by parasite species richness or parasite abundance .
next, when fish mhc was expressed as the type of dab lineage, nested anova  revealed a statistically significant effect exerted by fish mhc  and an insignificant effect exerted by reproduction strategy  on parasite load expressed as parasite species richness.
using the bonferonni post-hoc test, a significant difference was found in parasite species richness only between the dab <dig> and dab <dig> genotypes , where the dab <dig> genotype was more parasitized than the dab <dig> genotype.
no statistical difference between the genotype of combined lineages  and single genotypes  was found, demonstrating no significant link between increasing mhc variability and parasite load.
to test the red queen hypothesis, first, parasitism was compared between gynogenetic triploids and sexual diploids.
a higher mean parasite species richness was found in gynogenetic triploids compared to sexual diploids .
however, the difference in parasite species richness between gynogenetic triploids and sexual diploids was not statistically significant .
no significant difference in parasite abundance or parasite diversity was found between diploids and triploids .
no significant difference was found when comparing the variance in parasite abundance or parasite diversity .
next, we divided the mhc genotypes of triploid individuals into three groups: common genotype “a” , common genotype “b” , and rare genotype .
the parasitism of each common genotype was compared to the parasitism of gynogenetic triploids with the rare genotype and sexual diploids .
significantly higher parasite species richness was found in gynogenetic triploids expressing the common genotype “a” than in sexual diploids  and gynogenetic triploids with the rare genotype .
however, after bonferroni correction, only the difference between triploids expressing common genotype “a” and sexual diploids was significant .
no difference in parasite abundance or parasite diversity was found between gynogenetic triploids expressing the common genotype “a” and sexual diploids or gynogenetic triploids with the rare genotype .
a significantly higher parasite abundance was found for triploid individuals with common genotype “b” when compared to sexual diploids  or gynogenetic triploids with common genotype “a” .
after bonferroni correction, no relationship was significant .
dactylogyrus species represented 56% of total parasite species and 98% of total parasite abundance.
a significantly higher dactylogyrus abundance was found for triploids with common genotype “b” when compared to sexual diploids  or gynogenetic triploids with common genotype “a” .
after bonferroni correction, only the difference between gynogenetic triploids with common genotype “b” and sexual diploids was significant .
discussion
this study presents the first analysis of the genetic diversity of functional mhc class iib genes in gibel carp, a fish species with dual reproduction modes – sexual reproduction in diploids and gynogenesis in triploid females.
similarly to previous studies analyzing dab genes in cyprinid fish , high mhc iib diversity was also shown in this diploid-polyploid fish species.
both forms express the alleles of one locus  or the alleles of both loci , which was shown for diploid cyprinid species using genomic or expressed mhc iib genes .
our study revealed the presence of many alleles specific to gynogenetic triploids or specific to sexual diploids.
moreover, several alleles were found at high frequencies only in triploids  or only in diploids.
concerning the specific and frequent alleles found in gynogenetic triploids, we cannot distinguish if they are merely characteristics of our study population, or if they have persisted ever since the species invasion of the czech republic, or even since the origin of the triploid form .
such hypotheses could be evaluated using a large sample and time series from a wide range of gibel carp populations.
despite the reduced genotype diversity in triploids , similar overall nucleotide and amino-acid diversity at the population level was found for dab datasets in sexual diploids and gynogenetic triploids, which indicates the genetic variation among different gynogenetic lineages of gibel carp.
at the individual level, gynogenetic triploids expressed mainly  <dig> or  <dig> dab alleles , whilst sexual diploids expressed mainly  <dig> or  <dig> dab alleles.
this could be explained by polyploidization of the genome, where a higher number of gene copies is expected for polyploid individuals, which might give them a crucial advantage in pathogen recognition.
however, following the red queen hypothesis, asexually reproducing organisms should be very susceptible to pathogen infection because of low genotypic diversity which could annihilate the positive effect of having many mhc copies  <cit> .
when compared carassius gibelio to poecilia, between  <dig> and  <dig> mhc class iib sequences per individual were found in both asexual amazon molly  and sexual sailfin molly   <cit>  .
thus, it seems that the high number of mhc iib variants in gynogenetic individuals does not represent a general rule for the species where two forms with different reproduction modes coexist.
the number of mhc alleles per individual may be determined by the potential duplication of some mhc lineage found in cyprinid species e.g.
duplication of the dab <dig> lineage in common carp   <cit> , duplication of the dab <dig> lineage in chub   <cit> , or duplication of both dab <dig> and dab <dig> lineages found only for the diploid form of gibel carp in this study.
it is also possible that some alleles or allelic lineages are not detectable in cdna, as they are not expressed.
high mhc polymorphism is explained by two, though not mutually exclusive hypotheses, i.e.
sexual selection and parasite-mediated selection.
we tested whether or not the sexual form of gibel carp shows higher mhc polymorphism than the gynogenetic form, following the hypothesis that mhc diversity is maintained by sexual-mediated selection.
schaschl et al.
