Quantitative Analyses of Interbreeding in Populations of vannus-Morphotype Euplotes, with Special Attention to the Nominal Species E. vannus and E. crassus

ABSTRACT. Classical genetic techniques were applied to clonal cultures of the Euplotes vannus-crassus-minuta sibling species complex in an effort to provide some resolution to the species problem among these hypotrichs. Complex mating interactions were observed among clonal stock cultures derived from samples collected from sympatric and allopatric populations in a wide geographic survey. These results suggested that the classical model for the mating type inheritance and determination in these Euplotes is necessary but not sufficient to describe the mating mating interactions among populations of these ciliates. Successful conjugation between the nominal species E. crassus and E. vannus was observed routinely, and crosses between these two nominal species did not differ significantly from those among the other clonal stock cultures with respect to mating intensity and exconjugant survival. Data from backcrosses suggests that E. vannus and E. crassus can and do exchange genes. Based upon these data, we conclude that E. vannus and E. crassus comprise a single, highly polymorphic species with countless small populations, among which incomplete genetic exchange takes place.     

Assessing the impact of stocking northern-origin hatchery brook trout on the genetics of wild populations in North Carolina

The release of hatchery-origin fish into streams with endemics can degrade the genetics of wild populations if interbreeding occurs. Starting in the 1800s, brook trout descendent from wild populations in the northeastern United States were stocked from hatcheries into streams across broad areas of North America to create and enhance fishery resources. Across the southeastern United States, many millions of hatchery-origin brook trout have been released into hundreds of streams, but the extent of introgression with native populations is not well resolved despite large phylogeographic distances between these groups. We used three assessment approaches based on 12 microsatellite loci to examine the extent of hatchery introgression in 406 wild brook trout populations in North Carolina. We found high levels of differentiation among most collections (mean F′_ST = 0.718), and among most wild collections and hatchery strains (mean F′_ST = 0.732). Our assessment of hatchery introgression was consistent across the three metrics, and indicated that most wild populations have not been strongly influenced by supplemental stocking. However, a small proportion of wild populations in North Carolina appear to have been strongly influenced by stocked conspecifics, or in some cases, may have been founded entirely by hatchery lineages. In addition, we found significant differences in the apparent extent of hatchery introgression among major watersheds, with the Savannah River being the most strongly impacted. Conversely, populations in the Pee Dee River watershed showed little to no evidence of hatchery introgression. Our study represents the first large-scale effort to quantify the extent of hatchery introgression across brook trout populations in the southern Appalachians using highly polymorphic microsatellite markers.     

Discordant patterns of nuclear and mitochondrial introgression in Iberian populations of the European common frog (Rana temporaria)

Amphibians often show complex histories of intraspecific and interspecific genetic introgression, which might differ in mitochondrial and nuclear genes. In our study of the genetic differentiation of the European common frog, Rana temporaria (159 specimens from 23 populations were analyzed for 24 presumptive allozyme loci; 82 specimens were sequenced for a 540-bp fragment of the mitochondrial 16S rRNA gene), multilocus correspondence analysis (CA) and Bayesian assignment tests of the nuclear data were concordant in identifying 2 population groups corresponding to 1) the Pyrenees in the east and 2) the Galicia and Asturias regions in the west, the latter corresponding to the subspecies R. temporaria parvipalmata. Geographically intermediate populations were genetically intermediate in the allozyme CA and, less clearly in the Bayesian assignment, with mitochondrial haplotypes exclusively belonging to the parvipalmata group. This indicates different degrees of introgression in the mitochondrial and nuclear genomes. Although Pyrenean high-altitude populations are morphologically distinct from low-altitude populations, these 2 groups were not separate clusters in any analysis. This suggests that the morphological differences may be due to fast adaptation to elevational gradients, likely under maintenance of gene flow, and that the underlying genetic changes are not detectable by the analyzed markers. We argue that a parsimonious explanation for the observed pattern along the east-west axis in northern Spain may be competition between invading and resident populations, with no need to invoke selection. However, in order to conclusively rule out selective processes, additional and finer scale data are required to test for asymmetric mating preference/behaviour, sex-biased gene flow, or sex-biased survival of potential hybrids.     

