{"paper_id":"074fce5e-6b4e-4df6-a9a3-9a1dafd62b71","body_text":"1 \n \nWolbachia strengthens the match between pre-mating and early post-mating \nisolation in spider mites \nMiguel A. Cruz1, Sara Magalhães1,2, Murat Bakırdöven3, Flore Zélé4 \n \n1Centre for Ecology, Evolution and Environmental Changes (cE3c) & CHANGE - Global Change \nand Sustainability Institute, Department of Animal Biology, Faculdade de Ciências da Universidade \nde Lisboa, Edifício C2, 3° Piso, Campo Grande, Lisboa, Portugal \n2Departamento de Biologia Animal, Faculdade de Ciências da Universidade de Lisboa, Campo \nGrande, Lisboa, Portugal \n3Institute of Environmental Sciences, Boğaziçi University, Istanbul, Turkey \n4ISEM, Institut des Sciences de l’Évolution de Montpellier (ISEM), Université de Montpellier, \nCNRS, IRD, EPHE, Montpellier, France \nCorrespondence: flore.zele@cnrs.fr \n \nAuthors’ contributions \nMC, SM, and FZ conceived and designed the experiments. MC and MB performed the choice and \nno-choice tests, respectively. MC and FZ analysed the data. Funding agencies did not participate in \nthe design or analysis of experiments. MC and FZ wrote the manuscript with input from SM. All \nauthors read and approved the final version of the manuscript. \n \nAcknowledgements \nWe are grateful to Inês Santos for the maintenance of the spider mite populations and the plants, to \nLeonor Rodrigues and Élio Sucena for useful advises with experimental designs, and to Vitor Sousa \nand Alexandre Blanckaert for useful comments that improved the manuscript.  \n \nFunding \nThis work was funded by an FCT -ANR project (FCT -ANR//BIA-EVF/0013/2012) to SM and \nIsabelle Olivieri, and by an ERC Consolidator Grant (COMPCON, GA 725419) to SM. MC was \nfunded through an FCT PhD fellowship (SFRH/BD/136454/2018), and FZ through an FCT Post-Doc \nfellowship (SFRH/BPD/125020/2016) when experiments were performed. This is contribution \nISEM-2024-XXX of the Institute of Evolutionary Science of Montpellier (ISEM). \n \nConflict of interest \nThe authors declare that they have no conflict of interest with the content of this article. \n \nData availability \nAll datasets and R scripts are available at Zenodo (https://doi.org/10.5281/zenodo.11160702). \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n2 \n \nAbstract \nEndosymbiotic reproductive manipulators are widely studied as sources of post-zygotic isolation in \narthropods, but their effect on pre-zygotic isolation between genetically differentiated populations \nhas garnered less attention. We tested this using two partially isolated populations of the red and green \ncolour forms of Tetranychus urticae, either uninfected or infected with a Wolbachia strain inducing \nor not cytoplasmic incompatibility. We first investigated male and female  preferences, and found \nthat, in absence of infection, females were not choosy  but all males prefer red red-form females. \nWolbachia effects were more subtle, with only the CI-inducing strain slightly strengthening colour-\nform based preferences. We then performed a double -mating experiment to test how incompatible \nmatings affect subsequent mating behaviour and offspring production, as compared to compatible \nmating. Females mated with an incompatible male (infected and/or heterotypic) were more attractive \nand/or receptive to subsequent (compatible) matings, although analyses of offspring production \nrevealed no clear benefit for this remating behaviour ( i.e., apparently unaltered first male  sperm \nprecedence). Finally, by computing the relative contributions of each reproductive barrier to total \nisolation, we showed that pre-mating isolation matches both host-associated and Wolbachia-induced \npost-mating isolation, suggesting that Wolbachia could assist speciation processes in this system. \n \nKeywords: \nHaplodiploidy, mate choice, reproductive interference, sperm precedence, cytoplasmic \nincompatibility, reinforcement. \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n3 \n \nIntroduction \nUnderstanding the evolution of reproductive barriers between taxa ha s long been a major focus of \nevolutionary biology (Coyne and Orr 2004) . While speciation research has traditionally viewed \nspecies divergence as a process inevitably leading to full reproductive isolation (biological species \nconcept; Mayr 1942), recent evidence has shown that partial isolation occurring along the speciation \ncontinuum (Stankowski and Ravinet 2021)  can be reversible (Taylor et al. 2006; Bhat et al. 2014; \nKearns et al. 2018), or may even be selected in some circumstances (Servedio and Hermisson 2020). \nStudying population pairs for which reproductive barriers are incomplete is of great value  to \nunderstand these processes, as it can provide insight into which type of reproductive barrier is more \nlikely to evolve first , then drive the evolution of others  (Baack et al. 2015; Lackey and Boughman \n2017). On the one hand late post-zygotic barriers leading to costly hybridization can evolve first (e.g., \nin allopatry), then promote the evolution of  pre- and/or early post -zygotic barriers  at secondary \ncontact (i.e., reinforcement following the definition of Coughlan and Matute; 2020; but see Bank et \nal. 2012) . On the other hand , by limiting gene flow, pre -zygotic barriers should lead to faster \naccumulation of genetic differences between populations  in sympatry , thereby promoting the \nevolution of greater post-zygotic barriers (e.g., Lackey and Boughman 2017) . In addition , \nreproductive isolation may be driven not only by the genetics of the organisms themselves, but also \nby their endosymbionts (Brucker and Bordenstein 2012) . This is especially true for endosymbionts \nthat directly manipulate the reproduction of their hosts  (Duron et al. 2008; Engelstädter and Hurst \n2009; Brucker and Bordenstein 2012). \n \nWolbachia is a widespread endosymbiotic bacterium (Weinert et al. 2015)  that manipulates \nits host reproduction in different ways to increase its own transmission and invasiveness  (Werren et \nal. 2008; Engelstädter and Hurst 2009) . The most common of such manipulations is cytoplasmic \nincompatibility (CI), a conditional sterility phenotype which results in embryonic mortality of \noffspring from crosses between infected males and uninfected females (or females infected with an \nincompatible strain; Shropshire et al. 2020). Although the contribution of Wolbachia to post-zygotic \nisolation has been extensively studied  in different systems , its contribution to pre -zygotic isolation \n(both pre- and post-mating) between hosts has received comparatively less attention (see Shropshire \nand Bordenstein 2016; Bi and Wang 2020; Kaur et al. 2021) , especially when acting alongside host \ngenetic incompatibilities. \nTheory predicts that Wolbachia could drive reinforcement  between undifferentiated host \npopulations (i.e., females may evolve avoidance of incompatible males to escape CI ; Champion de \nCrespigny et al. 2005; Telschow et al. 2005), but empirical studies have produced contrasting results, \nmost of them showing no (or weak) evidence for CI -driven assortative mating (reviewed by \nShropshire and Bordenstein 2016; Bi and Wang 2020) . Such discrepancy could be explained by \nuneven abilities of hosts to detect Wolbachia infection in their mates (e.g., Wolbachia may alter the \nchemical profiles of some host species only; Richard 2017; Fortin et al. 2018; Schneider et al. 2019), \nor because avoidance of CI might be more likely when the infection is associated with pre -existing \nhost traits that can be used for mate recognition (Engelstädter and Telschow 2009). If such is the case, \nCI avoidance should be more commonly found between already differentiated populations.  