Anthony G E Mathiron
email: anthony.mathiron@etu.univ-tours.fr

Patrice Pottier

Ryan Earley

Marlène Goubault

Keep calm, we know each other: kin recognition affects aggressiveness and conflict resolution in a solitary parasitoid

Keywords: Eupelmus vuilleti, familiarity, genetic relatedness, phenotype matching, subjective resource value

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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Keep calm, we know each other: kin recognition affects aggressiveness and conflict resolution in a solitary parasitoid Abstract -Intraspecific competition for indivisible resources can trigger the expression of agonistic behaviour in individuals of many animal species. Aggressiveness and conflict resolution may be influenced by the value individuals place in the resource (subjective resource value) but also by genetic relatedness between competitors. The ability to differentiate genetically related from unrelated individuals (i.e., kin recognition) can play a key role in the dynamics of agonistic interactions between individuals. In this context, the theory of kin selection predicts that competitors should display fewer aggressive behaviours towards closely related individuals. Recognition of kin conspecifics can be driven by the perception of [START_REF] Goubault | Previous experience and contest outcome: winner effects persist in absence of evident loser effects in a parasitoid wasp[END_REF] genetically-linked phenotypic cues (phenotypic matching) and/or (2) environmental cues (familiarity). In the hymenopteran solitary parasitoid Eupelmus vuilleti, individuals develop on larvae and pupae of their host, Callosobruchus maculatus, which infest cowpea seeds (Vigna unguiculata). Eupelmus vuilleti females can fight for the host on which they lay their eggs.

Here, we investigated the effect of genetic relatedness (genotype) and familiarity (the seed containing the host on which they develop as juvenile) on aggressiveness and contests outcome over hosts in E. vuilleti females. We first demonstrated that the probability of a conflict escalating was affected by the interaction between genetic relatedness and familiarity among females. We then found that familiarity alone affected the likelihood of contest resolution. The occurrence of escalated conflicts was reduced between related and familiar females, and contests were more likely to be clearly resolved when occurring between familiar competitors.

INTRODUCTION

Intraspecific competition for an indivisible resource can lead to direct interactions between individuals (i.e., conflicts, Hardy & Briffa, 2013). To prevent other individuals from accessing the resource, animals often exhibit agonistic behaviour during contests [START_REF] Hack | Assessment strategies in the contests of male crickets, Acheta domesticus (L.)[END_REF][START_REF] Riechert | Game theory and animal contests[END_REF]. Winning a conflict can improve access to sexual partners [START_REF] West-Eberhard | Sexual selection, social competition, and speciation[END_REF], food resources [START_REF] Vogel | Rank differences in energy intake rates in white-faced capuchin monkeys, Cebus capucinus: The effects of contest competition[END_REF], territories [START_REF] Festa-Bianchet | Aggression among lekking male fallow deer (Dama dama): territory effects and relationship with copulatory success[END_REF] or dominance status [START_REF] Chase | Models of hierarchy formation in animals[END_REF], but being aggressive can potentially be costly in terms of energy expenditure [START_REF] Smith | The theory of games and the evolution of animal conflicts[END_REF], physical injuries [START_REF] Palombit | Lethal territorial aggression in a white-handed gibbon[END_REF][START_REF] Innocent | Lethal combat over limited resources: Testing the importance of competitors and kin[END_REF], or increased predation risk [START_REF] Jakobsson | Escalated fighting behaviour incurs increased predation risk[END_REF]. Animals should therefore evaluate the costs and benefits of exhibiting agonistic behaviours and adjust their behaviour accordingly [START_REF] Smith | The logic of animal conflict[END_REF]. In the case of dyadic interactions, game theory predicts that contest outcome should be influenced by the asymmetries that exist between contestants in terms of their intrinsic ability to obtain and hold access to a resource ('Resource Holding Potential', RHP; [START_REF] Smith | The theory of games and the evolution of animal conflicts[END_REF][START_REF] Parker | Assessment strategy and the evaluation of fighting behaviour[END_REF], and the value they place on the resource ('Subjective Resource Value', SRV; Maynard [START_REF] Smith | The logic of asymmetric animal contests[END_REF][START_REF] Hammerstein | The role of asymmetries in animal contests[END_REF][START_REF] Enquist | Evolution of fighting behaviour: The effect of variation in resource value[END_REF]. Individuals with higher RHP (e.g., bigger contestants) typically win the resource (Maynard Smith, 1982;[START_REF] Elias | Assessment during aggressive contests between male jumping spiders[END_REF]Arnott & Elwood, 2009), and individuals with higher SRV (e.g., with greater egg load or lower food intake) are expected to be more motivated to fight and have a higher probability of winning contests [START_REF] Stokkebo | The importance of being gravid: egg load and contest outcome in a parasitoid wasp[END_REF][START_REF] Dissanayake | Physiological condition and intraspecific agonistic behaviour in Carcinus maenas (Crustacea: Decapoda)[END_REF]. Moreover, these factors have been shown to influence aggressiveness, the most aggressive contestants being generally more likely to win conflicts (Goubault et al., 2007;[START_REF] Mohamad | Can subjective resource value affect aggressiveness and contest outcome in parasitoid wasps?[END_REF][START_REF] Mathiron | Let the most motivated win : resource value components affect contest outcome in a parasitoid wasp[END_REF].

Aggressiveness during conflict can also vary with the degree of genetic relatedness between competitors [START_REF] West | Cooperation and competition between relatives[END_REF]. Kin selection theory postulates that a trait that does not provide a direct fitness benefit (or to some extent, a negative effect) to the individual who expresses it, can be selected for if it improves the fitness of the individuals carrying the same genes as the protagonist (i.e., indirect fitness; Hamilton, 1964). In the context of conflicts, it would be advantageous for an individual to be less aggressive towards genetically related conspecifics [START_REF] Hamilton | Dispersal in stable habitats[END_REF]Griffin & West, 2002;[START_REF] Lizé | Two components of kin recognition influence parasitoid aggression in resource competition[END_REF] because this would maximize their inclusive fitness [START_REF] West | Testing Hamilton's rule with competition between relatives[END_REF]. However, avoiding conflicts between relatives implies kin recognition, i.e. the identification (neurophysiological process), and discrimination (the expression of different behaviours) of genetically related individuals.

Two types of recognition exist: (i) recognition based on the perception of genetically encoded phenotypic indices [START_REF] Gadagkar | Kin recognition in social insects and other animals-A review of recent findings and a consideration of their relevance for the theory of kin selection[END_REF] and (ii) familiarity, which refers to the recognition of individuals regardless of their genetic relatedness [START_REF] Lizé | Two components of kin recognition influence parasitoid aggression in resource competition[END_REF]. For instance, two individuals can be considered familiar if they meet often, or if they come from the same environment. The ability to recognize and discriminate genetically related individuals has been observed in various animal taxa, such as nematodes [START_REF] Kapranas | Composition of cuticular lipids in the pteromalid wasp Lariophagus distinguendus is host dependent[END_REF], anuran amphibians [START_REF] Blaustein | Kin recognition in anuran amphibians[END_REF] and mammals [START_REF] Heth | Kin recognition in golden hamsters: Evidence for phenotype matching[END_REF]. In social insects, for example, kin recognition and avoidance of competition between relatives have been demonstrated in many species, like in sweat bees Lasioglossum zephyrum, honey bees Apis mellifera [START_REF] West-Eberhard | Kin recognition in animals[END_REF], and in the socially polymorphic ant, Pheidole pallidula [START_REF] Fournier | Social structure and genetic distance mediate nestmate recognition and aggressiveness in the facultative polygynous ant Pheidole pallidula[END_REF].

In non-social insects, on the other hand, this phenomenon seems rarer (for reviews, see [START_REF] Fellowes | Do non-social insects get the (kin) recognition they deserve?[END_REF]Griffin &West, 2002) or even absent, like in the parasitoid wasps Aphidius matricariae [START_REF] Bourdais | Lack of behavioural evidence for kin avoidance in mate choice in a hymenopteran parasitoid (Hymenoptera: Braconidae)[END_REF] and Cotesia glomerata [START_REF] Ruf | No kin discrimination in female mate choice of a parasitoid with complementary sex determination[END_REF]. Theoretical models also predict that kin discrimination may not evolve in populations with a low dispersion of individuals due to the high risk of competition for resource between relatives [START_REF] West | Cooperation and competition between relatives[END_REF].