<cit>  presumed that high mhc diversity in the asexual form or comparable diversity between sexual and asexual forms indicates that sexual selection is not important for explaining mhc diversity.
less mhc diversity at the allelic as well as genotypic level for asexual species compared to sexual ones was also hypothesized by lampert et al.
<cit>  as an important prerequisite of the red queen hypothesis.
in our study, the high mhc genotype and allelic diversity  in sexual diploids compared to gynogenetic triploids living in the same habitat is consistent with the prediction that mhc diversity is driven by sexually-mediated selection .
on the other hand, reduced mhc genotype diversity in gynogenetic triploids is probably due to the lack of recombination and segregation in these asexually reproducing organisms, where only point mutations could generate new mhc alleles.
similar findings were previously shown in poecilia, i.e.
lower mhc genotype diversity and the reduced diversity of mhc class i and class iib alleles was found in asexual amazon molly compared to sexual sailfin molly  <cit> .
lower diversity in mhc class i genes was also found in a parthenogenetic gecko species compared to closely related sexual gecko species  <cit> .
the difference in genotype and allelic diversities between asexually and sexually reproducing gibel carp could be the result of sexual selection and/or selection from pathogens and parasites, or may be due to genetic drift or the bottleneck effect after introduction, as was suggested for asexual amazon mollies by schaschl et al.
<cit> .
our study revealed a similar number of positively selected sites in both dab1-like and dab3-like genes in sexual diploids, where the majority of pss were identical with human antigen binding sites.
however, when analyzing the selection pattern in dab genes of gynogenetic triploids, the different number of pss between dab1-like and dab3-like sequence alignments was shown.
this may suggest that sexual selection or parasite-mediated selection  contribute to the maintenance of mhc diversity in diploid-polyploid populations of gibel carp.
however, we should emphasize that only extensive experimental study could clarify the mechanisms of selection affecting mhc diversity in gibel carp.
in addition, we showed stronger positive selection in dab3-like genes in gynogenetic triploids compared to the selection in dab1-like genes in gynogenetic triploids or to the selection in sexual diploids, which suggests a potential functional difference between dab1-like and dab3-like genes , especially for triploids.
parasite-mediated selection  together with the coevolutionary “arms races” between hosts and parasites form the core idea of the red queen hypothesis.
we showed that, at the population level, mhc genotype and allelic diversities in asexual triploids are reduced compared to sexual diploids.
therefore, gibel carp clearly fulfills a prerequisite of the red queen hypothesis underlying the evolution of sex.
concerning parasites, their virulence and pathogenicity represent important components of the red queen hypothesis.
these features were demonstrated for some gill and skin monogeneans .
dactylogyrus parasitizing carassius species and closely related common carp are considered to be the most pathogenic monogenean species on the gills causing fish mortality  <cit> .
we evaluated parasite load, including metazoan parasite species with a high prevalence of dactylogyrus species that are host specific to carassius species or specific to phylogenetically closely related fish species .
in our study, the parasite load on gynogenetic and sexual forms was compared following the prediction of the red queen hypothesis, i.e.
sex provides an advantage to hosts by creating rare genotypes that confer resistance to parasites.
thus, in the case when the genetic diversity of asexual lineages is low, the asexually reproducing form is more intensely infected than the sexual form  <cit> .
our study showed that parasite species richness is slightly higher in the gynogenetic form compared to the sexual form.
the finding that the overall amino-acid diversity of dab genes in gynogens was comparable to the amino-acid diversity in the sexual form suggests that the analyzed gynogenetic lineages are genetically diverse.
therefore, the subsequent analyses were focused on the parasite load of common mhc iib genotypes in gynogenetic triploids, following the prediction that the parasite load of the sexual form should be lower than that of the more common clone, but that the parasite load of some infrequent clones might not necessarily be higher than that of the sexual form  <cit> .
two common mhc iib genotypes were more parasitized than the genotypes of sexual diploids and rare genotypes of gynogenetic triploid females, which is in line with the prediction of the red queen hypothesis.
the present study revealed that at the individual level high mhc diversity is not an important factor determining parasitism, using whichever of the two measures of individual mhc diversity – the number of expressed dab alleles or the type of dab lineage.
high diversity in mhc iib genes allows the recognition of more antigens derived from parasites in a population.
however, many mhc variants in the individual will also result in self-reactive t-cell elimination.
in line with this theory, nowak et al.
<cit>  predicted that an intermediate number of mhc alleles should be associated with minimal parasite load, which was supported by the study of three-spined sticklebacks  by wegner et al.
<cit> .
our study on gibel carp showed that higher parasite species richness is linked to the expression of the alleles of the dab <dig> lineage.
as the common triploid genotype “a” represents the genotype of the dab <dig> lineage, this finding simply reflects the high parasite species richness of the most common genotype rather than the importance of the dab <dig> lineage for parasitism.
moreover, it supports the study of lampert et al.