Restricted mating dispersal and strong breeding group structure in a mid-sized marsupial mammal (Petrogale penicillata)

Ecological genetic studies have demonstrated that spatial patterns of mating dispersal, the dispersal of gametes through mating behaviour, can facilitate inbreeding avoidance and strongly influence the structure of populations, particularly in highly philopatric species. Elements of breeding group dynamics, such as strong structuring and sex-biased dispersal among groups, can also minimize inbreeding and positively influence levels of genetic diversity within populations. Rock-wallabies are highly philopatric mid-sized mammals whose strong dependence on rocky terrain has resulted in series of discreet, small colonies in the landscape. Populations show no signs of inbreeding and maintain high levels of genetic diversity despite strong patterns of limited gene flow within and among colonies. We used this species to investigate the importance of mating dispersal and breeding group structure to inbreeding avoidance within a 'small' population. We examined the spatial patterns of mating dispersal, the extent of kinship within breeding groups, and the degree of relatedness among brush-tailed rock-wallaby breeding pairs within a colony in southeast Queensland. Parentage data revealed remarkably restricted mating dispersal and strong breeding group structuring for a mid-sized mammal. Breeding groups showed significant levels of female kinship with evidence of male dispersal among groups. We found no evidence for inbreeding avoidance through mate choice; however, anecdotal data suggest the importance of life history traits to inbreeding avoidance between first-degree relatives. We suggest that the restricted pattern of mating dispersal and strong breeding group structuring facilitates inbreeding avoidance within colonies. These results provide insight into the population structure and maintenance of genetic diversity within colonies of the threatened brush-tailed rock-wallaby.     

Patterns of nucleotide diversity in two species of Mimulus are affected by mating system and asymmetric introgression

The evolutionary transition from outcrossing to self-fertilization has far-reaching implications for patterns of intraspecific genetic diversity and the potential for speciation. Using DNA sequence variation at two nuclear loci, we examined the divergence history of two closely related species of Mimulus. To investigate the effects of mating system and introgressive hybridization on the outcrossing M. guttatus and the selfing M. nasutus, we inspected nucleotide diversity within and between natural populations spanning the species' geographic ranges. High sequence similarity among populations of the selfing M. nasutus points to a single evolutionary origin for the species. Consistent with their distinct mating systems, all genetic variation in M. nasutus is distributed among populations, whereas M. guttatus exhibits appreciable levels of nucleotide diversity within populations. Silent genetic diversity is extensive in M. guttatus (mean theta(sil)/site = 0.077) and greatly exceeds the predicted twofold elevation in neutral variation for outcrossers relative to selfers. The finding of several M. guttatus sequences that share complete identity with sequences from M. nasutus suggests that recent asymmetric introgression may have occurred. We argue that exceptionally high nucleotide diversity in M. guttatus is consistent with a long-term history of directional introgression from M. nasutus to M. guttatus throughout the divergence of these two species.     

On quantifying tolerance of herbivory for comparative analyses

As the evolutionary importance of plant tolerance of herbivory is increasingly appreciated, more and more studies are not just measuring a plant's tolerance, but are comparing tolerance among plant genotypes, populations, species, and environments. Here, we suggest that caution must be taken in such comparative studies in the choice of measurement scales (and data transformations) for damage levels and plant performance. We demonstrate with a simple scenario of two plant groups of equal tolerance how the choice of scales can lead one to infer that the first group is more tolerant, the second group is more tolerant, or the two groups are equally tolerant-using the identical dataset. We conclude that to make reliable, logically consistent inferences when comparing tolerances among groups of plants, damage and performance should both be on an additive scale or both on a multiplicative scale.     

ECOLOGICAL SEGREGATION IN A SMALL MAMMAL HYBRID ZONE: HABITAT‐SPECIFIC MATING OPPORTUNITIES AND SELECTION AGAINST HYBRIDS RESTRICT GENE FLOW ON A FINE SPATIAL SCALE

The degree to which closely related species interbreed is determined by a complex interaction of ecological, behavioral, and genetic factors. We examine the degree of interbreeding between two woodrat species, Neotoma bryanti and N. lepida, at a sharp ecological transition. We identify the ecological association of each genotypic class, assess the opportunity for mating between these groups, and test whether they have similar patterns of year‐to‐year persistence on our study site. We find that 13% of individuals have a hybrid signature but that the two parental populations and backcrosses are highly segregated by habitat type and use. Also, we find that adult hybrids are comparable to parental types in terms of year‐to‐year persistence on our site but that, among juveniles, significantly fewer hybrids reach adulthood on site compared to their purebred counterparts. Our analyses show that this hybrid zone is maintained by occasional nonassortative mating coupled with hybrid fertility, but that these factors are balanced by lower apparent survival of juvenile hybrids and habitat‐based preference or selection that limits heterospecific mating while promoting backcrossing to habitat‐specific genotypes. This system presents a novel example of the role that sharp resource gradients play in reproductive isolation and the potential for genetic introgression.     