In hymenopteran parasitoids (i.e. organisms that feed and develop at the expense of a host, resulting in its death), females may compete to feed and lay their eggs on (i.e. ectoparasitoid) or in (i.e. endoparasitoid) a host. Because offspring survival directly depends on the mother's ability to acquire and defend the host [START_REF] Petersen | The importance of being larger: parasitoid intruder -owner contests and their implications for clutch size[END_REF], agonistic interactions may occur between females that may, in some cases, be fatal, as described in some Melittobia species (Godfray, 1994;[START_REF] Hartley | The effect of body size on male-male combat in the parasitoid wasp Melittobia digitata Dahms (Hymenoptera : Eulophidae)[END_REF][START_REF] Innocent | Lethal combat over limited resources: Testing the importance of competitors and kin[END_REF]. In this context, four main factors acting on RHP and/or SRV appear to influence conflict resolution:

(1) relative size and age of the opponents, (2) order of arrival on the resource (resident or intruder), (3) egg load, and (4) previous experience. Bigger and older females usually win contests [START_REF] Petersen | The importance of being larger: parasitoid intruder -owner contests and their implications for clutch size[END_REF]Humphries et al., 2006;Tsai et al., 2014), as do residents [START_REF] Smith | The logic of asymmetric animal contests[END_REF][START_REF] Enquist | Evolution of fighting behaviour: The effect of variation in resource value[END_REF], even if they are smaller than intruders [START_REF] Bentley | The elusive paradox: owner-intruder roles, strategies, and outcomes in parasitoid contests[END_REF]. In addition, individuals with a greater egg load are generally advantaged during combat; these females value the resource more because a greater egg load would allow them to oviposit on the host sooner [START_REF] Stokkebo | The importance of being gravid: egg load and contest outcome in a parasitoid wasp[END_REF][START_REF] Mohamad | Can subjective resource value affect aggressiveness and contest outcome in parasitoid wasps?[END_REF]; but see Goubault et al., 2007). Finally, females that have experienced low quality hosts or low host densities are usually more aggressive and win more often in the presence of high quality hosts [START_REF] Mohamad | Can subjective resource value affect aggressiveness and contest outcome in parasitoid wasps?[END_REF][START_REF] Mathiron | Let the most motivated win : resource value components affect contest outcome in a parasitoid wasp[END_REF]. Although the ability to treat kin differentially may influence the dynamics of contests between parasitoids competing for hosts, this has been rarely tested empirically. To our knowledge, only two studies measured the effect of kin recognition on competition avoidance and aggression in hymenopteran parasitoids: [START_REF] Goubault | Previous experience and contest outcome: winner effects persist in absence of evident loser effects in a parasitoid wasp[END_REF] in the polyembryonic wasp (i.e. clonal multiplication of eggs within the host) Copidosoma floridanum [START_REF] Giron | Aggression by polyembryonic wasp soldiers correlates with kinship but not resource competition[END_REF] and (2) in the gregarious (i.e. several larvae develop on the same host) parasitoid Goniozus legneri [START_REF] Lizé | Two components of kin recognition influence parasitoid aggression in resource competition[END_REF]. In the latter study, [START_REF] Lizé | Two components of kin recognition influence parasitoid aggression in resource competition[END_REF] showed that adult females recognized their relatives through phenotypic cues and familiarity (development on the same host), adjusting their aggressiveness accordingly. Familiarity would thus act as a proxy for kinship. However, no study has measured the effect of genetic relatedness on aggression and conflict resolution in a solitary parasitoid species.

Here, we explored the effect of kin recognition on aggressiveness and resolution of conflict in females of the solitary ectoparasitoid wasp, Eupelmus vuilleti (Hymenoptera: Eupelmidae). Females parasitize and feed upon larvae and pupae of the cowpea seed beetle Callosobruchus maculatus (Coleoptera: Bruchidae), which infest seeds of Vigna unguiculata (Fabaceae). Eupelmus vuilleti is a solitary parasitoid, which implies that only one individual develops per host (supernumerary larvae are eliminated by larval competition; [START_REF] Fisher | A study in insect multiparasitism. I. Host selection and oviposition[END_REF].

When several females are simultaneously present on a patch, they tend to protect the host by directing agonistic behaviours towards conspecific competitors [START_REF] Mohamad | Can subjective resource value affect aggressiveness and contest outcome in parasitoid wasps?[END_REF][START_REF] Mathiron | Let the most motivated win : resource value components affect contest outcome in a parasitoid wasp[END_REF]. In sub-Saharan Africa, farmers traditionally stored cowpea seeds in granaries, sometimes resulting in populations of both hosts and parasitoids reaching high densities. Since seeds often contain several hosts, several individuals of E. vuilleti can emerge from the same seed, leading females to experience intense intraspecific competition with both kin and non-kin conspecifics.

We therefore tested whether wasps are able to adjust their aggressiveness during contests for host access depending on their level of genetic relatedness with their competitor.

Because reducing the costs associated with conflicts between kin may be beneficial in terms of inclusive fitness, we expected a decrease in aggressiveness and conflict escalation between related females. Moreover, if kin recognition is effective in E. vuilleti females, this recognition might be based on phenotypic matching, familiarity, or both. Females may use cues coming from genetically encoded phenotypic traits and/or from a common environment. For example, the seed containing hosts on which wasps developed may itself act as a way to identify kin.

Using a full factorial design, we made genetically related or unrelated juveniles develop on hosts within the same seed (familiar) or different seeds (unfamiliar). We then investigated the effects of ( 1) genetic relatedness among adult females (genetically related vs. unrelated), ( 2) the seed containing the hosts on which females developed (same vs. different seeds, i.e. familiar vs. unfamiliar), (3) the absolute difference in SRV (number of ready-to-lay eggs) of individuals, and (4) the interaction between these variables on aggressiveness, contest escalation and resolution. As contests are predicted to be clearly settled and less aggressive when contestants show a large difference in SRV (Maynard [START_REF] Smith | The logic of asymmetric animal contests[END_REF], we expected contests in which the absolute difference in egg load between contestants was large to be less aggressive and often won by the most gravid female. If females recognize kin and avoid fighting against them, this effect of SRV asymmetry should be stronger between unrelated/unfamiliar females.

MATERIAL AND METHODS

Laboratory breeding

We tested individuals of E. vuilleti that were collected on cowpea seeds from crops cultivated in Togo in 2007. Wasps were brought to the laboratory (Institut de Recherche sur la Biologie de l'Insecte, Université de Tours, France) and reared on juvenile stages of C. maculatus, as described by [START_REF] Jaloux | Interspecific relationships between the solitary ectoparasitoid, Eupelmus vuilleti (Crw.) (Eupelmidae), and its sympatric species, Dinarmus basalis (Rond.) (Pteromalidae), in the presence of their host, Callosobruchus maculatus pic (Coleoptera: Bruchidae)[END_REF]. We performed all experimental procedures in a climate room (temperature = 30 °C and photoperiod = 12:12 h light: dark).

Genotypes

To study the influence of genetic relatedness between females on competition for host access, we bred four genetically inbred strains. Working on individuals coming from more than two genotypes minimized the risk of bias due to contest behaviours specific to those genotypes.

To do so, we separately introduced four mating pairs from the same breeding in Petri dishes (diameter: 8.5 cm, height: 2.7 cm) with cowpea seeds infested by C. maculatus and a piece of cotton soaked in water. We isolated females at emergence so that they were virgin and we placed them in the presence of a male. We left couples in contact for one to two days so that females could mate and parasitize hosts. We then removed the wasps to allow offspring development (generation 1, G1). At the emergence of G1 offspring (about 15 days after hatching; [START_REF] Jaloux | Interspecific relationships between the solitary ectoparasitoid, Eupelmus vuilleti (Crw.) (Eupelmidae), and its sympatric species, Dinarmus basalis (Rond.) (Pteromalidae), in the presence of their host, Callosobruchus maculatus pic (Coleoptera: Bruchidae)[END_REF], we mated one son and one daughter from each original mating pair to produce the second generation (G2). We repeated this operation until the 17 th generation (G17). Theoretical models estimate that inbred lineages become fixed from G20 in diploid species [START_REF] Hamilton | Population Genetics[END_REF]. As E. vuilleti is a haplodiploid species, fixation process is likely to be more rapid; we therefore assumed that we had generated four different, highly inbred genotypes (referred as genotypes 'A', 'B', 'C' and 'D'). We then individually placed G17 females in Petri dishes with a brother male and supplied them with infested cowpea seeds for 7 days, in order to obtain G18 daughters that were (1) familiar (same seed) and related (same genotype), (2) familiar (same seed) and unrelated (different genotypes), (3) unfamiliar (different seeds) and related (same genotype) and ( 4) unfamiliar (different seeds) and unrelated (different genotypes). To obtain familiar -unrelated females (i.e., coming from the same seed but genetically different), we cut seeds into halves, placed each half in different Petri dishes and allowed G17 mothers from different genotypes to separately parasitize hosts contained in each half of the seeds (Fig. 1). The difficulty in obtaining wasps that emerge synchronously from each half seeds explains the relatively small sample size of contests performed between these unrelated-familiar females (see the 'experimental procedure' section).

At the emergence of G18 individuals, we isolated wasps to prevent them from having any competition experience and introduced them separately in Petri dishes supplied with a piece of cotton soaked in water (Fig. 1). During the 3 days preceding behavioural observations, we provided females with one C. maculatus pupa, which was replaced daily. To do so, we dissected cowpea seeds to collect pupae. We then individually placed them in standard transparent gelatine capsule (length: 2 cm, diameter: 0.6 cm). These artificial seeds allow for better observation of laid eggs, without altering females' oviposition behaviour [START_REF] Gauthier | Could the egg itself be the source of the oviposition deterrent marker in the ectoparasitoid Dinarmus basalis[END_REF][START_REF] Jaloux | Interspecific relationships between the solitary ectoparasitoid, Eupelmus vuilleti (Crw.) (Eupelmidae), and its sympatric species, Dinarmus basalis (Rond.) (Pteromalidae), in the presence of their host, Callosobruchus maculatus pic (Coleoptera: Bruchidae)[END_REF]. Moreover, as E. vuilleti is a synovigenic species (i.e. they possess a few mature eggs at emergence and egg maturation continues throughout adult life), we added a male to the females' petri dishes for 24h to stimulate oogenesis [START_REF] Terrasse | Distribution de la ponte et évitement du superparasitisme chez l'hyménoptère solitaire Bruchocida vuilleti Cwf. (Hym., Eupelmidae), parasite des stades larvaires de son hôte, Callosobruchus maculatus F. (Col., Bruchidae)[END_REF]). In addition, to facilitate individual identification during contests, we marked them on the dorsal part of their thorax with a dot of bright yellow or bright red acrylic paint. On the day of behavioural observations, we removed the hosts and deprived females of hosts for at least 2h before the start of the contest so that they were more motivated to oviposit. We then observed contests between females fighting for a 4 th instar larva of C. maculatus.