<cit>  suggesting that a higher allelic copy number in clonal fish might not convey an immune advantage .
thus, the present study may indicate that parasitism is one of the plausible candidates for explaining temporal changes in the population character of gibel carp, from the period of its introduction into czech rivers until today.
the first invasive populations were triploid females with gynogenetic reproduction; their mhc genotypes were probably infrequent in the new invaded habitats.
it is possible that triploid clones were able to become common and a potential target for parasite selection due to their high reproductive ability linked to gynogenesis.
what might explain the recent coexistence of gynogenetic and sexual forms of gibel carp?
hakoyama and iwasa  <cit>  showed that parasitism plays an important role in bringing about the coexistence of asexual and sexual forms in japanese crucian carp  by giving a frequency dependent benefit to the sexual population.
this supports the prediction that the coexistence of sexual and unisexual forms should be stabilized by parasites  <cit> .
the high susceptibility to diseases in the unisexual form could compensate the two-fold cost of sex in the sexual form.
as recent knowledge on the distribution of gibel carp in central europe shows that both sexual and gynogenetic forms coexist in many natural populations, we suggest that the higher parasite load in common gynogenetic triploid genotypes could represent one of the possible factors promoting the coexistence of this sexual-gynogenetic complex.
hakoyama et al.
<cit>  showed that the high prevalence of metagonnimus  in japanese crucian carp  is in part due to the lower immune activity of the phagocytes in the gynogenetic form.
therefore, for further studies of gibel carp, we suggest analyzing measurements of specific  and non-specific immunity  to investigate whether enhanced parasite load in gynogenetic females reflects an immune disadvantage.
however, the coexistence of sexual and unisexual forms may also be maintained by male mate choice  or spatial and temporal extinction and recolonization , factors that have not yet been analyzed in c. gibelio.
moreover, for a better understanding of whether the coexistence of sexual and gynogenetic forms in the populations of gibel carp is stable, or whether the evolution of gibel carp populations is directed toward strictly sexual reproduction, long-term studies are needed.
the latter possibility was demonstrated for sexual and asexual snails by jokela et al.
<cit> , where co-evolutionary dynamics predicted by the red queen hypothesis favor sexual reproduction in natural populations, i.e.
the most common clones were almost completely replaced by initially rare clones, while the sexual form persisted throughout a long period of investigation.
CONCLUSIONS
our study investigated the genetic diversity of mhc class iib genes and the selection mechanisms potentially driving mhc diversity in diploid-polyploid fish species with dual reproduction strategies.
in mixed populations of gibel carp, where gynogenetic triploids coexist with sexually reproducing diploids, the genotype and allelic diversity is reduced in triploids compared to diploids, which is in accordance with the hypothesis of sexually-mediated selection increasing the mhc diversity and fulfils a prerequisite of the red queen hypothesis.
we showed that the most common genotypes of gynogenetic triploids are the target of parasite selection.
this finding also supports the prediction of the red queen hypothesis.
on the other hand, the individual allelic copy number does not seem to be important for fish immunocompetence .
we propose that the evolutionary twofold cost of sexual reproduction in diploids is compensated by the limited ability of asexual forms to escape parasitism and, thus, that this process could facilitate the recent coexistence of sexual and gynogenetic forms of gibel carp.

 the coexistence of asexual and sexual forms in nature has been reported mainly in plants and invertebrates. concerning vertebrates, asexual reproduction in many forms has been found in fishes, amphibians and lizards. the mechanisms promoting the coexistence of asexual fish living in sympatry with sexual ones have been investigated e.g. phoxinus, poecilia, carasssius auratus. sexual and asexual forms do not coexist easily. sexual forms suffer from the twofold costs of sex and, therefore, they should be outcompeted by asexual forms using the same resources and producing eggs at twice the rate. on the other hand, the long-term costs associated with the accumulation of deleterious mutation leading to fitness decline in asexual forms and their potential extinction within many generations have been predicted. moreover, asexual forms have a limited ability to adapt to environmental changes, show reduced performance under stress, and exhibit a higher susceptibility to parasites when compared to sexual forms ). concerning the form of asexual reproduction termed gynogenesis, the offspring are formed parthenogenetically but egg development is induced by sperm of the same or closely-related sexual species. gynogenetic females can only persist in sympatry with closely-related bisexual forms. thus, this form of reproduction has some of the disadvantages of sexual and asexual reproduction. one of the most widely accepted hypotheses proposed to explain the maintenance and evolution of sex is the red queen hypothesis. this hypothesis postulates antagonistic coevolution between hosts and parasites leading to sustained oscillations in genotype frequencies, whereby hosts refine their defense strategies against parasites, and parasites, in turn, evolve counter-adaptations to these defense strategies. the core idea is that parasites are generally under selection to infect the most common genotypes in the local host population. parasite mediated frequency-dependent selection leads to genetic polymorphism in both the host and parasite. in species where asexual reproduction is possible, co-evolutionary interactions with parasites may select for sexual reproduction in hosts as a way of reducing the risk of infection in offspring. thus, the high level of parasite infection in common asexual clones could favor genetically diverse sexual individuals and promote the short-term coexistence of sexual and asexual populations. in fish, several studies were conducted to compare the level of parasitism between sympatric asexual and sexual forms or