Introgression between highly divergent fungal sister species

To understand how species evolve and adapt to changing environments, it is important to study gene flow and introgression due to their influence on speciation and radiation events. Here, we apply a novel experimental system for investigating these mechanisms using natural populations. The system is based on two fungal sister species with morphological and ecological similarities occurring in overlapping habitats. We examined introgression between these species by conducting whole genome sequencing of individuals from populations in North America and Europe. We assessed genome-wide nucleotide divergence and performed crossing experiments to study reproductive barriers. We further used ABBA–BABA statistics together with a network analysis to investigate introgression, and conducted demographic modelling to gain insight into divergence times and introgression events. The results revealed that the species are highly divergent and incompatible in vitro. Despite this, small regions of introgression were scattered throughout the genomes and one introgression event likely involves a ghost population (extant or extinct). This study demonstrates that introgression can be found among divergent species and that population histories can be studied without collections of all the populations involved. Moreover, the experimental system is shown to be a useful tool for research on reproductive isolation in natural populations.     

Patterns of mating call preferences in túngara frogs, Physalaemus pustulosus

We examine acoustic mating preferences of a focal population at four different scales of divergence: within the population, between populations in the same genetic group, between populations in different genetic groups and between different species. At all scales there is substantial genetic divergence, variation in mating signals and preferences are influenced by signal variation. There is, however, no support for the hypothesis that mating preferences accumulate predictably with genetic distance. Females preferred the local conspecific call to the foreign conspecific call in about one-third of the experiments, and preferred the local call to all of the heterospecific calls tested. But there was no significant relationship between the variation in the strength of preference and genetic distance either among conspecific populations, or among heterospecific species. Thus, in this study macroevolutionary patterns are not apparent at the microevolutionary scale.     

High domestic pig contribution to the local gene pool of free-living European wild boar: a case study in Poland

Rates of hybridization between wild and domesticated animals appear to be increasing worldwide. Recent results suggest that genetic introgression from domestic swine into European wild boar is much more common in local populations than expected, based on pan-European studies. Thus, we screened the genetic purity of 265 free-living wild boars from two hunting areas in Poland by genotyping the melanocortin receptor 1 gene (MC1R) for polymorphism. Unexpectedly, high numbers of individuals with domestic genes (24%) were identified. This suggests that mixed ancestry may be common in Polish wild boar. Among admixed individuals, backcrosses with domestic pig and/or introgressed wild boars were detected (2%). Multiple commercial domestic pig breeds are possibly involved in the introgression observed in the study populations. In addition, the absence of significant differences in the frequency of wild-type allele among two hunting areas suggests high dispersal of individuals and gene flow among populations. We conclude that further study is needed to better understand the mechanisms and sources of introgression in wild boars in Poland.     

Assortative mating by size: A meta-analysis of mating patterns in water striders

Summary Assortative mating by size is a common mating pattern that can be generated by several different behavioural mechanisms, with different evolutionary implications. Assortative mating is typically associated with sexual selection and has been regarded as an attribute of populations, species, mating systems or even higher order taxa. In most animal groups, however, appropriate analyses of assortative mating at these different levels are lacking and the causes and forms of assortative mating are poorly understood. Here, we analyse 45 different population level estimates of assortative mating and non-random mating by size in seven confamiliar species of water striders that share a common mating system. A hierarchical comparative analysis shows that virtually all the variance within the clade occurs among samples within species. We then employ meta-analysis to estimate the overall strength of assortative mating, to determine the form of assortative mating and to further assess potential differences among species as well as the probable causes of assortative mating in this group of insects. We found overall weak but highly significant positive assortative mating. We show that analyses of the degree of heteroscedasticity in plots of male versus female size are critical, since the evolutionary implications of true and apparent assortative mating differ widely and conclude that the positive assortative mating observed in water striders was of the true rather than the apparent form. Further, within samples, mating individuals were significantly larger than non-mating individuals in both males and females. All of these non-random mating patterns were consistent among species and we conclude that weak positive assortative mating by size is a general characteristic of those water strider species that share this mating system. We use our results to illustrate the importance of distinguishing between different forms of assortative mating, to discriminate between various behavioural causes of assortative mating and to assess potential sources of interpopulational variance in estimates of assortative mating. Finally, we discuss the value of using meta-analytic techniques for detecting overall patterns in multiple studies of non-random mating.     