Experimental procedure

At the start of the observation, we simultaneously introduced two parasitoid females into an arena consisting of a plastic block made of three chambers linked by a narrow channel (adapted from [START_REF] Petersen | The importance of being larger: parasitoid intruder -owner contests and their implications for clutch size[END_REF]. We placed females in the central chamber already containing a 4 th instar larva of C. maculatus previously placed in a capsule. [START_REF] Mathiron | Let the most motivated win : resource value components affect contest outcome in a parasitoid wasp[END_REF] showed that females with oviposition pre-experience on pupae were more motivated to gain access to a 4 th instar larva. Contestants were of the same age (3 days old), marked with different colours and visually matched for size (a posteriori analysis confirmed that contestants did not differ significantly in body mass; Wilcoxon signed-ranks test: T = 2463, N = 92, P = 0.14). We set up four different types of contests (Fig. 1), with different combinations of genotypes: (1) females emerged from the same seed that were from the same genotype (i.e. During all tests, we recorded oviposition and aggressive behaviours displayed by each female. When wasps detect a conspecific (identified by females raising their antennae in the direction of their opponent), they frequently stop their behavioural oviposition sequence [START_REF] Mohamad | Can subjective resource value affect aggressiveness and contest outcome in parasitoid wasps?[END_REF]. Females can either display defensive behaviour, kicking the opponent with their legs without taking their ovipositor out of the capsule, or they can exhibit a full attack, in which case one parasitoid usually hits her opponent with her head, chases her away from the capsule, or even mounts her. This hitting, chasing, or mounting can lead the loser to quit the central chamber of the arena. We therefore considered only these behaviours, collectively termed as 'attack', in the rest of the study. We conducted behavioural observations during 1h or stopped the contests when one female left the central chamber for at least 2 min. We also stopped contests when neither of the females touched the host nor displayed any aggressive interactions for at least 2min. We considered a contest as resolved when a female chased her opponent out of the central chamber, or when only one female displayed oviposition behaviour (i.e. the winning female) during the experiment time.

At the end of experiments, we immediately froze the wasps at -20°C. We then weighed them using an electronic balance (Ohaus Discovery® model, accuracy: 0.01mg) and we counted the number of mature eggs in their abdomen (i.e. egg load) by dissecting them. Finally, we noted for each contest the number of eggs laid on the host by the winning females to determine their initial egg load (i.e. at the start of the contest).

Statistical analysis

We analysed our data with the software R (R Development Core Team, 2016; version 3.5.0), using α = 0.05. Instead of transforming data to fit standard assumptions, we chose to perform parametric analyses in which the assumed distribution of residuals was matched to the data [START_REF] Wilson | Statistical analysis of sex ratios: an introduction[END_REF][START_REF] Briffa | Analysis of animal contest data[END_REF].

We first defined contest aggressiveness as a binary response: 0 = no aggressive behaviour occurred during a replicate and 1 = aggressive behaviours occurred (N = 92 contests).

We then performed a generalized linear model (GLM) with a binomial error distribution (link function = 'logit') to investigate the influence of absolute difference in initial egg load between contestants, genetic relatedness and familiarity among females on the probability that aggressive behaviours would be exhibited during contest. The model included the three main effects and two-way interactions between variables. We used the same procedure to explore the influence of absolute difference in initial egg load between contestants, genetic relatedness and familiarity among females on the probability of contest resolution. We thus defined contest resolution as a binary response: 0 = the contest was unresolved and 1 = there was a clear winner (N = 92 contests).

We finally examine the influence of absolute difference in initial egg load between contestants, genetic relatedness and familiarity among females on the total number of attacks displayed by both females during contest. To account for overdispersion, we ran a GLM assuming a quasi-Poisson distribution of residuals (link function = 'log'), considering only aggressive contests (i.e. contests where at least one attack was observed; N = 76). The model included main effects and two-way interactions between variables. We also included in the model the logarithm of contest duration as an offset term to account for differences in contest duration.

For all analyses, we started with the maximal model and removed non-significant interactions to test the significance of main effects. Likelihood-ratio chi-square and F-statistics were calculated using the 'Anova' function (package car), which performs type-II analysis of variance for GLM models, i.e. without a priori of the sequence order of explanatory variables [START_REF] Fox | An R companion to applied regression[END_REF]. Where we found significant interactions between factors, we ran Dunn's post-hoc multiple comparison tests to establish which modalities significantly differed from each other [START_REF] Dunn | Multiple comparisons among means[END_REF].

Ethical Note

We used 372 individuals in this study: 186 females for contests, and 186 males for female insemination. No animal ethics approval was required for this study. Nevertheless, we handled all individuals with care and we kept handling time to an absolute minimum. Acrylic paint used for marking does not cause injury to wasps. Subjects submitted to dissection were chilled in a freezer prior to decapitation.

RESULTS

Effect of genotype combination

We first verified that the genotype combination of the contestants did not affect the probability of contests being aggressive (GLM with binomial distribution of residuals: G9 = 12.72, P = 0.18), the probability of contest being resolved (GLM with binomial distribution of residuals: G9 = 9.91, P = 0.36), or the number of attacks during contest (GLM with quasi-Poisson distribution of residuals: F9,66 = 1.90, P = 0.07). However, this could reflect a lack of statistical power due to small sample sizes.

Escalation and resolution of conflicts

The probability of contests being aggressive was significantly influenced by the interaction between genetic relatedness and familiarity of females (GLM with binomial distribution of residuals: estimates ± S.E. = -17.29 ± 1312.5, G1 = 5.54, P = 0.02, Fig. 2a).

Contest escalation was indeed less likely when contestants were familiar and related than when they were familiar and unrelated (post-hoc Dunn's multiple comparison test: P = 0.02, Fig. 2a), but no difference was observed between unfamiliar-related and unfamiliar-unrelated females (post-hoc Dunn's multiple comparison test: P = 0.27; Fig. 2a). Moreover, no difference was observed between familiar-related and unfamiliar-related females (post-hoc Dunn's multiple comparison test: P = 0.05; Fig. 2a), and between unfamiliar-related and unfamiliar-unrelated females (post-hoc Dunn's multiple comparison test: P = 0.10; Fig. 2a). The probability of contest being aggressive did not significantly vary with the absolute difference in female egg load (GLM with binomial distribution of residuals: estimates ± S.E. = -0.115 ± 0.184, G1 = 0.38, P = 0.54). All other interactions were also non-significant.

The probability of a contest being clearly resolved varied significantly with familiarity (GLM with binomial distribution of residuals: estimates ± S.E. = 2.216 ± 0.802, G1 = 11.32, P < 0.001). Contests between familiar females had a greater chance of showing a clear winner than contests between unfamiliar females (Fig. 2b). However, we found no effect of genetic relatedness (GLM with binomial distribution of residuals: estimates ± S.E. = -0.332 ± 0.507, G1 = 0.430, P = 0.51), the absolute difference in female egg load (GLM with binomial distribution of residuals: estimates ± S.E. = -0.201 ± 0.158, G1 = 1.634, P = 0.2), or any 2-way interactions between variables.

Number of attacks during conflicts

The combined total number of attacks displayed by the two wasps during a contest was influenced by the absolute difference in female egg load, with contests being more aggressive when this difference was larger (GLM with quasi-Poisson distribution of residuals: estimates ± S.E. = 0.229 ± 0.041, F1,72 = 28.01, P < 0.001; Fig. 3). This total number of attacks did not vary with female genetic relatedness (GLM with quasi-Poisson distribution of residuals: estimates ± S.E. = -0.059 ± 0.148, F1,72 = 0.1593, P = 0.69), familiarity (GLM with quasi-Poisson distribution of residuals: estimates ± S.E. = -0.323 ± 0.170, F1,72 = 3.795, P = 0.06) or any 2way interactions between these variables.

DISCUSSION

The main goals of our study were: 1) to investigate whether E. vuilleti females adjust their aggressive behaviour during contests for host access according to the genetic relatedness with their competitor, and if so, 2) to explore on which components (phenotypic matching and/or familiarity) kin recognition is based. We observed that both genetic relatedness and familiarity influence the probability of conflict being aggressive: contest escalation was the least likely when contestants were both familiar and related. Moreover, familiarity affected conflict resolution, contests being more likely to be resolved when females were familiar. We thus highlighted kin discrimination in E. vuilleti, a solitary parasitoid with low dispersion ability [START_REF] Cortesero | Dispersal and parasitizing abilities of Eupelmus vuilleti (Hymenoptera: Eupelmidae) within a column of cowpea seeds[END_REF]. This evidence is all the more important that, in species with limited dispersion, the high risk of competition between kin may weaken the evolution of kin discrimination [START_REF] West | Testing Hamilton's rule with competition between relatives[END_REF][START_REF] West | Cooperation and competition between relatives[END_REF] and behaviours favouring relatives may not be selected for, as observed in fig wasps [START_REF] West | Testing Hamilton's rule with competition between relatives[END_REF].