species. some of them support the red queen hypothesis and showed that asexual fish suffer from a higher parasite load than sexual fish. however, no difference in parasitism between asexual and sexual forms of fish species was detected in several empirical studies. the mhc is a family of highly polymorphic genes occurring in vertebrates that encode cell-surface glycoproteins responsible for the recognition of peptide fragments of self or non-self-origin and for presenting them to t cells, which can initiate an appropriate immune response resulting in the destruction of antigen-presenting cells or in antibody production. the high polymorphism is most pronounced in the peptide-binding region, which is at specific amino-acid sites directly in contact with the bound peptides. mhc polymorphism is maintained by balancing selection driven by parasites or mating choice. for this reason, mhc is a suitable candidate for testing the mechanisms proposed for the coexistence of asexual and sexual species based especially on the red queen hypothesis. the difference in the specific immunity, of which mhc is representative, may cause negative density-dependence mortality in the asexual form and thus maintain sexual reproduction in the populations. the variability of mhc in asexual vertebrates has only been examined by a few studies. to our knowledge, parasite-mediated selection acting on mhc diversity has not yet been compared between sexual and asexual fish. gibel carp represents an interesting and unique species in which the coexistence of gynogenetic reproduction and gonochoristic reproduction is known. the origin of this species is thought to be eastern russia and it colonized europe successively during the 20th century. in a non-native gibel carp permeated into the czech hydrologic system by migration from the danube river and, with human help and by its own invasive activities, gradually colonized most of the suitable biotopes of the three czech basins. the former populations of c. gibelio were composed of triploid females with gynogenetic reproduction in which gynogenetic females used the sperm of males of other cyprinid fish species for the activation of egg development. the solely female character of populations had been recorded up to when the first males were recorded in the populations. a few years later, c. gibelio had formed stable mixed diploid-polyploid populations composed of sexually reproducing diploids, asexually reproducing dominant triploid females, and very rare triploid and tetraploid males. following the different advantages and disadvantages related to gynogenesis and sexual reproduction in gibel carp, a few studies were performed to compare growth as an important life trait between diploids and triploids expecting that gynogenetic triploids should exhibit fast growth than sexually reproducing diploids. however, no difference in growth patterns between two forms was found. the analyses of the control region and cytochrome b gene showed that both sexually reproducing diploids and gynogenetic triploid females of morphologically identified c. gibelio in the czech republic have the same origin. gibel carp is parasitized by a wide range of metazoan parasites ), many of them are specific to c. gibelio and the phylogenetically closely related species c. carassius. for host specific parasites, long-term host-parasite coevolution is hypothesized and such parasites are primarily responsible for the evolution of mhc polymorphism. in addition, gill and skin monogeneans represent the species richest group; they are pathogenic to gibel carp and closely-related fish species, causing fish mortality especially in fish aquaculture. this study was focused on the variability of mhc genes and parasitism in two forms of gibel carp with different modes of reproduction and ploidy level. first, we compared mhc variability and the selection pressure acting on mhc genes in sexual diploids and asexual triploids. we expected that differences between sexual and asexual forms would be due to the negative frequency dependent selection acting in asexually reproducing females and also due to the sexual selection acting on mhc diversity in sexual diploids. we addressed the following specific questions 1) do sexual gibel carp exhibit a higher diversity of mhc genotypes than asexual gibel carp? 2) do asexual gibel carp suffer from higher parasite loads than sexual gibel carp?  the mixed population of gibel carp in the locality studied was composed of 60% triploid individuals and 40% diploid individuals with no variation between two consecutive years. approximately the same number of triploid and diploid individuals, all of them with a similar total body size, were analyzed. variability of dab genes different genotype diversity was found for gynogenetic triploids and sexual diploids. diploids expressed genotypes whilst triploids expressed only genotypes. two genotypes were common in triploids, i.e. 53% of triploid females expressed the genotype including cagi-dab3* and cagi-dab3* alleles and 26% of triploid females expressed the genotype including cagi-dab1* cagi-dab3* and cagi-dab3* alleles. other triploids expressed rare genotypes. all genotypes of diploids were rare, i.e. expressed by only one or two individuals except for the genotype including one allele cagi-dab1* found in diploids. the individuals of both sexual diploids and gynogenetic triploids expressed genotypes including the alleles of a single lineage or the alleles of both lineages dab1-like and dab3-like. due to the expression of the common genotype “a” of the dab3-like lineage in gynogenetic triploids, 62% of triploid individuals expressed the genotypes belonging to a single dab3-like lineage. meanwhile, 32% of triploid individuals expressed the combined genotypes, whilst only 6% expressed the genotypes of the dab1-like lineage. a different ratio of genotypes was found in diploids, where 45% of diploid individuals expressed the genotypes of the dab1-like lineage, 25% of diploid individuals expressed dab3-like genotypes, and 30% expressed the combined genotypes. at population level, the total number of alleles expressed by sexual diploids and gynogenetic triploids was different. a total of different alleles were recorded in the population of gibel carp investigated, but only alleles were shared between the two forms. the majority of alleles were specific to either diploid or triploid genotypes. three alleles shared by sexual diploids and gynogenetic