In line \nwith this, the rare studies focusing on genetically differentiated hosts showed that pre-mating isolation \nwas strengthened (possibly even caused) by Wolbachia infection (e.g., Jaenike et al. 2006; Koukou \net al. 2006; Miller et al. 2010); but see Shoemaker et al. 1999). Finally, Wolbachia infection may also \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n4 \n \ndrive other forms of pre -zygotic isolation, including those occurring after mating . For instance, \nWolbachia infection can have deleterious effects on the production of sperm (Snook et al. 2000), the \ntransfer of fertile sperm (Lewis et al. 2011), the fertilization success (Bruzzese et al. 2021), and the \nreceptivity to and/or the effectiveness of re -mating; (De Crespigny and Wedell 2006; Champion De \nCrespigny et al. 2008; Liu et al. 2014; He et al. 2018) . However, to our knowledge, no study has \nspecifically disentangled the relative role of Wolbachia from that of host genetic factors on different \ntypes of post-mating pre-zygotic barriers. \n \nTetranychus spider mites are an excellent system to address the interplay between host -\nassociated and symbiont -induced incompatibilities (Cruz et al. 2021) . Indeed, Wolbachia is \nubiquitous in this genus (Breeuwer and Jacobs 1996; Gotoh et al. 2003; Xie et al. 2006; Zhang et al. \n2013, 2016; Zélé et al. 2018a), and its effects have been widely studied in the two-spotted spider mite \nT. urticae. In this host species, the bacterium induces highly variable degrees of different types of CI \n(mortality or development as male of fertilized eggs in incompatible crosses; e.g., Breeuwer 1997; \nPerrot-Minnot et al. 2002; Vala et al. 2002; Gotoh et al. 2007; Suh et al. 2015; Zélé et al. 2020; \nWybouw et al. 2023) , and has variable effects on pre-mating isolation (either no effect: Zhao et al. \n2013b; Rodrigues et al. 2022; or avoidance of infected males by uninfected females: Vala et al. 2004). \nHowever, in spider mites , as in many other arthropod species, its  contribution to post-mating pre-\nzygotic isolation has seldom been studied, which is at odds with the critical role that this symbiont \nmay play in the speciation processes currently ongoing in this group.  \nGiven the wide and overlapping distribution of m any spider mite species (Migeon and \nDorkeld 2023), as well as the high variability in genetic distances both between and within species \n(e.g., Matsuda et al. 2018; Villacis-Perez et al. 2021), spider mites commonly suffer various degrees \nof reproductive isolation . In particular, there is ample evidence of variation in all possible post-\nzygotic reproductive barriers  (zygote and juvenile hybrid mortality, hybrid sterility, hybrid \nbreakdown), both between (Keh 1952; Helle and Van de Bund 1962; Hill and O’Donnell 1991)  and \nwithin spider mite species (e.g., Van de Bund and Helle 1960; de Boer 1982a,b; Sugasawa et al. 2002; \nKnegt et al. 2017; Cruz et al. 2021) . Several studies also revealed variable post-mating pre-zygotic \nisolation in this group  (e.g., fertilization failure resulting from gametic or mechanical \nincompatibilities), as evidenced by a reduction in the production of female offspring in this system, \nbecause spider mites are arrhenotokous haplodiploids (haploid males develop from unfertilized eggs \nand diploid females from fertilized eggs; Helle and Bolland 1967) . Hence, whereas no female \noffspring are produced in crosses between well-formed species (e.g., Helle and Van de Bund 1962; \nHill and O’Donnell 1991; Chain-Ing and Sheuan-Ping 1995; Clemente et al. 2016, 2018), male-biased \nsex ratios are often reported in crosses between genetically differentiated ‘forms’ of the same species \nor even between genotypes of the same form (e.g., Gotoh et al. 1993; Navajas et al. 2000; Sugasawa \net al. 2002; Auger et al. 2013; Cruz et al. 2021; Villacis-Perez et al. 2021). In addition, because spider \nmites exhibit first male sperm precedence (only the first male that mates with a female sires all the \noffspring; Helle 1967; Rodrigues et al. 2020), females usually cannot restore their fitness through re-\nmating. Therefore, post-mating incompatibilities are particularly costly and should select for earlier \npre-zygotic barriers through reinforcement. Yet, highly variable degrees of pre-mating isolation can \nbe found both between (Sato et al. 2014, 2016; Clemente et al. 2016; Sato and Alba 2020) and within \nspecies (e.g., Murtaugh and Wrensch 1978; Gotoh et al. 1993). \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n5 \n \nTo improve our understanding of the role that Wolbachia can play in ongoing speciation \nprocesses of its hosts, we aimed at disentangling the relative contributions of Wolbachia and host \ngenetic factors to the strength of both pre - and post-mating pre-zygotic barriers between two colour \nforms, green and red, of the two spotted spider mite T. urticae (sometimes also referred to as T. urticae \nand T. cinnabarinus; Xie et al. 2006; Auger et al. 2013; Lu et al. 2017, 2018) . A previous study \nfocusing on the joint effects of Wolbachia-induced and host-associated post-mating incompatibilities \nbetween populations of these two forms revealed full reproductive isolation due to late post-zygotic \nbarriers (hybrid sterility and hybrid breakdown) that were independent of Wolbachia infection (Cruz \net al. 2021). However, this study also revealed partial and asymmetrical earlier post-mating barriers \n(pre- and/or post-zygotic), resulting from a combination of host -associated and Wolbachia-induced \nincompatibilities (Cruz et al. 2021). Host genetic incompatibilities led to an increased proportion of \nhaploid sons to the detriment of diploid daughters (‘male development’ or MD-type incompatibility, \nlikely due to fertilization failure ), whereas incompatibilities attributed to Wolbachia infection in \nmales were expressed as an increased embryonic mortality of daughters (‘female mortality’ or FM -\ntype CI). Furthermore, both types of incompatibility ha d additive effects and act ed in the same \ndirection of crosses  (Cruz et al. 2021) , which hint ed at a possible role of Wolbachia-induced \nincompatibilities in host population divergence and subsequent evolution of intrinsic reproductive \nbarriers, as found in Nasonia wasps (Bordenstein et al. 2001).  \n \nHere, we first performed male and female choice tests to determine their preference for \ninfected or uninfected mates from their own or a different colour -form population (i.e., test for pre-\nmating isolation). Second, we used a no-choice test to determine the effect of female mating history \n(virgin or previously-mated with a compatible vs incompatible male) on their mating behaviour, and \nto investigate whether eggs are more likely fertilized by compatible than incompatible sperm (i.e., \ntest for ‘homotypic’ sperm precedence). Finally, we used data gathered throughout all experiments \nstemming from this study and the previous one (Cruz et al. 2021) to estimate the relative contribution \nof each measured host-associated or Wolbachia-induced individual barrier to total reproductive \nisolation in this system. \n \n \nMaterials and Methods \nSpider mite populations \nTwo populations of spider mites, each belonging to a different colour form of T. urticae (‘red’ or \n‘green’), and either infected or uninfected with Wolbachia, were used in this study. These populations \nwere previously used to assess post-mating isolation caused by both host-associated incompatibilities \n(HI) and Wolbachia-induced reproductive barriers (Cruz et al. 2021; see also Zélé et al. 2018 for field \ncollection, and Zélé et al. 2020  for the effects of Wolbachia following laboratory maintenance). \nBriefly, the Wolbachia-infected population ‘Ri’ and its uninfected counterpart ‘Ru’ (‘Ri1’ and ‘Ru1’ \nin Cruz et al. 2021) belong to the red form of T. urticae, whereas the Wolbachia-infected population \n‘Gi’ and the uninfected population that derived from it, ‘Gu’, belong to the green form of T. urticae. \nThe Ru and Gu populations used in the present study were obtained from the antibiotic treatments \nperformed for Experiments 2 and 1 in Cruz et al. (2021) , respectively. All populations were \nsubsequently reared under the same standard laboratory conditions (24±2ºC, 16/8h L/D) at high \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n6 \n \nnumbers ( >1000 females per population) in mite -proof cages containing bean plants ( Phaseolus \nvulgaris, cv. Contender seedlings obtained from Germisem, Oliveira do Hospital, Portugal). All \nbehavioural observations were conducted during daytime at constant room temperature (25 ± 2°C). \n \nMate preference and behaviour of males and females in choice tests \nTo determine whether spider mites discriminate between mates to avoid Wolbachia-induced and/or \nhost-associated incompatibilities, individual males and females were provided two mates from \ndifferent populations and/or infection statuses. All combinations of choice tests performed are \ndescribed in Table 1. To obtain a large number of individuals of similar age, age cohorts were created \nfor each population twelve to fourteen days prior to the onset of each mate choice observation ( i.e., \neach cohort was used for two to three sequential days of observation). To this aim, 50 mated females \nor 50 virgin females (to obtain cohorts of females or males, respectively) from