Our results first indicate that E. vuilleti individuals were able to recognize both genetically related females and familiar females, and then decide whether to engage in an aggressive contest or not. Recognition of relatives in E. vuilleti agrees with what was found in the polyembryonic wasp genus Copidosoma, in which attack rates by the larval soldier caste was inversely correlated with competitor relatedness [START_REF] Giron | Aggression by polyembryonic wasp soldiers correlates with kinship but not resource competition[END_REF]. Moreover, in the gregarious parasitoid wasp, G. legneri, contests between adult females over hosts were less aggressive when competitors were more closely related and when females had developed on the same host (i.e., familiar, [START_REF] Lizé | Two components of kin recognition influence parasitoid aggression in resource competition[END_REF]. However, several other studies of parasitoid hymenopterans have reported that kin recognition did not affect fights between males [START_REF] West | Testing Hamilton's rule with competition between relatives[END_REF][START_REF] Innocent | Lethal combat over limited resources: Testing the importance of competitors and kin[END_REF], mate choice [START_REF] Bourdais | Lack of behavioural evidence for kin avoidance in mate choice in a hymenopteran parasitoid (Hymenoptera: Braconidae)[END_REF][START_REF] Ruf | No kin discrimination in female mate choice of a parasitoid with complementary sex determination[END_REF], or sex allocation behaviour [START_REF] Reece | Kin discrimination and sex ratios in a parasitoid wasp[END_REF]. Recognition of genetically encoded phenotypic cues can operate through two mechanisms: (1) allelic recognition (greenbeard effect), i.e., recognition of an allele that underlies the expression of a phenotypic trait [START_REF] Gardner | Greenbeards[END_REF] and (2) phenotypic matching, which is based on overall phenotypic similarity between related individuals [START_REF] Gadagkar | Kin recognition in social insects and other animals-A review of recent findings and a consideration of their relevance for the theory of kin selection[END_REF][START_REF] Mateo | Recognition systems and biological organization: The perception component of social recognition[END_REF]. The first mechanism leads to the recognition of similarity at the locus responsible for expression of the phenotypic trait [START_REF] Gardner | Greenbeards[END_REF], while the second mechanism leads to relatedness recognition at the genome scale if there is a strong correlation between both genetic and phenotypic similarity [START_REF] Mateo | Recognition systems and biological organization: The perception component of social recognition[END_REF][START_REF] Gardner | Greenbeards[END_REF]. Because allelic recognition may involve cooperation between unrelated individuals expressing the trait encoded by a given allele, fixation in a population is expected to be rare owing to invasion by cheaters [START_REF] Gardner | Greenbeards[END_REF]. As such, phenotypic matching is more plausible in the current case. Phenotype matching, in which the genotype of individuals is expressed through their phenotype, contrasts to familiarity in which phenotypic cues may derive from genotypes and/or environment [START_REF] Lizé | Two components of kin recognition influence parasitoid aggression in resource competition[END_REF]. Even if phenotypic matching is generally referred to as a different mechanism from familiarity, phenotypic matching may thus be seen as an extension of familiarity [START_REF] Mateo | Recognition systems and biological organization: The perception component of social recognition[END_REF]. Here, because the probability of a conflict being aggressive was influenced by the interaction between genetic relatedness and familiarity among females, this suggests that phenotypic matching mechanism may be an extension of familiarity in E. vuilleti females. In accordance with the study of [START_REF] Lizé | Two components of kin recognition influence parasitoid aggression in resource competition[END_REF] in G. legneri, our experiments highlighted that these two components of kin recognition affect contests for hosts in a parasitoid wasp. More strikingly, we showed for the first time that both components can interact to affect intraspecific competition for resources in a solitary insect species.

While we also expected the total number of aggressive behaviours displayed during contests to be influenced by both kin recognition components, only the difference in competitors' egg load had a significant effect: fights were less aggressive when the opponents had a similar number of mature eggs. This unexpected result seems in contradiction with theory models predicting that symmetry between individuals leads to more escalated contests of longer duration [START_REF] Parker | Assessment strategy and the evaluation of fighting behaviour[END_REF][START_REF] Smith | The logic of asymmetric animal contests[END_REF]). Here, one explanation is that the total number of attacks during a contest may have been driven by the behaviour of the contestant with the highest egg load. As egg load is known to influence competitors' subjective value of the resource [START_REF] Enquist | Evolution of fighting behaviour: The effect of variation in resource value[END_REF][START_REF] Stokkebo | The importance of being gravid: egg load and contest outcome in a parasitoid wasp[END_REF], the more gravid females (i.e. with more mature eggs in their ovaries) are expected to be more motivated to fight for winning access to the host. Opponent females can match their aggressiveness, resulting in an increase in the total number of aggressive behaviours during conflicts. Additional analyses seem to support this hypothesis: the total number of attacks displayed by the two wasps during contests significantly increased with the egg load of the most gravid contestant (GLM with quasi-Poisson distribution of residuals: estimates ± S.E. = 0.11 ± 0.04, F1,74 = 6.20, P < 0.02) and the number of attacks displayed by both females are positively correlated (GLM with quasi-Poisson distribution of residuals: estimates ± S.E. = 0.04 ± 0.01, F1,74 = 36.27, P < 0.001). Furthermore, our results add to the evidence that egg load is the major factor driving variation in aggressive behaviours during contests for hosts in E. vuilleti [START_REF] Mohamad | Can subjective resource value affect aggressiveness and contest outcome in parasitoid wasps?[END_REF][START_REF] Mohamad | Wait or fight? Ownership asymmetry affects contest behaviours in a parasitoid wasp[END_REF]. The underlying mechanism is still unknown, but the involvement of hormones has been suggested [START_REF] Stokkebo | The importance of being gravid: egg load and contest outcome in a parasitoid wasp[END_REF][START_REF] Goubault | Previous experience and contest outcome: winner effects persist in absence of evident loser effects in a parasitoid wasp[END_REF]. In particular, juvenile hormone is known to be involved in egg maturation in many insect species (reviewed by [START_REF] Nijhout | Insect Hormones[END_REF] and associated with aggressiveness in different hymenopteran species such as paper wasps [START_REF] Tibbetts | The challenge hypothesis in an insect: juvenile hormone increases during reproductive conflict following queen loss in Polistes wasps[END_REF] and honeybees [START_REF] Pearce | Juvenile hormone and aggression in honey bees[END_REF]. Ecdysteroids also play a major role in the regulation of oogenesis in E. vuilleti females [START_REF] Bodin | Reproduction in a variable environment: How does Eupelmus vuilleti, a parasitoid wasp, adjust oogenesis to host availability[END_REF], and may affect female aggressive behaviours. Nevertheless, additional work is required to investigate to what extent hormones may affect female aggressiveness during contest over host.

In contrast with [START_REF] Lizé | Two components of kin recognition influence parasitoid aggression in resource competition[END_REF] who found no effect of familiarity or genetic relatedness on the probability of contests being resolved in G. legneri, we showed that conflicts between familiar females were more likely to result in a clear winner and loser. In parasitoid hymenopterans, offspring survival, hence females' direct fitness, depends directly on their ability to acquire, parasitize and defend hosts [START_REF] Petersen | The importance of being larger: parasitoid intruder -owner contests and their implications for clutch size[END_REF]. However, exploiting a host, and in particular drilling through the seed to access it, has been shown to be highly energetically costly in this species [START_REF] Boisseau | The metabolic costs of fighting and host exploitation in a seed-drilling parasitic wasp[END_REF]. Quitting a host, to the benefit of another female, is thus a waste of potential fitness return, but may save energy. Nevertheless, if both contestants are relatives, the increase in inclusive fitness may counterbalance the cost of surrendering the host to a conspecific female, as predicted by kin selection theory (Hamilton, 1964). Behavioural mechanisms that allow higher probabilities of contest resolution between familiar females in E. vuilleti would therefore be adaptive, as long as emerging from hosts that developed in the same seed is a good predictor of genetic relatedness in this species. In E. vuilleti, mothers often monopolize a cowpea seed infested by several juvenile C. maculatus [START_REF] Mohamad | Wait or fight? Ownership asymmetry affects contest behaviours in a parasitoid wasp[END_REF], and wasps that emerge from the same seed are therefore highly likely to be sisters.

Altogether, we observed that E. vuilleti females variably rely on genetic relatedness and familiarity cues depending on the contest stage: they first rely on both types of cues when deciding whether to escalate the conflict or not. Then, during escalated contests, aggressiveness varied only with SRV asymmetry between contestants. Finally, contest resolution was based on familiarity cues only. If this may be surprising at first glance, the type of information used by animals for decision-making is classically known to change over contests (see Arnott & Elwood, 2009 for a review of 'assessment' models). For example, [START_REF] Elias | Assessment during aggressive contests between male jumping spiders[END_REF] showed that males of the jumping spider, Phidippus clarus, use multimodal signals during aggressive interactions: the duration of pre-contact phases was based on differences in vibration behaviour between males, while motivation to escalate towards contact phases was mainly based on body size. Moreover, in the mangrove rivulus fish, Kryptolebias marmoratus, males use different assessment strategies during conflicts. Fish usually adopt 'mutual assessment' to decide whether to escalate the contest, and then change to a 'self-assessment' to whether persist the escalation [START_REF] Hsu | Switching assessment strategy during a contest: fighting in killifish Kryptolebias marmoratus[END_REF]. Overall, our results demonstrated that wasps are able to use different cues at each phase of the conflict to make their behavioural decisions.