Hybridization and reproductive isolation among syntopic populations of the topminnows Fundulus notatus and F. olivaceus

Fundulus notatus and Fundulus olivaceus are two closely related topminnow species that exhibit similar ecological niches and broad, largely overlapping, North American ranges extending throughout much of the Mississippi River drainage as well as the coastal drainages of the Gulf of Mexico. Previous studies have suggested that these two species are reproductively compatible despite cytogenetic differences and will hybridize when syntopic. We used nuclear and mtDNA loci to assess levels of hybridization and test for introgression in syntopic populations of these two species in four drainages in southern Illinois. Although hybridization was detected in all syntopic populations, an assessment of the proportion of hybrid individuals indicated a deficiency of hybrids relative to expectations under random mating. We determined that, although mtDNA introgression was prevalent and extended beyond the zones of contact, evidence of nuclear introgression was limited to the zone of sympatry.     

Viral Richness is Positively Related to Group Size,but Not Mating System,in Bats

Characterizing host traits that influence viral richness and diversification is important for understanding wildlife pathogens affecting conservation and/or human health. Behaviors that affect contact rates among hosts could be important for viral diversification because more frequent intra- and inter-specific contacts among hosts should increase the potential for viral diversification within host populations. We used published data on bats to test the contact-rate hypothesis. We predicted that species forming large conspecific groups, that share their range with more heterospecifics (i.e., sympatry), and with mating systems characterized by high contact rates (polygynandry: multi-male/multi-female), would host higher viral richness than species with small group sizes, lower sympatry, or low contact-rate mating systems (polygyny: single male/multi-female). Consistent with our hypothesis and previous research, viral richness was positively correlated with conspecific group size although the relationship plateaued at group sizes of approximately several hundred thousand bats. This pattern supports epidemiological theory that, up to a point, larger groups have higher contact rates, greater likelihood of acquiring and transmitting viruses, and ultimately greater potential for viral diversification. However, contrary to our hypothesis, there was no effect of sympatry on viral richness and no difference in viral richness between mating systems. We also found no residual effect of host phylogeny on viral richness, suggesting that closely related species do not necessarily host similar numbers of viruses. Our results support the contact-rate hypothesis that intra-specific viral transmission can enhance viral diversification within species and highlight the influence of host group size on the potential of viruses to propagate within host populations.     

Conspecific crop-weed introgression influences evolution of weedy rice (Oryza sativa f. spontanea) across a geographical range

Background

Introgression plays an important role in evolution of plant species via its influences on genetic diversity and differentiation. Outcrossing determines the level of introgression but little is known about the relationships of outcrossing rates, genetic diversity, and differentiation particularly in a weedy taxon that coexists with its conspecific crop.

Methodology/Principal Findings

Eleven weedy rice (Oryza sativa f. spontanea) populations from China were analyzed using microsatellite (SSR) fingerprints to study outcrossing rate and its relationship with genetic variability and differentiation. To estimate outcrossing, six highly polymorphic SSR loci were used to analyze >5500 progeny from 216 weedy rice families, applying a mixed mating model; to estimate genetic diversity and differentiation, 22 SSR loci were analyzed based on 301 weedy individuals. Additionally, four weed-crop shared SSR loci were used to estimate the influence of introgression from rice cultivars on weedy rice differentiation. Outcrossing rates varied significantly (0.4∼11.7%) among weedy rice populations showing relatively high overall Nei''s genetic diversity (0.635). The observed heterozygosity was significantly correlated with outcrossing rates among populations (r² = 0.783; P<0.001) although no obvious correlation between outcrossing rates and genetic diversity parameters was observed. Allelic introgression from rice cultivars to their coexisting weedy rice was detected. Weedy rice populations demonstrated considerable genetic differentiation that was correlated with their spatial distribution (r² = 0.734; P<0.001), and possibly also influenced by the introgression from rice cultivars.