triploids were expressed in common genotypes of triploids, and one shared allele was the most frequent allele found in diploid genotypes. even if the diploids expressed a wide range of different genotypes, three alleles were more frequent than others i.e. cagi-dab1* cagi-dab1* and cagi-dab3*. no significant influence of year of collection on the number of alleles expressed by individuals was found. therefore, data from two years were pooled for the next analyses. the number of alleles per individual ranged from to dab alleles. the number of alleles per individual expressed by triploids was significantly higher than the number of alleles expressed by diploids. even the total number of different dab sequences was higher for sexual diploids than for gynogenetic triploids at the population level; the mean overall nucleotide diversity was similar for both forms. after translating the nucleotide sequences into amino-acid sequences, the alignment reflected the high level of non-synonymous substitutions in dab1-like and dab3-like genes in both forms. the rate of nonsynonymous substitutions was significantly higher compared to synonymous ones. standard errors estimates shown in parentheses were obtained from bootstrap replicates. numbers following dab gene groups shown in parentheses indicate the number of different allelic variants in the alignment. mean amino-acid variability calculated for the total data set at the population level was slightly higher for sexual diploids than for gynogenetic triploids. a similar nucleotide and amino-acid diversity for the dab1-like gene and for the dab3-like gene was found when comparing both forms. however, the means of nucleotide and amino-acid diversity were higher in the dab3-like data set than in the dab1-like data set. at the individual level, gynogenetic triploids had a significantly higher amino-acid diversity than sexual diploids. however, when corrected for the number of alleles no significant difference in amino-acid diversity was found between gynogenetic triploids and sexual diploids. nucleotide diversity was not significantly different between the two forms of gibel carp. however, the variance in both amino-acid diversity and nucleotide diversity was significantly higher in sexual diploids compared to gynogenetic triploids without correction for the number of alleles and also using correction for the number of alleles. positive selection in dab sequences maximum likelihood estimates of parameters under different codon models of variable ω across sites and the number of pss for both forms of gibel carp are included in table  the lrt statistic comparing the two models indicates that the alternative models m2a, m and m fit the data significantly better than the simpler models m1a, m and m, which indicates the action of positive selection at specific sites in dab sequences. bayes identification of sites under positive selection calculated using the m model is included in figure  the number following the model code given in parentheses represents the number of free parameters for the ω ratios. parameters indicating positive selection are presented in bold type. parameters in parentheses are presented for clarity only but are not free parameters; for example, under m p1 = 1-p0; pss are the positive selected sites identified using the beb method at *: p > 95%; **: p > 99%; ω is the selection parameter; pn is the proportion of sites that fall into the ωn site classes; p and q are the shape parameters of the β function. the estimates from the m model suggest a different pattern of selection in dab1-like and dab3-like genes between both forms, i.e. a different number and different positions of pss in sexual diploids and gynogenetic triploids were found. concerning sexual diploids, a similar number of pss sites were under positive selection in the dab1-like and dab3-like sequences. out of pss identified in dab1-like sequences and out of pss identified in dab3-like sequences corresponded to human antigen binding sites identified using crystallography by brown et al.. for dab sequence variants of gynogenetic triploids, sites were identified as targets of positive selection in dab1-like alignment, of them identical to human abs. a higher number of pss were detected in dab3-like sequences of gynogenetic females compared to the pss identified in dab1-like sequences in this form or pss identified in dab1-like or dab3-like sequences of sexual diploids. out of pss identified in dab3-like sequences of gynogenetic triploids corresponded to human abs. all pss found in dab3-like sequences in sexual diploids were also identified as pss in dab3-like sequences of gynogenetic triploids. pss found in dab1-like sequences of gynogenetic triploids were identified in dab1-like sequences of diploids. mhc and parasitism no significant difference in parasite species richness was found between the fish investigated in and. therefore, the parasitological data were pooled for all analyses. a total of metazoan parasite species were found in the gibel carp studied. among them, monogenea was the species richest group. the prevalences of five dactylogyrus species were high and the prevalences of another three ectoparasite species d. vastator, g. sprostonae and glochidum were moderate. the presence of other parasite species was rare. means of parasite species richness, total parasite abundance, abundance of dactylogyrus, and parasite diversity are shown in table  when comparing infection by parasite species between the two forms of gibel carp, the prevalences of the five dominant dactylogyrus species were similar. the intensities of infection for four of the five dominant dactylogyrus species tended to be higher in triploids than in diploids. moreover, triploids were occasionally parasitized by four rare endoparasite species that were absent in diploids. sc – specific parasite i.e. parasitizing two congeneric c. gibelio and c. carassius, sph – specific parasite i.e. parasitizing phylogenetically related species c. gibelio, c. carassius and cyprinus carpio. g – generalist parasite species. host specificity was taken from moravec. first, when fish mhc was expressed as the number of dab alleles, nested anova revealed no significant effects of fish mhc and reproductive strategy on parasite load measured by parasite species richness or parasite abundance. next, when fish mhc was expressed as the type of dab lineage, nested anova revealed a statistically significant effect exerted by fish mhc and an