The mechanisms underlying the identification of genetic relatedness and familiarity remain to be explored in E. vuilleti. However, E. vuilleti females are known to adjust their behaviour based on the detection of conspecific cuticular hydrocarbons (CHC; [START_REF] Darrouzet | Parasitoids modify their oviposition behaviour according to the sexual origin of conspecific cuticular hydrocarbon traces[END_REF]. Because the wasps, in our study, discriminated competitors of the same genotype and those from the same seed, they may have detected genetically encoded or environmentally influenced differences in the CHC profile of their opponent. Studies in parasitoid hymenoptera suggest that CHC may be characteristic of related individuals [START_REF] Höller | Evidence for a two-component external marking pheromone system in an aphid hyperparasitoid[END_REF][START_REF] Howard | Cuticular hydrocarbons of adult Pteromalus cerealellae (Hymenoptera: Pteromalidae) and two larval hosts, angoumois grain moth (Lepidoptera: Gelechiidae) and cowpea weevil (Coleptera: Bruchidae)[END_REF]. Several studies have also measured biochemical convergence between the surface lipids of host plants and cuticular compounds of insect herbivores and their predators or parasitoids (reviewed in [START_REF] Blomquist | Insect hydrocarbons: biology, biochemistry, and chemical ecology[END_REF]. Kühbandner et al. (2012) found, in the pteromalid wasp Lariophagus distinguendus, that shifting host during only one generation led to distinguishable CHC profiles among females. Moreover, since familiar E. vuilleti females came from the same seed on which the hosts fed, it is possible that females developing on hosts that exploit the same resource might express a similar biochemical profile that allows for recognition of familiar individuals [START_REF] Ode | Brood-mate avoidance in the parasitic wasp Bracon hebetor Say[END_REF][START_REF] Singer | Roles of hydrocarbons in the recognition systems of insects[END_REF]). For example, von Beeren et al. (2011) showed that ant parasites Malayatelura ponerophila acquire cuticular hydrocarbons directly from their host Leptogenys distinguenda. However, E. vuilleti females may have simply acquired seed-specific compounds by friction when emerging out of the seed.

Physical contacts can also generate variation in the chemical cuticular hydrocarbon profile: when stroking their abdomens on the nest surface of their host, both parasite species Sulcopolistes sulcifer and Polistes semenowi acquire host hydrocarbons from the comb [START_REF] Turillazzi | Invasion of the Nest of Polistes dominulus by the Social Parasite Sulcopolistes sulcifer (Hymenoptera, Vespidae)[END_REF]Zacchi, 1995). Moreover, in social wasps, a hydrocarbon layer similar to that of resident wasps covers nest paper, so that physical contact between parasite cuticle and nest hydrocarbons may favour the transfer of odours between parasites and comb [START_REF] Lorenzi | The role of cuticular hydrocarbons in social insects: is it the same in paper wasps[END_REF][START_REF] Singer | Roles of hydrocarbons in the recognition systems of insects[END_REF]. We call future studies to test these hypotheses and identify the cues involved in the different components of kin recognition.

Conclusion

By investigating the effects of both genetic relatedness and common developmental environment on contest behaviour and resolution in E. vuilleti, we were able to demonstrate that two kin recognition components (phenotypic matching and familiarity) mediate the occurrence of escalated conflicts and contest resolution in a parasitoid wasp. We showed for the first time that both components can interact to affect intraspecific competition in a solitary insect species. Additionally, in species with limited dispersion like E. vuilleti [START_REF] Cortesero | Dispersal and parasitizing abilities of Eupelmus vuilleti (Hymenoptera: Eupelmidae) within a column of cowpea seeds[END_REF], high competition risk for resources between kin may weaken the evolution of reducing aggressive behaviours toward related conspecifics [START_REF] West | Testing Hamilton's rule with competition between relatives[END_REF][START_REF] West | Cooperation and competition between relatives[END_REF]. Our study therefore shows that kin discrimination during intraspecific competition can also evolve in a species with limited dispersion.

Figure captions

  familiar -related , N = 19; genotype combinations: 'AA', N = 7; 'BB', N = 5; 'CC', N = 2; 'DD', N = 5), (2) females emerged from the same seed that were from different genotypes (i.e. familiar -unrelated, N = 9; genotypes combinations: 'AD', N = 1; 'BC', N = 2; 'BD', N = 1; 'CD', N = 5), (3) females emerged from different seeds that were from the same genotype (i.e. unfamiliar -related, N = 25; genotype combinations: 'AA', N = 9; 'BB', N = 5; 'CC', N = 7; 'DD', N = 4) and (4) females emerged from different seeds that were from different genotypes (i.e. unrelated -unfamiliar, N = 39; genotypes combinations: 'AB', N = 6; 'AC', N = 5; 'AD', N = 8; 'BC', N = 6; 'BD', N = 7; 'CD', N = 7). Wasps from the same genotype were full sisters.

Figure 1 .

 1 Figure 1. Experimental design that allowed us to investigate the role of genetic relatedness and

Figure 2 .

 2 Figure 2. (a) Probability (±S.E.M.) of a contest being aggressive when familiar contestants were

Figure 3 .

 3 Figure 3. Combined total of number of aggressive behaviours observed during contest according to the

FiguresFigure 1 .

 1 Figures

  Figure 2.

Figure

  Figure 3.


S Wang

E Strandberg

Å Viklund

J J Windig

S Malm

T Lewis

D Laloë

G Leroy

Genetic improvement of canine hip dysplasia through sire selection across countries

Keywords: Canine, Estimated breeding value, Hip dysplasia, International breeding programme, Stochastic simulation

Breeding against canine hip dysplasia (HD) may benefit from the importation of foreign sires. When foreign sires are evaluated on a different HD scale, this may diminish the efficacy. Using stochastic simulations, we evaluated genetic change and inbreeding levels for different scenarios of importing sires with high genetic merit for HD. Population size and genetic parameters (e.g. heritability, accuracy of selection, genetic correlation) were based on actual data for HD in Golden retrievers and Labrador retrievers in the UK and Sweden.

For countries with different HD scales and an estimated breeding value (EBV) evaluation in place, the importation was useful if imported sires had EBV rankings in the top 50% and if genetic correlations between EBV systems were above 0.85. When importing sires with EBV rankings in the top 10%, moderate accuracies of EBVs (> 0.40) and moderately strong genetic correlations (> 0.70) were needed. Selection against HD without the importation of sires may increase inbreeding levels, while the importation of sires can decrease inbreeding levels. For national genetic evaluation and selection programmes, importing sires with high genetic merit can be an effective breeding strategy, but care is needed to estimate reliable EBVs.

Introduction

Canine hip dysplasia (HD) is an abnormal development of the coxofemoral joint in dogs, and as it progress can lead to lameness, arthritis and hip pain. HD is one of the most common orthopaedic disorders/diseases in dogs and has been reported in more than 188 dog breeds, with a prevalence varying from 1.0 to 71.2%1 . Unlike the control of undesirable single-gene traits where genetic testing (where the gene is known) can be used to guide breeding, the control of undesirable complex traits (i.e. those determined by multiple genes and interactions with environment, such as HD), through genetic screening is more difficult.

Worldwide, three different scoring schemes for HD are widely used: the BVA/KC (British Veterinary Association/The Kennel Club) scheme in the UK, Australia and New Zealand; the OFA (Orthopedic Foundation for Animals) scheme in the USA; and the FCI (Fédération Cynologique Internationale) scheme in Europe, Asia and Latin America. Furthermore, veterinary training for the measurement of hip joint laxity using other schemes, e.g. PennHIP (The University of Pennsylvania Hip Improvement Program), developed by [START_REF] Smith | New concepts of coxofemoral joint stability and the development of a clinical stress-radiographic method for quantitating hip joint laxity in the dog[END_REF], are also commercially available.

Selection after the evaluation of estimated breeding values (EBVs) is the most effective way to genetically improve hip joint status [START_REF] Wilson | Selection against canine hip dysplasia: Success or failure?[END_REF]Lewis and Windig 2016). Genetic evaluation routines have been implemented in some countries, such as Denmark, Finland, Germany, Norway, Sweden and the UK [START_REF] Wang | Breeding policies and management of pedigree dogs in 15 national kennel clubs[END_REF]. In most countries, however, selection to improve hip joint status is based solely on phenotypic assessments. Meanwhile, in recent years, the exchange of breeding animals and semen between countries has increased (Wang et al., 2017a). This exchange can increase the risk of importing dogs with unknown or low genetic merit for HD, thereby reducing the efficacy of the breeding programme. However, genetic progress towards improvement in HD may be accelerated by genetic evaluation across countries via an enlarged pool of breeding candidates [START_REF] Fikse | Opportunities for international collaboration in dog breeding from the sharing of pedigree and health data[END_REF].

The potential for improvement in the genetic progress against HD was demonstrated for the Golden retriever and Labrador retriever subpopulations in the UK and Sweden (Wang et al., 2017b). However, the grading systems used to indicate the severity of hip joint were not the same; the UK uses the BVA/KC scheme with scores ranging from 0 (no HD) to 106 (severe HD), while FCI countries, such as Sweden, follow a specific FCI scheme with fivelevel grades: A and B are normal, C is mild and D is moderate and E is severe. Imported dogs evaluated using a different scale may not be of expected genetic merit if the (genetic) correlation between traits measured on these different scales is substantially below 1.0, and a further reduction in efficacy may occur if the accuracy of the EBVs are low (Wang et al. 2017b). The consequence may be that a higher selection intensity is needed to allow only the importation of dogs with a (very) high genetic merit.

Evaluating the efficacy of various strategies for importing sires with high genetic merit has not been explored as a breeding strategy, and the influence of genetic correlations across countries and the accuracies of EBVs has also not been examined. Computer simulation can be an effective tool to predict genetic changes and inbreeding levels, especially for complex situations, such as overlapping generations and exchange between different subpopulations. In dogs, computer simulation has been used to study strategies aimed at reducing single-gene defects and high inbreeding rates in pedigree dogs [START_REF] Leroy | Mating practices and the dissemination of genetic disorders in domestic animals, based on the example of dog breeding[END_REF][START_REF] Windig | Genetic management of Dutch golden retriever dogs with a simulation tool[END_REF]. Based on estimated genetic parameters for the Golden retrievers and Labrador retrievers in the UK and Sweden (Wang et al. 2017b), a computer simulation study was used to study the efficacy of international breeding strategies to import sires with high genetic merit.