each population laid \neggs during 3 days on detached bean leaves placed on water -soaked cotton in p etri dishes under \nstandard laboratory conditions (24±2ºC, 16/8h L/D). Ten to twelve days later, female and male \ndeutonymphs undergoing their last moulting stage ( i.e., teleiochrysalids) were randomly collected \nfrom each of the female and male cohorts, respectively, and placed separately on bean leaf fragments \n(ca. 9cm2) to obtain virgin adult females and males of similar age two days later. As conversely to \nfemales, males cannot easily be identified based on their body colouration, they were painted before \neach observation with either blue or white water-based paint (randomized across treatments) using a \nfine brush. Previous experiments showed no effect of this paint on spider mite mate choice and \nbehaviour (Rodrigues et al. 2017, 2022) . Subsequently, a pair of virgin mates was installed on a \n0.4cm2 leaf disc (called ‘arena’ hereafter) and each observation started when the focal individual (a \nvirgin female or a virgin male) was introduced to the arena. The colour of the mate that first copulated \nwith each focal individual was registered, and later ass igned to the corresponding treatment (thus \nensuring that the observer was blind to the treatment to which mites belonged). Simultaneously, the \ntime until the beginning of copulation (‘latency to copulation’) and its duration (‘copulation duration’) \nwere recorded using an online chronometer ( http://online-stopwatch.chronme.com/). Each \nobservation lasted until the end of a first copulation or for 30 minutes if no mating occurred. Male \nand female choice tests were performed separately, each with one replicate of each treatment observed \nsimultaneously per session and four sessions of observations carried out per day. In total, 60 replicates \nper treatment were obtained over the course of 15 days for each of the two tests.  \n \n(Re)mating behaviour and offspring production in the no-choice test \nMating behaviour in the first mating event \nSpider mites may possess pre-zygotic mechanisms other than mate discrimination to avoid and/or \nreduce the cost of incompatibilities. For instance, an incompatible mate could be rejected after a \ncopulation has started (e.g., in Littorina snails; Rolán-Alvarez et al. 1999). Moreover, as copulations \nlasting for less than 30 seconds can be insufficient to fully fertilize a spider mite female (Potter and \nWrensch 1978; Satoh et al. 2001), shorter copulations might explain the excessive production of male \noffspring (i.e., arising from unfertilized eggs) to the detriment of female offspring ( i.e., arising from \nfertilized eggs) previously observed in crosses between green females and red males (Cruz et al. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n7 \n \n2021). To test whether such post -copulatory mechanisms of avoidance of incompatibilities occur in \nspider mites, we performed a no -choice test, where the mating behaviour of virgin females placed \nwith a single male was observed. Given the workload involved in this experiment, we only performed \nthe crosses allowing to test for the single and combined effects of host -associated and Wolbachia-\ninduced incompatibility, along with their respective controls  (cf. Table 2). Also, because only few \nindividuals could be test ed per day  in this experiment , producing age cohorts was not necessary. \nInstead, male and female teleiochrysalids were directly sampled from the base populations two days \nprior to observation, and isolated on bean leaf fragments to ensure their virginity. For each treatment, \none male and one female were installed together on a 0.5cm 2 bean leaf disc and observed for 60 \nminutes. During that time, multiple mating could occur. Thus, in addition to the mating propensity \n(i.e., the probability that mating occurred at least once), copulation frequency ( i.e., the number of \ncopulations during the observation period) was also registered. The latency to the first copulation and \nthe duration of each copulation occurring during the observation period were recorded as in the mate \nchoice experiment. The cumulative copulation duration of each couple was subsequently computed. \nAt the end of the observation period, females for which at least one copulation occurred were \nindividually placed on a 2cm2 bean leaf disc and kept for the next step ( cf. below), while non-mated \nfemales and all males were discarded.  \n \nMating behaviour in the second mating event \nIn species with first-male sperm precedence such as T. urticae (i.e., the first male that had mated with \na female sires all of her offspring; Rodrigues et al., 2020), females usually have low receptivity to a \nsecond mate (Clemente et al. 2016) . However, if the first copulation is interrupted or (at least \npartially) ineffective, females may show increased receptivity to second matings that could effectively \ncontribute to fertilization (Helle 1967; Clemente et al. 2016; Costa et al. 2023). To test this, females \nfor which at least one copulation occurred  during the first mating event were placed with a second \ncompatible male 24 hours later (cf. Table 2) and their mating behaviour was recorded. Behavioural \nobservations were carried out for 60 minutes as in the first mating event. The mating propensity, \nmating frequency, latency to first copulation and cumulative copulation duration with the second \nmale were simultaneously registered. At the end of the observation period, males were discarded and \nfemales were kept individually on 2 cm2 bean leaf discs placed on water-soaked cotton in petri dishes \nin a climatic chamber (25 ± 2°C, 60% RH, 16/8 h L/D). Given the workload and the multiplicity of \ntasks involved in this experiment , only 9 couples were observed simultaneously per session  of \nobservation, corresponding to one or two replicates per treatment. Four sessions of observation were \nperformed per day (hence 6  replicates of each treatment per day), each day corresponding to an  \nexperimental ‘block’. In total, 19 blocks, each separated by 3 days, were performed to obtain ca. 100 \nreplicates per treatment.  \n \nOffspring production and strength of post-mating incompatibilities \nTo test whether the second copulation with a compatible male could restore female offspring  \nproduction, the offspring produced over 3 days of oviposition by females mated with either a single \nor two different males was compared , and female mortality during that period also registered . The \nnumber of unhatched eggs was counted 6 days later (day 9), and the numbers of dead juveniles, adult \nmales and females were counted 3 and 6 days later (days 12 and 15). Then, to determine the proportion \nof offspring affected by host-associated MD-type incompatibility (i.e., “Male Development”), and/or \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n8 \n \nWolbachia-induced FM-type incompatibility (i.e., “Female Mortality”), we computed two indexes as \nfully described in Cruz et al. (2021): the 𝑀𝐷!\"## index, which calculates the overproduction of sons \nin the brood, and the 𝐹𝑀!\"## index, which calculates the embryonic mortality of fertilized offspring \n(i.e., only females in haplodiploids), both relative to the control crosses (thereby accounting for \nbackground variation). Finally, as in Cruz et al. (2021) , we also computed the proportion of F1 \nfemales over the total number of eggs (FP) to determine the combined effect of  FM- and MD-type \nincompatibilities on the total proportion  of daughters in each cross . Raw data are shown in the \nSupplementary Figure S1. \n \nStrength and contribution of each reproductive barrier to total isolation \nStrength of reproductive isolation for each reproductive barrier (RIn)  \nTo estimate the strength of pre- or post-mating reproductive barriers identified for each type of cross \nwithin and between the green - and red-form populations, we used a combination of the pre -mating \ndata obtained in the present study and the post-mating data obtained in the previous study (Cruz et al. \n2021), respectively. A ll reproductive barriers found to play a role in reducing gene flow among the \nspider mite populations were considered: mate preference  (RI1), fertilization failure ( RI2), hybrid \ninviability (RI3), hybrid sterility ( RI4), and hybrid breakdown ( RI5). As we found no evidence for \nhomotypic sperm precedence in the no -choice test ( cf. Results), this barrier was not considered. \nSimilarly, female choice data were not used when computing RI1, as females overall showed no mate \npreferences in the choice test (cf. Results).  \n To determine the strength of pre -mating isolation (RI1), we applied a sexual isolation index,  \nwhich varies between zero and one, to the male choice data. This index, adapted from Bateman (1949) \nand Merrell (1950) by Malogolowkin-Cohen et al. (1965), is given by: \n𝑅𝐼$(&) = \t (𝑛&& − \t 𝑛(& )\n(𝑛&& + \t 𝑛(& ) \nwhere nxx is the number of copulations observed between females and males of a population x, and \nnyx is the number of copulations observed between females of a population y and males of the \npopulation x. As RI1 represents the degree to which a population x is isolated from a population y due \nto mating preferences, it was set to 0 in the case of preference for heterotypic mates (i.e., no negative \nimpact on gene flow).  \nTo determine the strength of post -mating barriers, we used the data published in Cruz et al. (2021), \nas late reproductive barriers ( i.e., hybrid sterility and hybrid breakdown) were not measured here. \nMoreover, earlier post -mating barriers (fertilization failure and hybrid inviability) have been \nestimated more precisely in the previous study than in the current one (i.e., larger sample sizes). For \nfertilization failure (RI2) and hybrid inviability ( RI4), we used the median values of the MD corr and \nFMcorr indexes, which correspond to the percent increase in non -fertilized eggs and in embryonic \nmortality o f fertilized eggs, respectively ( cf. ‘Offspring production and strength of post -mating \nincompatibilities’ above). For hybrid sterility ( RI5) and hybrid breakdown ( RI6), we computed the \npercent decrease in ovipositing F1 females and increase in embryonic mortality of F1 females’ eggs \nrelative to compatible crosses, respectively. \nContribution of each reproductive barrier (Cn) to total isolation (T)  \nWe employed a method previously adapted from Coyne and Orr (1989, 1997)  by Ramsey and \ncolleagues (2003), in which total (cumulative) reproductive isolation between two populations or \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n9 \n \nspecies is computed as a multiplicative function of the strength of each reproductive barrier (𝑅𝐼); see \nabove), so that the contribution of each barrier to reducing gene flow at a stage 𝑛 in life history  is \ncalculated as: \n𝐶) = \t 𝑅𝐼) .1 − 0 𝐶*\n)+$\n*,$\n1 \nThus, a given reproductive barrier eliminates gene flow that has not been prevented by earlier barriers, \nand for 𝑚 reproductive barriers, total reproductive isolation is given by: \n𝑇 = \t 0 𝐶*\n-\n*,$\n \n \n \nStatistical analyses  \nAnalyses were carried out using the R statistical software (v3.6.1). The general procedure for building \nall statistical models is detailed in the Supplementary Tables S1 and S2. Time-to-event data (latency \nto copulation and copulation duration) were analysed using Cox proportional hazards mixed models \n(coxme procedure; coxme package), a non -parametric method that does not assume any particular \nerror distribution (Crawley 2007) . All other  data were analysed using generalized linear mixed \nmodels (glmmTMB procedure; glmmTMB package ; Brooks et al. 2017) . P roportion data were \ncomputed either as binary response variables (e.g., mated or not, chosen mate) or using a concatenated \nresponse variable binding the number of successes and failures with the function cbind to account for \nthe number of eggs in the analyses of female proportion (i.e., number of daughters vs. number of eggs \n– number of  daughters). F or the corrected variables 𝑀𝐷!\"## and 𝐹𝑀!\"##, which are continuous \nvariables bounded between 0 and 1, we weighted each individual datum by  the number of \nobservations (i.e., a “weights” argument was added to the model s). These data were subsequently \nanalysed with a binomial or (zero -inflated) betabinomial error distribution when errors were  \noverdispersed. Count data were analysed with a Poisson error distribution, and continuous data with \na Gaussian, except for daily oviposition which was Box-Cox transform ed (λ=0.549) to improve \nnormality (Crawley 2007) and analysed with a zero-inflated Gaussian.  \nFor the analyses of each response variable of the choice tests, the ‘type’ of the focal individual \n(i.e., combination of population and infection status), and either the combination of provided mates \n(for the analyses of mating propensity and mate preference) or the chosen mate (for  the analyses of \nlatency to copulation and copulation duration), were fit as fixed explanatory variables, whereas the \nday and session (nested within day) of observation, the colour with which chosen males were painted \n(in the female choice test), and the combination of provided mates (for  the analyses of  latency to \ncopulation and copulation duration) were fit as random explanatory variables. In addition, f or the \nanalyses of mate preference, the intercept of the models was forced to zero  to obtain the estimate of \nthe fixed factor as the difference to a 0.5 probability (particularity of models with categorical factors \nand binomial distribution; Crawley 2007).  \nFor the analyses of the no -choice test, mating propensity data obtained in the two mating \nevents were analysed altogether, and the mating event was fit as fixed explanatory variable, along \nwith the type of cross (i.e., female x first male) and the interaction between these two factors, whereas \nthe day and the session (nested within day) of observation were fit as random explanatory variables. \nTo avoid pseudoreplication i n the analyses of copulation frequency,  latency to copulation and \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n10 \n \ncopulation duration, data obtained in each mating event were analysed separately and only the type \nof cross was fit as fixed explanatory variable.  Subsequently, to determine whether  these response \nvariables differed when females mated with the first or with the second male, the differences in \ncopulation frequency, latency, and duration between 1st and 2nd mating events were computed for \neach individual female that mated at least once in both events . For that, the type of cross was fit as \nfixed explanatory variable, the day and session (nested within day) were fit as random explanatory \nvariables, and the intercept was forced to zero to obtain the estimate of the fixed factor relative to no \ndifference between mating events . Finally, for the analyses of offspring data, the type of cross, the \nmating status of the females (i.e., mated with only the first or both males), and the interaction between \nthese two factors, were fit as fixed explanatory variables, whereas the day and session (nested within \nday) during which they mated were fit as random explanatory variables.  \nFor all analyses, maximal models containing the complete set of explanatory variables were \nsubsequently simplified by sequentially eliminating non -significant terms to establish a minimal \nmodel (Crawley 2007). The significance of the explanatory variables was established using chi-square \ntests with the Anova function (car package; Fox et al. 2019). The significant values given in the text \nare for the minimal model, whereas non -significant values correspond to those obtained before \ndeletion of the variable from the model (Crawley 2007). When explanatory variables with more than \ntwo levels were found significant in the analyses of behavioural data, a posteriori contrasts between \nfactor levels were carried out by aggregating factor levels together and testing the fit of the simplified \nmodel using a likelihood ratio test (function anova; Crawley 2007). When interactions between two \nexplanatory variables were significant, the two variables were concatenated (e.g., crosses and mating \nevents) and a posteriori contrasts between the factor levels of the concatenated variable were carried \nout as described above . In all cases, Holm-Bonferroni corrections ( i.e., classical chi -squared Wald \ntest for testing the global hypothesis H0; Holm 1979)  were subsequently applied to account for \nmultiple testing. Note that contrast analyses were not carried out for offspring production in the no -\nchoice test because the results did not differ qualitatively from th ose of the previous study (Cruz et \nal. 2021). Finally, to determine the difference to random mating in the analyses of mate choice , as \nwell as changes in mating behaviour between 1 st and 2 nd mating event s in the no -choice test , \ncoefficients (estimated as the difference with the zero -intercept) obtained from the maximal models \nfor each combination of focal individual and pair of provided mates  (mate choice test), or for each \ncross (no-choice test), were analysed with Z -tests using the function test (emmeans package; Lenth \net al. 2018).  \n \nResults \nMale and female mating behaviour in choice tests  \nOverall, the propensity of females to mate with one of two provided males depended on their own \npopulation (‘focal’: χ23=10.06, p=0.018; Model 1.1 in Table S1; Figure 1a), with green females being, \non average, ca. 20% less likely to mate than red females (Tables S3 and S4). However, their mating \npropensity was unaffected by the type of males they were offered (‘mates’: χ23=2.70, p=0.44; Model \n1.1), and none of them showed any clear mating preference ( ‘focal’: χ24=2.91, p=0.57, and ‘mates’: \nχ24=3.47, p=0.48; Model 1.2; Figure 1b  and Table S5 ). Conversely, the mating propensity and the \nmate choice of males were independent of their population ( ‘focal’: χ23=4.13, p=0.25 and χ24=5.01, \np=0.29 in Model 1.5 and 1.6, respectively ), but strongly affected by the type of females provided \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n11 \n \n(‘mates’: χ23=15.72, p=0.001 and χ24=50.24, p<0.0001 in Model 1.5 and 1.6, respectively; Figures 1c \nand 1d). Indeed, males that were given the choice between two green females were less likely to mate \nthan those that were given the chance to mate with a red female ( ca. 40% vs 68% mated males on \naverage; Figure 1c, Tables S3 and S4), and males of either colour form showed a preference for red \nfemales (ca. 60% to 80% preference; Tables S3 and S4).  \nIn contrast to the mite colour  form, Wolbachia infection had no effect on mite mating \npropensity and only a small effect on their mate preference  (cf. Figure 1, Table S3 and contrasts in \nTable S4). Neither uninfected females nor infected males showed any preference between infected or \nuninfected mates of the same colour (cf. category I in Figures 1b and 1d), but Wolbachia infection in \nred males (CI -inducing Wolbachia strain; Cruz et al. 2021)  strengthened their preferences for red \nfemales. Indeed, although the mate preference  of Ru and Ri males did not differ significantly, Ru \nmales showed no significant difference from random mating (cf. category II in Figure 1d) whereas Ri \nmales significantly preferred red females over green females (cf. categories III and IV; see also Table \nS5). In addition, whereas red females overall did not differ in their mate preference, and showed no \npreference between males of either colour form  when these were from the same infection status as \nthemselves (cf. categories II and III in Figure 1b ), Ru females preferentially mated with Gi males  \nover Ri males , suggesting avoidance of the CI induced by  Wolbachia infection in red males  (cf. \ncategory IV; see also Table S5). Conversely, the non-CI-inducing Wolbachia strain infecting green \nmales (Cruz et al. 2021) had no effect on the strength of mate preference of both males and females.  \nFinally, latencies to copulation did not differ significantly among focal females or chosen \nmales in the female choice test (χ23=6.76, p=0.08 and χ23=1.35, p=0.72, respectively; Model 1.3), nor \namong focal males or chosen females in the male choice test (χ23=1.33, p=0.72 and χ23=1.03, p=0.79, \nrespectively; Model 1.7, Figure 2a,b), but copulation duration differed between males of different \ncolours (Figure 2c)  and between females of different infection status  (Figure 2d) . Regardless of \nWolbachia infection (although Ru males showed intermediate copulation durations in the female \nchoice test; Figure 2c; Table S6 and S4), green males copulated on average 37 and 40 seconds longer \nthan red males in the female and male choice test, respectively (‘chosen’: χ23=7.92, p=0.048, and \n‘focal’: χ23=27.09, p<0.0001; Model 1.4 and 1.8, respectively ). Conversely, the copulation duration \nof females was not affected by  their colour form (although Gi females showed intermediate \ncopulation durations in the female choice test; Figure 2d and Table S6; cf. contrasts in Table S4), but \nthat of infected females was, on average, ca. 29 and 34 seconds shorter than that of uninfected females \nin the female and male choice test, respectively (‘focal’: χ23=10.64, p=0.014, and ‘chosen’: χ23=24.70, \np<0.0001 in Model 1.4 and 1.8, respectively).  \n \n(Re)mating behaviour in the no-choice test  \nOn average, 58% of the virgin females placed on a leaf disc with a single male mated within 1 hour, \nwhereas less than 20% of those mated females re-mated when placed with another male 24 hours \nlater. In line with this, a reduced copulation frequency ( 1.6±0.1 vs 2.1±0.1 copulations per couple) \nand copulation duration (118±13 vs 252±8 seconds) and an increased latency to copulation (1582±108 \nvs 986±46 seconds) , were observed, on average , between the first and second mating events for \ncouples that mated at least once (Figures 3,4 and Table S7). However, this reduction in the willingness \nto mate varied across types of crosses for all behavioural traits tested but copulation frequency (no \nstatistically significant differences found among crosses for either o r among the two mating events; \ncf. models 2.2 to 2.4 in Table S2). \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n12 \n \n For the mating propensit ies observed  during the first mating event, we found  the same \ntendencies as in the choice tests: Gu females were less likely to mate than Ru females (except when \npaired with Ru males  here), and  Wolbachia infection in red males seemed to promote mate \ndiscrimination (Ri males were less willing to mate with Gu females  than Ru males , whereas both \ntypes of males  mated as much with Ru females ; Figure 3; Table s S7 and  S8). Then, although no \nstatistically significant differences were found among crosses in the second event, not all crosses led \nto the same reduction in mating propensity between the two mating events ( cross x mating event \ninteraction: χ25=16.56, p=0.005; model 2.1; Figure 3; Table S 8): Gu females showed a lower \nreduction in their tendency to re-mate than Ru females, especially when they were previously mated \nwith an incompatible Ri male (hence when both types of incompatibilities were at play; Figure 3; \nTables S7 and S8).  \nIn this experiment, conversely to the previous experiment in which virgin individuals could \nchoose their mate and were given only half an hour to mate, we found significant differences among \nlatencies to copulation of  couples that mated at least once during the first mating event  (χ25=13.19, \np=0.02; model 2.5) . Gu females took, on average, 5 more minutes than Ru females to engage in \ncopulation with their first partner, regardless of the form or infection status of the latter (although Ru \nx Ru crosses had  intermediate latencies to copulation ; cf. circles in Figure 4a; Tables S7 and S9). \nAlso, in this first mating event, as in the previous experiment (choice tests), the cumulative time spent \ncopulating was longer for green males than for red males regardless of their infection status and the \nfemale they mated with (ca. 39 seconds difference; χ25=21.19, p<0.001; model 2.4; Figures 2c,d and \n4c; Tables S7 and S9). Then, when females that mated during the first mating event were placed with \na second male 24 hours later, their latency to copulation increased by almost 10 minutes, and their \ncopulation duration was more than 2 minutes shorter, than when they were virgin ( cf. diamonds in \nFigure 4a,c; Table S7). Despite no significant differences being found among types of crosses for  \nboth latency to copulation and cumulative copulation duration in the second mating event (χ25=5.16, \np=0.40; model 2.6; Figure 4a, and χ25=2.78, p=0.73; model 2.9; Figure 4c, respectively), behavioural \nchanges between the two mating events at the female level (for those who mated in both events) \nvaried depending on the type of cross  (χ26=12.47, p=0.05; model 2.7 ; Figure 4b , and χ 26=43.73, \np<0.0001; model 2.10 ; Figure 4d , for latency to copulation and cumulative copulation duration,  \nrespectively). Thus, i n line with the mating propensity observations , differences in latency to \ncopulation and copulation duration between mating events tended to disappear for females that had \nfirst mated with an incompatible male (except for the copulation duration of Gu females mated with \nRi males; Table S10). \n \nEffect of remating on offspring production in the no-choice experiment \nThe pattern of offspring production for females