Conclusions/Significance

Outcrossing rates can significantly affect heterozygosity of populations, which may shape the evolutionary potential of weedy rice. Introgression from the conspecific crop rice can influence the genetic differentiation and possibly evolution of its coexisting weedy rice populations.     

Genetic structure in a dynamic baboon hybrid zone corroborates behavioural observations in a hybrid population

Behaviour and genetic structure are intimately related: mating patterns and patterns of movement between groups or populations influence the movement of genetic variation across the landscape and from one generation to the next. In hybrid zones, the behaviour of the hybridizing taxa can also impact the incidence and outcome of hybridization events. Hybridization between yellow baboons and anubis baboons has been well documented in the Amboseli basin of Kenya, where more anubis-like individuals tend to experience maturational and reproductive advantages. However, it is unknown whether these advantages are reflected in the genetic structure of populations surrounding this area. Here, we used microsatellite genotype data to evaluate the structure and composition of baboon populations in southern Kenya. Our results indicate that, unlike for mitochondrial DNA, microsatellite-based measures of genetic structure concord with phenotypically based taxonomic distinctions and that the currently active hybrid zone is relatively narrow. Isolation with migration analysis revealed asymmetric gene flow in this region from anubis populations into yellow populations, in support of the anubis-biased phenotypic advantages observed in Amboseli. Populations that are primarily yellow but that receive anubis gene flow exhibit higher levels of genetic diversity than yellow populations far from the introgression front. Our results support previous work that indicates a long history of hybridization and introgression among East African baboons. Specifically, it suggests that anubis baboons are in the process of gradual range expansion into the range of yellow baboons, a pattern potentially explained by behavioural and life history advantages that correlate with anubis ancestry.     

Molecular and morphological patterns of introgression between two large white-headed gull species in a zone of recent secondary contact

Incomplete reproductive isolation promotes gene flow between diverging taxa. However, any gene encoding for traits involved in the reproductive barriers will be less prone to introgression than neutral markers. Comparing introgression rates among loci is thus informative of the number and functions of loci involved in the reproductive barriers. This study aimed at identifying possible mechanisms of restriction to gene flow across a zone of recent secondary contact between Larus argentatus and Larus cachinnans by comparing introgression patterns for nine microsatellite loci, a fragment of mitochondrial DNA and a set of phenotypic traits. The low linkage disequilibrium between neutral nuclear markers indicated introgression without any barrier to gene flow. However, asymmetric introgression of mitochondrial DNA suggested that interspecific crosses may be more successful in one direction. The introgression rate for phenotypic traits was variable and low compared to neutral molecular markers. This was particularly evident in colouration of bare parts: individuals with intermediate colouration were scarcer in sympatry than expected if the genomes recombined freely. We hypothesized that one of these variables, the orbital ring colour, may play a role in mate choice, acting as an incomplete premating barrier through assortative mating. This study emphasizes that multilocus approaches are useful to discriminate among possible mechanisms responsible for the maintenance of hybrid zones.     

Regulation of Anguillicola crassus (Nematoda) infrapopulations in their definitive host, the European eel, Anguilla anguilla

The parasitic nematode Anguillicola crassus was recently introduced into populations of the European eel, Anguilla anguilla. We investigated, under experimental conditions, the regulation of A. crassus infrapopulations. We tested the effects of (1) the resource-limited habitat of the parasite and (2) the coexistence of several developmental stages in its niche (the swim-bladder) on the composition of the infrapopulations. The results revealed that the respective effects of these factors differed substantially during the course of the infection. Third-stage larvae (L3s) establishment would not be constrained by the size of the swim-bladder. Their moult to fourth-stage larvae (L4s) would be accelerated as the number of L3s increased. The moulting time of L4s to adults would be reduced by males and would be constrained by the size of the swim-bladder. However, the moult of L4s to adults and their further development would be synchronized with those of the opposite sex. At the time of mating, the number of males and the body weight of adults would depend on the size of the swim-bladder. Soon after the laying of eggs, the developmental constraint on the late L3s would decrease. When adults die, constraints would cease and late larval stages would moult to become adults.     