Materials and methods

Simulation program

A FORTRAN program 'GenManSim' [START_REF] Windig | Genetic management of Dutch golden retriever dogs with a simulation tool[END_REF] was used and extended with the facility to simulate subpopulations [START_REF] Windig | Limits to genetic rescue by outcross in pedigree dogs[END_REF]. The specific functions needed in our study were added to the original simulation workflow of 'GenManSim', including generation of breeding values and phenotypes for individual animals and selection based on EBVs and/or phenotypes of HD. Because genetic evaluation across two countries with different HD scales was to be simulated, all animals had two sets of true breeding values (TBVs) and EBVs simulated: TBV1 and EBV1 for the scale used in country 1 (country 1 scale) and TBV2 and EBV2 used in country 2 (country 2 scale). The method about how TBVs and EBVs for HD were calculated is shown in Appendix (see Supplementary document 1). The objective of this study was to simulate genetic change as a result of selection across two countries having two HD grading systems, with possible importation from country 1 to country 2. Given the gene flows previously observed from the UK to Sweden [START_REF] Wang | Merging pedigree databases to describe and compare mating practices and gene flow between pedigree dogs in France, Sweden and the UK[END_REF], country 1 therefore corresponds to the UK, and country 2 corresponds to Sweden.

Input values for the simulation parameters are shown in Table 1, including those necessary to run the simulation: population size and structure; phenotypes and genetic parameters; breeding selection and mating restrictions options. In countries using FCI scale, grade A and B indicate normal status of hip joint in dogs, which corresponds to BVA/KC scores below 26 in the UK [START_REF] Flückiger | Scoring radiographs for canine hip dysplasia-The big three organisations in the world[END_REF]. However, in order to perform the genetic evaluation required, the FCI grades A, B, C, D and E were replaced with 1, 2, 3, 4 and 5, respectively (Wang et al. 2017b), while the BVA/KC scores 0-106 (with an addition of 1) were transformed by natural logarithms [START_REF] Lewis | Genetic evaluation of hip score in UK Labrador Retrievers[END_REF]. In this study, transformed phenotypic mean (SD) of HD measures in country 1 and country 2 were set as 1.67 (0.6) and 2.66 (0.7) as in Wang et al. (2017b). Therefore, to differentiate between dogs being affected (20%) or unaffected (80%) by HD in country 1 and country 2, respectively, 2.259 and 3.165 of transformed phenotypic values were set as threshold assuming a normal distribution (Table 1). Additional details regarding the function of the simulation program and how input values for the simulation parameters were chosen are shown in the Appendix (see Supplementary document 1).

Simulation scenarios

Eight scenarios with varying importation of sires and use of EBVs in selection were studied (Table 2). No selection occurred in the scenario 1, while in all other scenarios, only dogs without HD were allowed to reproduce. Comparison to this strategy allowed us to determine the effect of selection in the other models. In scenarios 1-3, no foreign sires are imported, while in scenarios 4-5, 20% of the sires used in breeding were foreign. This allowed us to determine the effect of the importation of sires.

In the scenarios without importation, selection of sires differed: either (1) no sire selection (random mating), abbreviated as NoSel; (2) sire selection on phenotype only (Pheno2); or (3) sire selection on both phenotype and EBV, i.e. sires must have EBV2 in the top 50% (Pheno2+DomEBV2). Phenotypes and EBVs for scenarios 2 and 3 were evaluated on the domestic scale (country 2). In all scenarios with importation of sires, all sires were selected based on their phenotype using the scale of their country of birth (e.g. scale 2 for domestic sires, scale 1 for foreign sires). The scenarios differed in the use of EBVs in the selection of sires: (4) No selection on EBV (Pheno+IntAll); (5) selection on EBV for foreign sires only, i.e. EBV1 in the top 50% (Pheno+IntEBV1); (6) selection on EBV for domestic sires only, i.e., EBV2 in the top 50% (Pheno+DomEBV2+IntAll); (7) selection on EBV for both foreign and domestic sires, both on the scale of their own country of birth (Pheno+DomEBV2+IntEBV1); (8) was as (7) but foreign sires were evaluated on the scale of the country of importation (Pheno+DomEBV2+IntEBV2). These scenarios enabled us to evaluate the effect of using EBVs for selection, either on the domestic or on the foreign scale, when importing sires.

Each scenario was run for 10 years with 40 replicates. Four statistics of newborn pups in country 2 were calculated: average TBV2, percentage of dogs with HD, average inbreeding and coancestry coefficients. In all scenarios, in country 1 phenotypic selection and selection on EBV (Pheno1+DomEBV1) was employed. This strategy enabled us to evaluate the importation of sires from a country with selection based on EBV to countries either with or without selection based on EBV. Restriction on relatedness between mating partners (<0.125) was applied for all the scenarios. Scenario 7 Pheno+DomEBV2+IntEBV2 was regarded as the basic standard scenario to evaluate the efficacy of importing sires in this study. This scenario was further explored with various selection intensities of sires, percentages of foreign sires used in breeding, accuracies, and genetic correlations. The selection intensities regarding the EBVs of imported sires were varied from the top 10% to top 50% (with an interval of 10%); the percentages of litters produced by foreign sires were varied from 0% to 60% (with an interval of 20%); the accuracy of EBV2 was varied from 0.45 to 0.75 (with an interval of 0.1); and the genetic correlation between the two HD scales was varied from 0.65 to 0.95 (with an interval of 0.1).

To evaluate what parameters are worthwhile for imported dogs, the outcomes of the above alternatives were compared with a purely national breeding programme in country 2 (referred to as scenario Pheno2+DomEBV2).

Results

Because the study focused only on changes in the populations of countries importing foreign sires, and not on countries exporting sires, only results for country 2 are reported, i.e., the TBVs below are actually TBV2, unless otherwise indicated. Note that TBV scores describe the severity status of hip joint, with lower scores being better.

Breeding strategies

As expected, neither genetic improvement nor reduction in the prevalence of HD was achieved by random mating in scenario NoSel (Table 3). In contrast, the scenario without the importation of foreign sires but with selection of only healthy dogs within country 2 in scenario Pheno2, reduced the average TBVs to -0.23 and the prevalence of HD to 11.2% at year 10. Adding a restriction on domestic dogs' EBVs (use of top 50% only, scenario Pheno2+DomEBV2) further reduced the average TBVs to -0.62 and the prevalence of HD to 3.0%. In country 1, under the same strategy for each scenario focusing on TBV1, an average TBV1 of -0.62 and an average TBV2 of -0.46 were achieved at year 10 (see Appendix: Supplementary Table 1).

When 20% of the newborn litters were produced by foreign sires each year, and only healthy dogs within country 2 were used, but with no restriction on imported dogs (Pheno+IntAll), the average TBV decreased to -0.24 and the prevalence of HD decreased to 11.0% (Table 3). When a restriction was added to use only foreign sires from the top 50% (based on the EBV in the exporting country), similar genetic progress was achieved (average TBVs of -0.27 and prevalence of HD of 10.2% (Pheno+IntEBV1). Compared with scenario Pheno+IntAll, also including a restriction on the EBVs of domestic dogs, (Pheno+DomEBV2+IntAll) resulted in substantial improvement in the genetic progress, from -0.24 to -0.53 in average TBVs and from 11.0% to 4.3% in HD prevalence. However, the addition of a restriction on the EBVs of imported dogs (on either scale) resulted in only a slight improvement in genetic gain; average TBV -0.56 and prevalence of HD 3.8% in scenario Pheno+DomEBV2+IntEBV1, and average TBV -0.57 and prevalence of HD 3.8% in scenario Pheno+DomEBV2+IntEBV2. In short, for scenarios Pheno+IntAll and Pheno+DomEBV2+IntAll, the importation of sires (regardless of EBV) resulted in genetic progress, however the genetic progress was always lower compared to similar scenarios including selection on EBV using domestic sires only (Pheno2 + DomEBV2). The trends of the genetic progress made in the first 10 years for each breeding strategy are shown in Fig. 1.

Inbreeding reached a level of 0.30% after 10 years without importation or selection (NoSel). Selection on phenotype only slightly increased the inbreeding level (0.37%), while selection on EBV resulted in a notable increase in the inbreeding level to 1.28%. The importation of foreign animals decreased inbreeding levels to 0.22% when there was only phenotypic selection, and to an average of 0.70% with additional selection on EBV in domestic animals. Coancestry levels followed the same pattern but reached values of approximately four times the inbreeding levels. This observed difference between coancestry and inbreeding levels was probably linked to restrictions on mating partners that were close relatives.

Selection intensity of importing sires

The average TBVs of newborn pups in year 10 improved from -0.57 to -0.65 when the selection intensity was increased from importing top 50% sires to top 10% under Pheno+DomEBV2+IntEBV2 (Table 4). The prevalence of HD decreased from 3.8% to 2.9%.

The increase in the inbreeding level was only slight when the required EBV rankings of sires changed from the top 50% to the top 20% (from 0.70% to 0.74%), while it increased to 0.99% when the required EBV rankings of sires were the top 10%.