that mated only with one male (Figure 5a) was \nconsistent with that described in our previous study (Cruz et al. 2021) . Briefly, (i) we found an \noverproduction of males (MD -type incompatibility) in crosses between green females (Gu) and red \nmales (Ru or Ri ) as compared to the other crosses (χ25=76.30, p<0.0001; model 2.12; Figure 5b) . \nMoreover, because copulations were observed in the present study, it further unambiguously revealed \na high variability for this barrier: among the  66 Gu females that mated only with a Ru or Ri mate and \noviposited (i.e., 85 Gu females mated with a Ru or Ri male subsequently refused to mate wi th a \nsecond male; Table S7, and 19 of these females did not lay a single egg), 20 produced only sons (i.e., \nfull incompatibility), whereas 18 did not produce a more male-biased sex ratio than the controls (i.e., \nno incompatibility); (ii) we found an increased female embryonic mortality (FM -type CI quantified \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n13 \n \nas a decreased hatching rate of fertilized eggs) in crosses between uninfected females (Gu or Ru) and \nmales infected with a CI -inducing Wolbachia strain (Ri males), as compared to the other crosses  \n(χ25=76.78, p<0.0001; model 2.13; Figure 5c); and (iii) we found a reduction in the proportion of \ndaughters (FP) in crosses affected by either (or both) type(s) of incompatibility (i.e., Ru x Ri, Gu x \nRu and Gu x Ri, female x male crosses ) as compared to compatible crosses ( χ25=87.65, p<0.0001; \nmodel 2.14; Figure 5d). However, no difference in offspring production was found between females \nthat mated with only one or two different males (daily fecundity: χ 21=3.19, p=0.07; model 2.11; \nMDcorr: χ21=0.63, p=0.43; model 2.12; FM corr: χ21=0.14, p=0.71; model 2.13; FP: χ 21=0.17, p=0.68; \nmodel 2.1 4), regardless of whether the first male was compatible or not ( i.e., no significant \ninteractions between the type of cross and whether females mated with one or two males; daily \nfecundity: χ25=4.57, p=0.47; model 2.11; MD corr: χ25=0.89, p=0.97; model 2.12; FM corr: χ25=9.33, \np=0.10; model 2.13; FP: χ25=7.17, p=0.21; model 2.14; Figure 5; see also Figure S1 and Table S7). \n \nContribution of intrinsic and Wolbachia-induced reproductive barriers to reducing gene flow \nAlthough hybrid breakdown is the strongest reproductive barrier in both directions of crosses between \nthe two studied spider mite populations (100% F2 embryonic mortality; Cruz et al. 2021), it ultimately \ncontributes very little to total isolation  due to the occurrence of earlier barriers  (Figure 6 and Table \nS11). Red females and green males are mainly isolated due to hybrid sterility (98 to 100% isolation  \nregardless of Wolbachia infection), as no other reproductive barrier exist s in this cross direction. \nHowever, despite having the same strength in both directions of heterotypic crosses, hybrid sterility \nacts along with other reproductive barriers in crosses between green females and red mal es, which \nstrongly reduced its contribution to total isolation (ca. 12% and 29% in crosses with infected and \nuninfected males, respectively). In this cross direction , our estimations revealed that assortative \nmating and fertilization failure are in fact the main sources of reproductive isolation, contributing to \n27-61% and 23-71% of total isolation, respectively. Moreover, although hybrid inviability caused by \nthe CI-inducing Wolbachia strain infecting red males only has a weak contribution to total isolation \nin heterotypic crosses (as compared to homotypic crosses: ca. 5.5 to 6.4% in crosses between green \nfemales and Ri males vs 32% in crosses between Ru females  and Ri males; Table S11), infection of \nmales with this Wolbachia strain clearly potentiates pre-mating isolation (Figure 6) . Indeed, t he \nstrength of assortative mating increases from ca. 27% in crosses between Gu females  and Ru males \n(non-significantly different from random mating; cf. Figure 1 and above)  to ca. 61% in crosses \nbetween Gu females and Ri  males (Figure 6) and to ca. 49% in crosses between Gi females and Ri \nmales (Table S11). \n \nDiscussion \nIn this study, we sought to shed light on the potential role played by Wolbachia as an agent of pre -\nzygotic isolation between genetically differentiated colour forms of the spider mite Tetranychus \nurticae. Our results revealed that Wolbachia infection had no effect on the mating preference of both \nmales and females in homotypic crosses, but the CI-inducing strain infecting the red form exacerbated \npre-existing colour-based mate preferences. Whereas both types of males showed a preference for \nred females , this preference seemingly disappeared when red males were cured from Wolbachia \ninfection. In line with this, females showed no mate preferences in the absence of Wolbachia \ninfection, but uninfected red females showed a preference for  green infected males  (which do not \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n14 \n \ncarry a CI-inducing Wolbachia strain) over red infected ones  (which do carry a CI -inducing \nWolbachia strain). Then, we also found that (i) females that had engaged in matings where both types \nof incompatibility occur red (Wolbachia-induced and host -associated) were more likely to re -mate \nwith a compatible male, and (ii) females exposed to either type of incompatibility did not significantly \nincrease their latency to re-mate, nor reduce their copulation duration when re-mating, as compared \nto their first matings. Yet, the significance of such behaviour is puzzling, as we found no evidence of \nsperm contribution by the second (compatible) males following copulations with incompatible mates \n(i.e., adult offspring production and sex -ratios were not more similar to  the controls). This further \nindicated that ‘homotypic sperm precedence’ was not a reproductive barrier at play in our experiment. \nFinally, our estimations of the  relative contribution of each reproductive barrier  to reproductive \nisolation between the studied populations clearly illustrate the strong asymmetries that occur in this \nsystem: red females are isolated from green males due to late intrinsic post-zygotic barriers (hybrid \nsterility and hybrid breakdown), whereas green females are isolated from red males by a combination \nof early and late reproductive barriers (pre-mating, post-mating pre-zygotic, and post-zygotic), either \ndirectly caused (hybrid inviability  due to  CI) or strengthened (assortative mating) by Wolbachia \ninfection in males. \n \nA system driven by male rather than female mate preferences  \nIn most tested scenarios, T. urticae females did not choose between mates with different colour forms. \nThis corroborates earlier results, where females from different morphs or infection status did not \ndiscriminate between different types of males (Murtaugh and Wrensch 1978, Zhao et al. 2013b; \nRodrigues et al. 2022, but see Vala et al. 2004; Rohrscheib et al. 2015) , and indicates no differences \nin male competitive ability  as well (Murtaugh and Wrensch 1978 ; Wagner 1998) . In fact, several \nother studies revealed an absence of mate choice in spider mite females (e.g., Magalhães et al. 2009; \nZhou et al. 2020). This is surprising, as females invest more energy than males in their reproduction \n(Kokko et al. 2006 ), and spider mites have  first-male sperm precedence  (Helle 1967; Satoh et al. \n2001; Rodrigues et al. 2020) , hence the choice of the first male has enormous consequences for \nfemales (Wittenberger and Tilson 1980; Howlett 1988; Griffith et al. 2011) . Possibly, this weak \nfemale choice is a consequence of male guarding of females just before their emergence as virgin \nadults (Potter et al. 1976), leading to little opportunity for females to choose their mate (Everson and \nAddicott 1982; Oku 2014). In contrast, we found strong mate preferences in males, which is also in \nline with earlier studies on spider mites (e.g., Everson and Addicott 1982; Rodrigues et al. 2017), and \nin other arthropods in which males invest time and energy in pre - and/or post-copulatory guarding \n(reviewed in Bonduriansky 2001). \n \nAsymmetric reinforcement could explain the match between pre- and early post-mating \nbarriers  \nIn this system, one might expect assortative mating ( i.e., homotypic preference in both cross \ndirections) to be selected for due to severe costs of hybridization in both cross directions (Cruz et al. \n2021). Instead, our results  revealed an asymmetry in pre-mating isolation  (only