insignificant effect exerted by reproduction strategy on parasite load expressed as parasite species richness. using the bonferonni post-hoc test, a significant difference was found in parasite species richness only between the dab and dab genotypes, where the dab genotype was more parasitized than the dab genotype. no statistical difference between the genotype of combined lineages and single genotypes was found, demonstrating no significant link between increasing mhc variability and parasite load. to test the red queen hypothesis, first, parasitism was compared between gynogenetic triploids and sexual diploids. a higher mean parasite species richness was found in gynogenetic triploids compared to sexual diploids. however, the difference in parasite species richness between gynogenetic triploids and sexual diploids was not statistically significant. no significant difference in parasite abundance or parasite diversity was found between diploids and triploids. no significant difference was found when comparing the variance in parasite abundance or parasite diversity. next, we divided the mhc genotypes of triploid individuals into three groups: common genotype “a”, common genotype “b”, and rare genotype. the parasitism of each common genotype was compared to the parasitism of gynogenetic triploids with the rare genotype and sexual diploids. significantly higher parasite species richness was found in gynogenetic triploids expressing the common genotype “a” than in sexual diploids and gynogenetic triploids with the rare genotype. however, after bonferroni correction, only the difference between triploids expressing common genotype “a” and sexual diploids was significant. no difference in parasite abundance or parasite diversity was found between gynogenetic triploids expressing the common genotype “a” and sexual diploids or gynogenetic triploids with the rare genotype. a significantly higher parasite abundance was found for triploid individuals with common genotype “b” when compared to sexual diploids or gynogenetic triploids with common genotype “a”. after bonferroni correction, no relationship was significant. dactylogyrus species represented 56% of total parasite species and 98% of total parasite abundance. a significantly higher dactylogyrus abundance was found for triploids with common genotype “b” when compared to sexual diploids or gynogenetic triploids with common genotype “a”. after bonferroni correction, only the difference between gynogenetic triploids with common genotype “b” and sexual diploids was significant. discussion this study presents the first analysis of the genetic diversity of functional mhc class iib genes in gibel carp, a fish species with dual reproduction modes – sexual reproduction in diploids and gynogenesis in triploid females. similarly to previous studies analyzing dab genes in cyprinid fish, high mhc iib diversity was also shown in this diploid-polyploid fish species. both forms express the alleles of one locus or the alleles of both loci, which was shown for diploid cyprinid species using genomic or expressed mhc iib genes. our study revealed the presence of many alleles specific to gynogenetic triploids or specific to sexual diploids. moreover, several alleles were found at high frequencies only in triploids or only in diploids. concerning the specific and frequent alleles found in gynogenetic triploids, we cannot distinguish if they are merely characteristics of our study population, or if they have persisted ever since the species invasion of the czech republic, or even since the origin of the triploid form. such hypotheses could be evaluated using a large sample and time series from a wide range of gibel carp populations. despite the reduced genotype diversity in triploids, similar overall nucleotide and amino-acid diversity at the population level was found for dab datasets in sexual diploids and gynogenetic triploids, which indicates the genetic variation among different gynogenetic lineages of gibel carp. at the individual level, gynogenetic triploids expressed mainly or dab alleles, whilst sexual diploids expressed mainly or dab alleles. this could be explained by polyploidization of the genome, where a higher number of gene copies is expected for polyploid individuals, which might give them a crucial advantage in pathogen recognition. however, following the red queen hypothesis, asexually reproducing organisms should be very susceptible to pathogen infection because of low genotypic diversity which could annihilate the positive effect of having many mhc copies. when compared carassius gibelio to poecilia, between and mhc class iib sequences per individual were found in both asexual amazon molly and sexual sailfin molly. thus, it seems that the high number of mhc iib variants in gynogenetic individuals does not represent a general rule for the species where two forms with different reproduction modes coexist. the number of mhc alleles per individual may be determined by the potential duplication of some mhc lineage found in cyprinid species e.g. duplication of the dab lineage in common carp, duplication of the dab lineage in chub, or duplication of both dab and dab lineages found only for the diploid form of gibel carp in this study. it is also possible that some alleles or allelic lineages are not detectable in cdna, as they are not expressed. high mhc polymorphism is explained by two, though not mutually exclusive hypotheses, i.e. sexual selection and parasite-mediated selection. we tested whether or not the sexual form of gibel carp shows higher mhc polymorphism than the gynogenetic form, following the hypothesis that mhc diversity is maintained by sexual-mediated selection. schaschl et al. presumed that high mhc diversity in the asexual form or comparable diversity between sexual and asexual forms indicates that sexual selection is not important for explaining mhc diversity. less mhc diversity at the allelic as well as genotypic level for asexual species compared to sexual ones was also hypothesized by lampert et al. as an important prerequisite of the red queen hypothesis. in our study, the high mhc genotype and allelic diversity in sexual diploids compared to gynogenetic triploids living in the same habitat is consistent with the prediction that mhc diversity is driven by sexually-mediated selection. on the other hand, reduced mhc genotype diversity in gynogenetic triploids is probably due to the lack of recombination and segregation in these