Genetic structure of wild sweet chestnut (<Emphasis Type="Italic">Castanea sativa</Emphasis> Mill.) populations in northwest of Spain and their differences with other European stands

The genetic structure of 15 wild populations of Castanea sativa located in Galicia (northwest of Spain) were compared with another 10 Spanish, 2 Italian, and 2 Greek populations using 9 nuclear microsatellites. Additionally, 41 Castanea crenata, 19 Castanea mollissima and 54 accessions of varieties were used to detect the presence of Asiatic germplasm and grafted varieties in wild stands. Bayesian methods detected two main clusters in the European populations of sweet chestnut: a Northern Iberian Peninsula cluster included the northern and northwestern Spanish populations and a Mediterranean cluster contained central and southern Spanish, Italian and Greek populations. Further subdivisions were also detected. The Northern Iberian Peninsula cluster was divided into Cantabrian and Atlantic clusters and the Mediterranean cluster was divided into western and eastern Mediterranean clusters. Several populations were highly affected by domestication because they were classified as admixed among the four defined groups. Negative values for the fixation index were detected in northern and northwestern Spain showing an excess of heterozygotes that could be explained by the introduction of grafted varieties and its introgression with local populations or by natural selection in favor of heterozygotes produced by ink disease. The combined analysis with the Asiatic species also indicated their presence and possible introgression in the wild populations of northwestern Spain. The results of this study suggest that the current genetic structure of sweet chestnut can be explained by existence of several refugia during Pleistocene and also by human activity that changes the structure of the populations.     

Broad geographic covariation between floral traits and the mating system in Camissoniopsis cheiranthifolia (Onagraceae): multiple stable mixed mating systems across the species' range?

Background and Aims

Plants vary widely in the extent to which seeds are produced via self-fertilization vs. outcrossing, and evolutionary change in the mating system is thought to be accompanied by genetic differentiation in a syndrome of floral traits. We quantified the pattern of variation and covariation in floral traits and the proportion of seeds outcrossed (t) to better understand the evolutionary processes involved in mating system differentiation among and within populations of the short-lived Pacific coastal dune endemic Camissoniopsis cheiranthifolia across its geographic range in western North America.

Methods

We quantified corolla width and herkogamy, two traits expected to influence the mating system, for 48 populations sampled in the field and for a sub-sample of 29 populations grown from seed in a glasshouse. We also measured several other floral traits for 9–19 populations, estimated t for 16 populations using seven allozyme polymorphisms, and measured the strength of self-incompatibility for nine populations.

Key Results

Floral morphology and self-incompatibility varied widely but non-randomly, such that populations could be assigned to three phenotypically and geographically divergent groups. Populations spanned the full range of outcrossing (t = 0·001–0·992), which covaried with corolla width, herkogamy and floral life span. Outcrossing also correlated with floral morphology within two populations that exhibited exceptional floral variation.

Conclusions

Populations of C. cheiranthifolia seem to have differentiated into three modal mating systems: (1) predominant outcrossing associated with self-incompatibility and large flowers; (2) moderate selfing associated with large but self-compatible flowers; and (3) higher but not complete selfing associated with small, autogamous, self-compatible flowers. The transition to complete selfing has not occurred even though the species appears to possess the required genetic capacity. We hypothesize that outcrossing populations in this species have evolved to different stable states of mixed mating.     

Extinction and introgression in a community of partially cross-fertile plant species

We develop a model to study the demography and genetics of an encounter between two partially cross-fertile plant species. We assume prezygotic reproductive isolation between the species, a common situation when the species differ by their phenology or floral traits that cause assortative mating. Three outcomes are possible: coexistence of both species with minimal introgression; domination by one species, with the other becoming extinct or surviving only through recurrent migration; or domination of the community by hybrid derivatives, with both species surviving but with a rather high level of introgression between them. The first situation is reached when interfertility is low, while the third requires high interfertility to develop. Occurrence of the second situation is observed with intermediate values of interfertility. Gene flow from nearby monospecific populations can prevent both introgression and the domination of the community by one species. Conversely, increasing the number of loci that determine the reproductive isolation between species or decreasing the degree of nonadditive interactions (epistasis and/or dominance) between alleles and loci makes introgression more likely. We found that hybridization can create positive frequency dependence and make extinction possible, even when hybrid individuals have no intrinsic fitness advantage.