Percentages of litters produced by foreign sires

Increasing the percentage of litters produced by foreign sires from 20% to 60% reduced the rate of improvement in genetic progress in both scenarios Pheno+DomEBV2+IntAll and Pheno+DomEBV2+IntEBV2 (Table 5). The absolute difference of average TBVs between scenarios Pheno+DomEBV2+IntEBV2 and Pheno+DomEBV2+IntAll was 0.04 in the pups born in year 10 when the percentages of litters was 20%, and the absolute differences were 0.07 and 0.09 when percentages of litters were 40% and 60%, respectively. Additionally, using more foreign sires in breeding also reduced inbreeding.

Accuracy of selection

The accuracy of selection is the correlation between EBV and TBV and indicated how well the EBV represented the TBV. When the accuracy of selection increased from 0.45 to 0.75, genetic progress improved (Fig. 2): from -0.53 to -0.61 (from -0.55 to -0.69) when the requirement was set to sires in the top 50% (top 10%). Regardless of which level of accuracy was used for selection, the TBVs achieved by importing sires with EBVs ranking in the top 50% (scenario Pheno+DomEBV2+IntEBV2) were always lower than the genetic progress made in scenario Pheno2+DomEBV2. Compared to the genetic progress made in scenario Pheno2+DomEBV2, the TBVs achieved by importing sires with EBVs ranking in the Top 10% (scenario Pheno+DomEBV2+IntEBV2) was higher only when the accuracy of selection was over 0.40.

Genetic correlations

When the genetic correlations between TBV1 and TBV2 increased from 0.65 to 0.95 in scenario Pheno+DomEBV2+IntEBV2, the average TBV changed from -0.54 to -0.65 when the required sire rankings were in the top 50%, and the average TBV changed from -0.60 to -0.72 when the required sire rankings were in the top 10% (Fig. 3). Compared to the genetic progress made in scenario Pheno2+DomEBV2 (average TBV was -0.62), the TBV achieved by importing sires with EBVs ranking in the top 50% (top 10%) were higher when the genetic correlation was over 0.85 (0.70).

Discussion

Controlling the prevalence of inherited disorders constitutes an important issue for the health and welfare of pedigree dogs. The use of sires with high genetic merit in an international context has been viewed as a promising breeding strategy to improve the health and welfare of dogs [START_REF] Wilson | Empowering international canine inherited disorder management[END_REF][START_REF] Fikse | Opportunities for international collaboration in dog breeding from the sharing of pedigree and health data[END_REF], and the simulation of such a breeding strategy is informative to national kennel clubs and may assist in the genetic management of various breeds. This study underlines how the international exchange of breeding animals may be used to improve the situation for a complex disease such as HD.

A posterior evaluation of HD trends over recent decades, where selection was implemented based solely on phenotypic observations, has shown favourable trends in the long run [START_REF] Malm | Genetic variation and genetic trends in hip and elbow dysplasia in Swedish Rottweiler and Bernese Mountain Dog[END_REF][START_REF] Lewis | Comparative analyses of genetic trends and prospects for selection against hip and elbow dysplasia in 15 UK dog breeds[END_REF][START_REF] Oberbauer | Long-term genetic selection reduced prevalence of hip and elbow dysplasia in 60 dog breeds[END_REF]. In some cases, the magnitude of the detected improvement for HD has been notable, for example in Swedish Rottweilers and Bernese mountain dogs, genetic progress corresponded to 0.67 and 0.57 genetic SD units, respectively, over the last 10 years by only breeding from dogs without HD [START_REF] Malm | Genetic variation and genetic trends in hip and elbow dysplasia in Swedish Rottweiler and Bernese Mountain Dog[END_REF]. This observed trend is in agreement with the trend of the simulation results in our study, showing that phenotypic selection can reduce the prevalence of HD when applied for a long period. In our simulation, the genetic change corresponded to 0.61 genetic SD units in the first 10 years of the breeding programme when only considering phenotypic selection (scenario Pheno2) (Table 3). In Europe, most countries have phenotypic restrictions for breeding but do not have genetic evaluation routines of HD for domestic selection.

Nevertheless, they can make use of foreign stock for breeding which is evaluated by scenario Pheno+IntAll in this study. For countries without EBVs, it can be recommended that breeders import sires from countries that already have EBV evaluation in place, thus choosing sires with good EBVs, as illustrated by comparing scenario Pheno+IntEBV1 to scenario Pheno+IntAll.

Our simulation study confirmed that implementation of international genetic evaluation can bring further genetic improvement of HD by the importation of sires with higher genetic merit (Table 4). When more litters were born by foreign sires, the difference in the genetic progress between scenarios with and without international evaluations of foreign sires became larger (Table 5), which means that more benefits can be expected using EBVevaluated foreign sires with an increased exchange of breeding animals. For countries such as the UK and Sweden, genetic evaluation has been implemented and is publicly available and there is migration of dogs between the two countries, which can be considered similar to scenario Pheno+DomEBV2+IntAll (average TBVs of -0.53, Table 3) assuming Swedish breeders importing UK sires do not consider their UK EBVs. Without international evaluation implemented, but considering UK EBVs, more genetic progress (average TBVs of -0.56) could be achieved by scenario Pheno+DomEBV2+IntEBV1. Once the international evaluation is implemented, as in scenario Pheno+DomEBV2+IntEBV2, some further genetic progress can be achieved (average TBVs of -0.57). However, the additional genetic gain achieved from scenario Pheno+DomEBV2+IntEBV2 (average TBVs of -0.57) compared to Pheno+DomEBV2+IntAll (average TBVs of -0.53) was small, if not negligible. More genetic progress would be achieved by scenario Pheno+DomEBV2+IntEBV2 if either the accuracy of selection or genetic correlation is higher than the default values (0.65 and 0.75, respectively) that were set in the simulation (Figs. 2 and3). In practice, reducing the average TBVs from -0.53 to -0.57 in the first 10 years corresponded to changing the genetic SD units of HD scores from 1.39 to 1.50 based on the transformed phenotype on the scale of country 2, which was an important genetic improvement and was reflected in the reduced prevalence of HD from 20% to 3.77 -4.28% (Table 3).

The simulation also demonstrated the benefits of importing sires with very high genetic merit (sires with EBV rankings in the top 10%; average TBVs of -0.65) compared to using sires with EBV rankings just in the top 50% (average TBVs of -0.57). Nevertheless, a moderate level of accuracy of selection was necessary to ensure the efficacy of importing sires based on EBV rankings. When the accuracy of selection was lower than 0.40, the genetic progress achieved by importing sires with EBV rankings in the top 10% (scenario Pheno+DomEBV2+IntEBV2) was not higher than from a purely national breeding programme, as in scenario Pheno2+DomEBV2 (Fig. 2). Accuracy depends on the heritability and the number of (close) relatives available. Similar to sire selection based on progeny testing in dairy cattle, sires that have HD screening records and have multiple offspring with HD screening records have higher accuracies in genetic evaluation. For instance, the accuracy of sires with only their own HD records can reach 0.55 when the heritability is 0.30, while at the same heritability the accuracy of sires without their own HD records can reach 0.44 once two offspring are tested. Hence, we highly recommend importing sires with their own HD records that have had several offspring screened.

A strong genetic correlation is also required to make the importation of sires worthwhile. The genetic correlation should be over 0.85 to ensure that more genetic progress is made from importing sires with EBVs in the top 50% (scenario Pheno+DomEBV2+IntEBV2) compared with the progress that could be achieved from a purely national selection programme, as in scenario Pheno2+DomEBV2 (Fig. 3). This may also be a reason for the small difference in genetic progress between scenarios Pheno+DomEBV2+IntAll (average TBVs of -0.53) and Pheno+DomEBV2+IntEBV2 (average TBVs of -0.57) when the genetic correlation was 0.75 (Table 4). This finding is similar to simulation studies carried out by varying genetic correlations in dairy cattle breeding programmes. A genetic correlation as high as 0.80-0.90 was required to obtain an extra 15% improvement in genetic gain [START_REF] Mulder | Benefits of cooperation between breeding programs in the presence of genotype by environment interaction[END_REF].

Theoretically, the genetic correlation between HD in the UK and Sweden should be close to 1.0, which has been confirmed by bootstrap simulation [START_REF] Fikse | Opportunities for international collaboration in dog breeding from the sharing of pedigree and health data[END_REF]). However, because the BVA/KC and FCI schemes evaluating the severity of hip joint status used in the UK and Sweden are measured on different scales, genetic correlations >0.90 may not be reached. Therefore, consensus across countries in screening protocols for HD would be useful to improve genetic correlations. Moreover, the genotype-environment interaction (G x E) in the genetic evaluation of HD may also exist across countries due to differences in veterinary care for HD, which could also prevent genetic correlations from reaching unity.

In all breeding scenarios in our study, the relatedness between mating partners was required to be less than 0.125. This result was because mating between close relatives, e.g. between a father and daughter or between half siblings, is not recommended or allowed in most European countries. Comparing inbreeding levels after 10 years in scenarios Pheno2 (0.33%) and Pheno2+DomEBV2 (1.28%), which modelled breeding in a closed population, to scenarios Pheno+IntAll (0.22%) and Pheno+DomEBV2+IntEBV2 (0.70%), which modelled the use of foreign sires, shows that the rate of inbreeding can be effectively controlled by importing sires. This finding meets the expectation of Wang et al. (2017a) that the management of inbreeding can benefit from the international exchange of breeding animals.

However, if high-EBV ranking foreign sires are heavily used, inbreeding levels will also increase more quickly. This observation is clearly shown when changing the use high-EBV sires from the Top 20% to the Top 10%, increasing the inbreeding level from 0.74 to 0.99 in 10 years (Table 3). This phenomenon has been largely illustrated in Holstein cattle, with a decrease in genetic variability related to the worldwide use of the best bulls through artificial insemination and frozen semen [START_REF] Brotherstone | Artificial selection and maintenance of genetic variance in the global dairy cow population[END_REF][START_REF] Doekes | Trends in genome-wide and region-specific genetic diversity in the dutch-flemish holstein-friesian breeding program from 1986 to 2015[END_REF].