asexually reproducing organisms, where only point mutations could generate new mhc alleles. similar findings were previously shown in poecilia, i.e. lower mhc genotype diversity and the reduced diversity of mhc class i and class iib alleles was found in asexual amazon molly compared to sexual sailfin molly. lower diversity in mhc class i genes was also found in a parthenogenetic gecko species compared to closely related sexual gecko species. the difference in genotype and allelic diversities between asexually and sexually reproducing gibel carp could be the result of sexual selection and/or selection from pathogens and parasites, or may be due to genetic drift or the bottleneck effect after introduction, as was suggested for asexual amazon mollies by schaschl et al.. our study revealed a similar number of positively selected sites in both dab1-like and dab3-like genes in sexual diploids, where the majority of pss were identical with human antigen binding sites. however, when analyzing the selection pattern in dab genes of gynogenetic triploids, the different number of pss between dab1-like and dab3-like sequence alignments was shown. this may suggest that sexual selection or parasite-mediated selection contribute to the maintenance of mhc diversity in diploid-polyploid populations of gibel carp. however, we should emphasize that only extensive experimental study could clarify the mechanisms of selection affecting mhc diversity in gibel carp. in addition, we showed stronger positive selection in dab3-like genes in gynogenetic triploids compared to the selection in dab1-like genes in gynogenetic triploids or to the selection in sexual diploids, which suggests a potential functional difference between dab1-like and dab3-like genes, especially for triploids. parasite-mediated selection together with the coevolutionary “arms races” between hosts and parasites form the core idea of the red queen hypothesis. we showed that, at the population level, mhc genotype and allelic diversities in asexual triploids are reduced compared to sexual diploids. therefore, gibel carp clearly fulfills a prerequisite of the red queen hypothesis underlying the evolution of sex. concerning parasites, their virulence and pathogenicity represent important components of the red queen hypothesis. these features were demonstrated for some gill and skin monogeneans. dactylogyrus parasitizing carassius species and closely related common carp are considered to be the most pathogenic monogenean species on the gills causing fish mortality. we evaluated parasite load, including metazoan parasite species with a high prevalence of dactylogyrus species that are host specific to carassius species or specific to phylogenetically closely related fish species. in our study, the parasite load on gynogenetic and sexual forms was compared following the prediction of the red queen hypothesis, i.e. sex provides an advantage to hosts by creating rare genotypes that confer resistance to parasites. thus, in the case when the genetic diversity of asexual lineages is low, the asexually reproducing form is more intensely infected than the sexual form. our study showed that parasite species richness is slightly higher in the gynogenetic form compared to the sexual form. the finding that the overall amino-acid diversity of dab genes in gynogens was comparable to the amino-acid diversity in the sexual form suggests that the analyzed gynogenetic lineages are genetically diverse. therefore, the subsequent analyses were focused on the parasite load of common mhc iib genotypes in gynogenetic triploids, following the prediction that the parasite load of the sexual form should be lower than that of the more common clone, but that the parasite load of some infrequent clones might not necessarily be higher than that of the sexual form. two common mhc iib genotypes were more parasitized than the genotypes of sexual diploids and rare genotypes of gynogenetic triploid females, which is in line with the prediction of the red queen hypothesis. the present study revealed that at the individual level high mhc diversity is not an important factor determining parasitism, using whichever of the two measures of individual mhc diversity – the number of expressed dab alleles or the type of dab lineage. high diversity in mhc iib genes allows the recognition of more antigens derived from parasites in a population. however, many mhc variants in the individual will also result in self-reactive t-cell elimination. in line with this theory, nowak et al. predicted that an intermediate number of mhc alleles should be associated with minimal parasite load, which was supported by the study of three-spined sticklebacks by wegner et al.. our study on gibel carp showed that higher parasite species richness is linked to the expression of the alleles of the dab lineage. as the common triploid genotype “a” represents the genotype of the dab lineage, this finding simply reflects the high parasite species richness of the most common genotype rather than the importance of the dab lineage for parasitism. moreover, it supports the study of lampert et al. suggesting that a higher allelic copy number in clonal fish might not convey an immune advantage. thus, the present study may indicate that parasitism is one of the plausible candidates for explaining temporal changes in the population character of gibel carp, from the period of its introduction into czech rivers until today. the first invasive populations were triploid females with gynogenetic reproduction; their mhc genotypes were probably infrequent in the new invaded habitats. it is possible that triploid clones were able to become common and a potential target for parasite selection due to their high reproductive ability linked to gynogenesis. what might explain the recent coexistence of gynogenetic and sexual forms of gibel carp? hakoyama and iwasa showed that parasitism plays an important role in bringing about the coexistence of asexual and sexual forms in japanese crucian carp by giving a frequency dependent benefit to the sexual population. this supports the prediction that the coexistence of sexual and unisexual forms should be stabilized by parasites. the high susceptibility to diseases in the unisexual form could compensate the two-fold cost of sex in the sexual form. as recent knowledge on the distribution of gibel carp in central europe shows that both sexual and gynogenetic forms coexist in many natural populations, we suggest that the higher parasite load in common gynogenetic triploid genotypes could represent one of the possible factors promoting the coexistence of this sexual-gynogenetic complex. hakoyama et al. showed that the high prevalence of metagonnimus in japanese crucian carp is in part due to the lower immune activity of the phagocytes in the gynogenetic form. therefore, for further studies of gibel carp, we suggest analyzing measurements of specific and non-specific immunity to investigate whether enhanced parasite load in gynogenetic females reflects an immune disadvantage. however, the coexistence of sexual and unisexual forms may also be maintained by male mate choice or spatial and temporal extinction and recolonization, factors that have not yet been analyzed in c. gibelio. moreover, for a better understanding of whether the coexistence of sexual and gynogenetic forms in the populations of gibel carp is stable, or whether the evolution of gibel carp populations is directed toward strictly sexual reproduction, long-term studies are needed. the latter possibility was demonstrated for sexual and asexual snails by jokela et al., where co-evolutionary dynamics predicted by the red queen hypothesis favor sexual reproduction in natural populations, i.e. the most common clones were almost completely replaced by initially rare clones, while the sexual form persisted throughout a long period of investigation.  our study investigated the genetic diversity of mhc class iib genes and the selection mechanisms potentially driving mhc diversity in diploid-polyploid fish species with dual reproduction strategies. in mixed populations of gibel carp, where gynogenetic triploids coexist with sexually reproducing diploids, the genotype and allelic diversity is reduced in triploids compared to diploids, which is in accordance with the hypothesis of sexually-mediated selection increasing the mhc diversity and fulfils a prerequisite of the red queen hypothesis. we showed that the most common genotypes of gynogenetic triploids are the target of parasite selection. this finding also supports the prediction of the red queen hypothesis. on the other hand, the individual allelic copy number does not seem to be important for fish immunocompetence. we propose that the evolutionary twofold cost of sexual reproduction in diploids is compensated by the limited ability of asexual forms to escape parasitism and, thus, that this process could facilitate the recent coexistence of sexual and gynogenetic forms of gibel carp. 
at the population level, genotype and allelic diversity of mhc were reduced in gynogenetic triploids when compared to sexual diploids. in this study, we investigated mhc variability and the selection pressure acting on mhc genes in sexual diploids and asexual triploids. following the red queen assumption, asexual organisms are, due to their low genetic diversity, targets for parasite adaptation. a weak difference in parasite species richness was found between sexual fish and gynogens. the differences in mhc diversity between gynogenetic triploids and sexual diploids are in accordance with the hypothesis of sexually-mediated selection increasing mhc diversity and fulfil a prerequisite of the red queen hypothesis. we showed that the most common mhc genotypes of gynogenetic triploids are the target of parasite selection. because mhc polymorphism is maintained by selection from parasites and sexual selection, mhc genes are considered as a suitable candidate for testing the rq hypothesis. the gibel carp is a fish species with dual reproduction modes, gynogenesis and sexual reproduction, coexisting in mixed diploid-polyploid populations. in addition, we tested whether the asexual form of gibel carp suffers from higher parasite loads than the sexual form. different patterns in positively selected sites between gynogens and sexual gibel carp were also found. the different patterns in pss between gynogens and sexual gibel carp also suggest the potential role of sexual selection and supports parasite-mediated selection maintaining mhc diversity. our results suggest that the mhc genotype in gibel carp is more important than allelic number for immunocompetence. at the individual level, the higher number of alleles was not associated with higher parasitism in either sexual diploids or gynogens. however, two common clones of gynogens were significantly more parasitized than sexual diploids or other gynogens with rare mhc genotypes.
 the gibel carp is a fish species with dual reproduction modes, gynogenesis and sexual reproduction, coexisting in mixed diploid-polyploid populations. following the red queen assumption, asexual organisms are, due to their low genetic diversity, targets for parasite adaptation. because mhc polymorphism is maintained by selection from parasites and sexual selection, mhc genes are considered as a suitable candidate for testing the rq hypothesis. in this study, we investigated mhc variability and the selection pressure acting on mhc genes in sexual diploids and asexual triploids. in addition, we tested whether the asexual form of gibel carp suffers from higher parasite loads than the sexual form.  at the population level, genotype and allelic diversity of mhc were reduced in gynogenetic triploids when compared to sexual diploids. different patterns in positively selected sites between gynogens and sexual gibel carp were also found. a weak difference in parasite species richness was found between sexual fish and gynogens. however, two common clones of gynogens were significantly more parasitized than sexual diploids or other gynogens with rare mhc genotypes. at the individual level, the higher number of alleles was not associated with higher parasitism in either sexual diploids or gynogens.  the differences in mhc diversity between gynogenetic triploids and sexual diploids are in accordance with the hypothesis of sexually-mediated selection increasing mhc diversity and fulfil a prerequisite of the red queen hypothesis. the different patterns in pss between gynogens and sexual gibel carp also suggest the potential role of sexual selection and supports parasite-mediated selection maintaining mhc diversity. we showed that the most common mhc genotypes of gynogenetic triploids are the target of parasite selection. our results suggest that the mhc genotype in gibel carp is more important than allelic number for immunocompetence. gynogenesissexual reproductionpolyploid fishmhccoexistenceparasitism 