Therefore, although dog breeders and owners are generally free to choose mating sires, national kennel clubs should develop breeding management strategies to ensure that no sires are used too extensively, especially when importing foreign sires with high genetic merits.

Future research could focus more on the systematic design of breeding programmes.

For instance, research may simulate genetic progress by considering the removal of recessive alleles and selection on complex traits at the same time, as well as considering the willingness of breeders to participate. Moreover, a benefit not shown from this study is that the reliability of estimation could be improved after receiving more phenotypic data from joint evaluation.

In addition, genomic prediction of HD-related traits was performed in UK Labrador retrievers [START_REF] Sánchez-Molano | Genomic prediction of traits related to canine hip dysplasia[END_REF], and joint genomic evaluation of HD between UK and US Labrador retrievers has also been reported [START_REF] Edwards | Joint Genomic Prediction of Canine Hip Dysplasia in UK and US Labrador Retrievers[END_REF], demonstrating the possibility of performing genomic prediction for HD. This could be the basis for future improvement of HD with faster genetic progress.

Conclusions

Our simulation study demonstrated that, for countries that do not have an EBV evaluation system, it would be beneficial to import dogs with high genetic merit from countries with EBV systems. Additionally, for countries with EBV systems, importing sires with high genetic merit could be an effective breeding strategy to improve the status of hip joints in pedigree dogs, although the outcome depends on the accuracies of EBVs, genetic correlations of HD across countries, selection intensities, and percentage of litter produced by foreign sires. When considering using foreign sires with EBV rankings in the top 50%, which is the selection intensity suggested by the British Kennel Club, strong genetic correlations (> 0.85) are necessary to ensure genetic progress equivalent to selection within an individual country. When considering the use of sires with high EBV rankings, e.g. in the top 10%, more moderate EBV accuracy (> 0.40) and moderately high levels of genetic correlation (> 0.70) are needed to improve on genetic progress achievable using domestic breeding stock alone. Assuming that 20% of litters are produced by foreign sires, scenario 'Pheno+DomEBV2+IntEBV2' was used to run the comparison of cases that only allowing healthy dogs to reproduce; using the top 50% domestic dogs and the top 10-50% of foreign dogs based on EBVs (country 2 scale). 'IntAll' and 'IntEBV2' herein are 'Pheno+DomEBV2+IntAll' and 'Pheno+DomEBV2+IntEBV2' and the difference is with and without using top 50% foreign dogs based on EBVs (country 2 scale). comparing eight breeding scenarios in country 2. Indicating specific breeding strategies under 4 various scenario, 'NoSel' is random mating, 'Pheno2' is only dogs without hip dysplasia 5 (HD; country 2 scale) that were allowed to reproduce, 'Pheno' is only domestic dogs without 6 HD (country 2 scale) and foreign dogs without HD (country 1 scale) that were allowed to 7 reproduce, 'DomEBV2' is domestic sires were used in the top 50% based on EBV (country 2 8 scale), 'IntAll' is no EBV requirement of using foreign sires, 'IntEBV1' is using foreign sires 9 in the top 50% based on EBV (county 1 scale), 'IntEBV2' is using foreign sires in the top 10

Figure legends 1 2 Fig. 1 .

 21 Figure legends 1 2 Fig. 1. Evolution of average true breeding values (TBVs) of newborn pups in 10 years 3

Fig. 2 .

 2 Fig. 2. Average true breeding values (TBVs) of newborn pups at year 10 as a function of 15 accuracy of selection in country 2. 'Pheno2+DomEBV2'is scenario using only healthy dogs 16 and top 50% domestic sire dogs (no foreign sire dogs used); 'Pheno+DomEBV2+IntEBV2 17 (Top 50%)' and 'Pheno+DomEBV2+IntEBV2 (Top 10%)' are scenario 18 'Pheno+DomEBV2+IntEBV2' that using only healthy dogs, using top 50% domestic sire dogs 19 based on estimated breeding value (EBV; country 2 scale) for 80% new born litters, and using 20 top 50% or top 10% of foreign sire dogs based on EBV (country 2 scale) for 20% newborn 21 litters in each year. Dotted lines indicate the simple linear trend for each simulation scenario. 22 23 Fig. 3. Average true breeding values (TBVs) of newborn pups at year 10 as a function of 24 genetic correlation in country 2. 'Pheno+EBV2'is scenario using only healthy dogs and top 25

Table 1 .

 1 Parameters used in breeding strategies simulation.

	Scenario	Item

Table 3 .

 3 Average true breeding values (TBVs), percentage of hip dysplasia (HD), coefficients of inbreeding (F) and coancestry (C) of newborn pups at year 10 comparing eight breeding scenarios in country 2 (standard error).

		No importation		With importation
				Pheno+	Pheno+DomEBV2+
		NoSel Pheno2 Pheno2+DomEBV2 IntAll IntEBV1 IntAll IntEBV1 IntEBV2
	TBVs	0.00 -0.23	-0.62	-0.24 -0.27 -0.53 -0.56	-0.57
		(0.01) (0.01)	(0.04)	(0.03) (0.03) (0.01) (0.02)	(0.02)
	HD (%) 20.40 11.21	3.04	11.04 10.19	4.28	3.82	3.77
		(0.38) (0.19)	(0.12)	(1.10) (1.11) (0.23) (0.17)	(0.16)
	F (%)	0.30 0.37	1.28	0.22	0.26	0.70	0.69	0.70
		(0.01) (0.01)	(0.03)	(0.13) (0.14) (0.09) (0.10)	(0.10)
	C (%)	1.37 1.57	4.42	1.11	1.17	2.81	2.72	2.81
		(0.01) (0.01)	(0.06)	(0.39) (0.42) (0.26) (0.28)	(0.25)
	NoSel -Random mating,
	Pheno2 -Only dogs without HD (country 2 scale) allowed to reproduce
	Pheno -Only domestic dogs without HD (country 2 scale) and foreign dogs without HD (country 1 scale) allowed to reproduce
	DomEBV2 -Using domestic sires in the top 50% based on EBV (country 2 scale)
	IntAll -No EBV requirement when using foreign sires
	IntEBV1 -Using foreign sires in the top 50% based on EBV (country 1 scale)
	IntEBV2 -Using foreign sires in the top 50% based on EBV (country 2 scale)
	No importation: 100% of litters produced by domestic sires
	With importation: 80% and 20% of litters produced by domestic and foreign sires respectively

Table 4 .

 4 Average true breeding values (TBVs), percentage of hip dysplasia (HD), coefficients of inbreeding (F) and coancestry (C) of new born pups at year 10 comparing different estimated breeding value (EBV) rankings of foreign sires used in breeding in country 2 (standard error).

	EBV ranking of foreign sires a Top 50% Top 40% Top 30% Top 20% Top 10%
	TBVs	-0.57	-0.58	-0.61	-0.61	-0.65
		(0.02)	(0.02)	(0.02)	(0.02)	(0.02)
	HD (%)	3.77	3.52	3.31	3.10	2.91
		(0.16)	(0.15)	(0.18)	(0.17)	(0.15)
	F (%)	0.70	0.71	0.73	0.74	0.99
		(0.10)	(0.10)	(0.10)	(0.10)	(0.09)
	C (%)	2.81	2.84	2.90	2.95	3.92
		(0.25)	(0.28)	(0.30)	(0.30)	(0.21)
	a

Table 5 .

 5 Average TBVs, percentage of hip dysplasia (HD), coefficients of inbreeding (F) and coancestry (C) of newborn pups at year 10 comparing with and without estimated breeding value (EBV) requirements of using foreign sires by different percentages of litters produced by foreign sires in country 2 (standard errors within brackets).

	Percentage of litters produced by foreign sires a 0%	20%		40%		60%
		IntAll IntEBV2 IntAll IntEBV2 IntAll IntEBV2
	TBVs	0.00 -0.53 -0.57 -0.48 -0.55 -0.41 -0.50
		(0.01) (0.01) (0.02) (0.01) (0.01) (0.01) (0.01)
	HD (%)	20.40 4.28	3.77	5.39	4.21	6.74	4.92
		(0.38) (0.23) (0.16) (0.40) (0.22) (0.59) (0.35)
	F (%)	0.30 0.72	0.70	0.47	0.57	0.41	0.64
		(0.01) (0.09) (0.10) (0.12) (0.12) (0.12) (0.10)
	C (%)	1.37 2.81	2.81	1.99	2.27	1.76	2.37
		(0.01) (0.26) (0.25) (0.36) (0.37) (0.38) (0.34)

a Full descriptions of

See: OFA -Orthopedic Foundation for Animal.

(Available at https://www.ofa.org/diseases/breedstatistics#detail (Accessed 10 March 2019)

Acknowledgement

Shizhi Wang benefited from a joint grant from the Erasmus Mundus European Graduate School in Animal Breeding and Genetic (EGS-ABG). We are grateful to EGS-ABG for funding and grateful to the Société Centrale Canine (SCC, France), Svenska Kennelklubben (SKK, Sweden), and The Kennel Club (KC, UK) for funding and data provision. Besides, we are also grateful to the valuable comments on the manuscript of Freddy Fikse and two anonymous reviewers.

Conflict of interests

None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.

Appendix: Supplementary material

Supplementary data associated with this article can be found, in the online version, at doi: ...