red males prefer \nhomotypic females) . Possibly,  post-mating pre -zygotic barriers ( e.g., fertilization failure due to \ncytonuclear incompatibility) first evolved incidentally between green -form females and red -form \nmales in allopatric populations. The resulting asymmetrical maladaptive hybridization may have \nsubsequently ( i.e., at secondary contact) led to asymmetrical levels of reinforcement in areas of \nsympatry (Noor 1999; Servedio and Noor 2003; Coyne and Orr 2004), thereby driving the evolution \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n15 \n \nof homotypic mate preferences by red males only (e.g., as observed between Drosophila recens and \nD. subquinaria populations due to unidirectional CI induced by Wolbachia; Jaenike et al. 2006). This \nmay explain the match between pre-mating and early-acting post-mating barriers in this system (sex \nratio distortion likely due to fertilization failure in crosses between red males and green females; Cruz \net al. 2021), as found in other systems (reviewed in Ortiz-Barrientos et al. 2009; see also Giesbers et \nal. 2013; Yukilevich et al. 2018). Alternatively, asymmetric barriers acting in the same cross direction \ncould as well be due to genetic linkage between barriers ( e.g., Merrill et al. 2011) , a possibility not \nyet investigated in spider mites . Subsequently, the two forms  might have further diverged due to \nlimited gene flow, leading to the establishment of strong late post-zygotic barriers in both directions \n(Servedio and Sætre 2003) . In line with this , previous work has shown that barriers acting early in \nreproduction tend to evolve faster than those acting later (Coyne and Orr 1989; Servedio 2001; \nTurissini et al. 2018).  \nAside from pre-mating isolation, reinforcement could also drive the evolution of other types \nof pre-zygotic barriers, including those occurring after mating, such as conspecific sperm precedence \n(Coughlan and Matute 2020). Although preferential use of sperm from conspecific (or ‘homotypic’) \nmales would reduce the negative effects of mating with heterospecifics (e.g., Price 1997; Fricke and \nArnqvist 2004; Noriyuki et al. 2012) , we did not find any evidence for such reproductive barrier . \nNevertheless, we observed that  the latencies to copulation and copulation duration s of green-form \nfemales previously mated with red-form males tended to remain the same as when they were virgins. \nThis contrasts with the behaviour of females mated with fully-compatible males, which become less \nreceptive to subsequent males (increased latency and reduced copulation duration), in line with the \nfirst-male sperm precedence pattern (Helle 1967; Rodrigues et al. 2020). Other studies also found that \nspider mites first mated with (fully or partially) incompatible males behave as virgins in subsequent \nmatings (Clemente et al. 2016; Costa et al. 2023) . Moreover , when the incompatibilities are not \ncaused by fertilization failure (as in the case of Wolbachia-induced CI), a reduced latency to \ncopulation and increased copulation duration with a second male could increase the likelihood that \nthe sperm of the latter outcompete the sperm of the first male (Potter and Wrensch 1978; Satoh et al. \n2001). However, contrarily to these earlier studies, the results obtained here do not indicate any use \nof the sperm from second males, which could rescue limited offspring production resulting from  \nincompatible matings. Yet, this pattern may be jeopardized under other conditions than those tested \nin the current study. Here, we allowed for several copulations with the first male, and mated females \nwere exposed to a second male only 24 hours later . This was done to detect potential issues with \nsperm transfer or storage when an excess of male offspring is found ( i.e., in crosses between green \nfemales and red males) . If this was the case, significant effects of double mating on offspring \nproduction could not be unambiguously attributed to changes in the sperm precedence patterns \n(García‐González 2004) . However, the timing used might have been excessive  to enable sperm \ncompetition/cryptic sperm choice (Potter and Wrensch 1978; Satoh et al. 2001) , and future studies \nare necessary to uncover potential benefits of the behaviours observed here.  \n \nWolbachia-induced CI might strengthen asymmetrical reinforcement \nOur results concerning t he effect of Wolbachia infection on mate preferences between mite colour \nforms corroborate those of previous studies showing that Wolbachia infection strengthens assortative \nmating between genetically differentiated hosts (e.g., Jaenike et al. 2006; Koukou et al. 2006; Miller \net al. 2010). In particular, the fact that Wolbachia infection alone (i.e., in homotypic crosses) has no \nsignificant effect on mate choice (as in Rodrigues et al. 2022), supports the hypothesis that avoidance \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n16 \n \nof CI might evolve more readily when the infection is associated with pre-existing host traits that can \nbe used for mate recognition  (Engelstädter and Telschow 2009) . Moreover, the fact that mating \npreferences contribute more to total reproductive isolation when infected red males , which carry  a \nCI-inducing Wolbachia strain, were involved ( cf. fig. 6) , suggests that CI could be  a mechanism \ndriving asymmetrical reinforcement between spider-mite colour forms.  Consistent with a previous \nstudy on incompatibilities between different geographic strains of green-form T. urticae, in which the \nonly females receptive to a second mate were those previously mated with a genetically incompatible \nmale carr ying a CI-inducing Wolbachia strain (Navajas et al. 2000) , we also found that only \nuninfected green females previously mated with a red infected male (hence carrying a CI -inducing \nWolbachia strain) were as likely to mate with a second male as when they were virgins. In line with \nthis, only when uninfected females (both red and green) had mated with an infected red male (with \nthe CI-inducing strain) did their latency to copulation and copulation duration remain as when they \nwere virgin . Hence, together these findings revealed that Wolbachia can affect other mating \nbehaviours beyond mating preferences ( as in other systems; reviewed in Bi and Wang 2020) , and \nsuggest that Wolbachia-induced CI could assist reinforcement processes in this system. \n \nNot just a missing barrier: Heterotypic mate preference may be an adaptive strategy  \nAlthough reinforcement is a seductive hypothesis to explain why red-form males prefer red females, \nit does not explain why green-form males also prefer these females. The occurrence of such seemingly \nmaladaptive behaviour suggests that other, or additional, evolutionary forces are at play.  \nOne possibility could be  that heterotypic mating preference is a by-product of  inbreeding \navoidance in the green -form population. Spider mites effectively avoid related individuals (Tien et \nal. 2011; Bitume et al. 2013; Yoshioka and Yano 2014) , but it is not clear whether this extends to \nmore distantly-related individuals. For instance, males of both T. evansi and T. urticae preferentially \nmate with T. urticae females (Sato et al. 2016 ; but see (Clemente et al. 2016) , but this occurs even \nwhen T. evansi females are non-kin (Sato et al. 2016) . In line with this, e vidence that inbreeding \navoidance can drive the evolution of disassortative mating is also lacking in other systems  (van den \nBerg et al. 1984; Juola and Dearborn 2012; Huchard et al. 2013; Galaverni et al. 2016).  \nAnother possibility could be that preference of both types of males for red-form females is \ndue to these females being more attractive. For instance, a new trait (e.g., a pheromone profile) may \nhave evolved in red females in response to intense female competition  (i.e., their sex ratio is more \nfemale-biased than that of green mites when they oviposit in groups; unpublished data), and this trait \nmay then be fortuitously preferred by green males  if it stimulates the same coding system as the \nancestral trait (Endler and Basolo 1998) . Alternatively, both types of male may have conserved an \nancestral preference for a trait that has been lost or diverged in green-form females (Endler and Basolo \n1998). This could occur if the rate of evolution of male preference is slower than that of the  female \ntrait. The observed male preferences may also be caused by differences in female reluctance and male \nvigour (e.g., van den Berg et al. 1984)  in response to stronger sexual conflicts in the green -form \npopulation. This hypothesis is supported by the fact that green females are less likely to mate  than \nred females  even in the absence of choice , whereas green males spend longer periods of time \ncopulating than red males do (suggesting longer post-copulatory guarding; (Satoh et al. 2001). In line \nwith this, theory predicts that sexual conflicts can drive the evolution of mate preferences, increasing \nreproductive isolation and, consequently, the rates of speciation (Parker and Partridge 1998). \n Finally, building upon the recent idea that partial reproductive isolation may be an adaptive \noptimum (Servedio and Hermisson 2020) , we considered the possibility that heterotypic mating \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n17 \n \npreference might be selected for under reproductive interference (Gröning and Hochkirch 2008), as \nthe two colour forms have overlapping distribution and host plant range (Migeon and Dorkeld 2023), \nand often co -occur on the same individual host plant  (Lu et al. 2017, 2018; Zélé et al. 2018b) . \nAlthough most conditions that have been theoretically considered to  promote the evolution of \n‘disassortative mating’, such as a heterozygote advantage (e.g., Maisonneuve et al. 2021), are not met \nin our system  (hybrids are sterile or suffer breakdown ; Cruz et al. 2021) , heterotypic mating \npreference may still confer higher benefits than costs to the green-form population in the presence of \nred-form competitors. Indeed, this behaviour should be highly costly for red females due to first male \nsperm precedence, but green males may only pay relatively small cost s as they can mate multiply \n(Krainacker and Carey 1989). Thus, similarly to how CI induced by Wolbachia increases the relative \nfitness of infected females, the ‘spiteful’ behaviour of green males might be selected for as it confers \nan indirect fitness advantage to their green sisters  (Hamilton 1970; Gardner and West 2004; \nEngelstädter and Charlat 2006) . Disassortative mating may thus act synergistically with sex-ratio \ndistortion (i.e., the overproduction of sons) in crosses between green females and red males (cf. Cruz \net al. 2021) to promote the exclusion of the red form population  (cf. Grether et al. 2017; Cruz et al. \n2023). Conversely, homotypic mating preference by red males  should decrease the strength of \nreproductive interference for the red population, as it reduces the prevalence of crosses between green \nfemales and red males (hence the overproduction of green males stemming from these crosses)  and \nshould prevent (non-choosy) red females from having a higher chance to mate with a green male . \nFollowing this hypothesis , the CI -inducing Wolbachia strain naturally infecting the red -form \npopulation seems to favour its own host population by increasing the likelihood that red males mate \nwith compatible (red) females, whereas it has no control over heterotypic mating preference by green \nmales. Testing whether such an ‘escalating arms race’ could indeed occur in response to reproductive \ninterference (involving or not Wolbachia-induced CI)  is of high relevance for future speciation \nstudies. \n \nConclusion \nIn this study, we identified a mechanism through which Wolbachia could assist host speciation \nprocesses. Our results show that Wolbachia infection in T. urticae males indirectly contributes to pre-\nmating isolation  between genetically differentiated T. urticae  colour forms  by strengthening pre-\nexisting preferences. These preferences match early post -mating barriers in the system , as crosses \nthat are affected both by host-associated and Wolbachia-induced incompatibilit ies are generally  \navoided. Our results also further highlight the importance of pre-mating isolation in this system , as \nthey revealed that, in our experimental conditions, females of either form are unable to compensate \nfor incompatible crosses by re -mating. Overall, our comprehensive study of pre - and post-zygotic \nreproductive barriers allowed identify ing asymmetries in patterns of isolation between the two \npopulations, hinting at a possible history of reinforcement followed by an interruption of gene flow . \nThese findings also open new research avenues, such as to study  the impact of complex patterns of \nisolation on population dynamics, and of the resulting selection pressures on the evolutionary \ntrajectories of populations.  \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n18 \n \nReferences \nAuger, P., A. Migeon, E. A. Ueckermann, L. Tiedt, and M. Navajas. 2013. 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Chen, and Q. Wang. 2020. No evidence for inbreeding depression and \ninbreeding avoidance in a haplodiploid mite Tetranychus ludeni  Zacher. Syst Appl Acarol \n25:1723–1728.\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n26 \n \nTable 1. Choice tests to assess the mating behaviour and preference of male s or females that \nwere given the choice between two mates of different colour forms and/or Wolbachia infection \nstatus. CI: cytoplasmic incompatibility; HI: host-associated incompatibility. \nCategory  Choice Female tests Male tests \nI – Avoidance of Wolbachia-\ninduced CI \nMates with different infection \nstatuses \nRu♀ x (Ri♂ vs Ru♂) \nGu♀ x (Gi♂ vs Gu♂) \nRi♂ x (Ri♀ vs Ru♀) \nGi♂ x (Gi♀ vs Gu♀) \nII – Avoidance of HI in \nabsence of Wolbachia \nUninfected mates from different \npopulations \nRu♀ x (Ru♂ vs Gu♂)  \nGu♀ x (Ru♂ vs Gu♂) \nRu♂ x (Ru♀ vs Gu♀) \nGu♂ x (Ru♀ vs Gu♀) \nIII – Avoidance of HI in \npresence of Wolbachia \nInfected mates from different \npopulations \nRi♀ x (Ri♂ vs Gi♂) \nGi♀ x (Ri♂ vs Gi♂) \nRi♂ x (Ri♀ vs Gi♀) \nGi♂ x (Ri♀ vs Gi♀) \nIV – Avoidance of HI, \nWolbachia-induced CI, or \nboth \nMates both with different \ninfection statuses and from \ndifferent populations \nRu♀ x (Ri♂ vs Gi♂) \nGu♀ x (Ri♂ vs Gi♂) \nRi♂ x (Ru♀ vs Gu♀) \nGi♂ x (Ru♀ vs Gu♀) \n \n \nTable 2. No-choice tests to assess the behaviour and offspring production of virgin females (♀) \nplaced with a compatible or incompatible male (1 st ♂ of a similar or different colour form \nand/or Wolbachia infection status, respectively), then (for those that had mated with the first \nmale) with a second compatible male (2 nd ♂ from their own population).  CI: cytoplasmic \nincompatibility; HI: host-associated incompatibility. \nCategory First crosses  ♀ 1st ♂ 2nd ♂ \nA – Controls Intra-population crosses between \nuninfected ♀ and ♂  \nRu Ru Ru \nGu Gu Gu \nB – HI but no effect on F1 production1 Inter-population crosses between \nuninfected red ♀ and green ♂ Ru  Gu Ru \nC – HI with reduced F1 production Inter-population crosses between \nuninfected green ♀ and red ♂ Gu Ru Gu \nD – Wolbachia-induced CI2 Intra-population crosses between red \nuninfected ♀ and infected ♂ Ru Ri Ru \nE – HI and Wolbachia-induced CI2  Inter-population crosses between green \nuninfected ♀ and red infected ♂ Gu Ri Gu \n1HI in this cross direction leads to F1 female sterility and hybrid breakdown (i.e., late post-zygotic isolation). \n2Only Ri males were used as only the Wolbachia strain infecting the red population induces CI (Cruz et al. 2021). \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n27 \n \nFigure 1. Mating propensity and mate choice of spider mites of different colour forms and/or Wolbachia infection status. For each type of choice \ntest, bars represent mean (± s.e.) proportion of mated (a) females and (c) males, and of mates chosen by (b) females and (d) males ( dotted: Wolbachia-\ninfected mates; plain: uninfected mates; orange: red mates; blue: green mates). Identical or absent superscripts indicate non-significant differences at the \n5% level among treatments, asterisks indicate a difference to random mating (white dotted line).  \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n28 \n \nFigure 2. Latency to copulation (a, b) and copulation duration (c, d) of virgin mites during the \nchoice and no-choice tests. Dots represent mean time (± s.e.) in seconds observed for males (a, c) \nand females (b, d) in the male and female choice tests (white and grey dots, respectively), and in the \nfirst mating event of the no-choice test (black dots). Overall, no significant differences were found \namong males or females for latency to copulation in the choice tests (statistical results are not given \nfor the no-choice test as latencies to copulation exceeding 30 minutes were excluded from the means \ndisplayed in this figure to allow comparisons across experiments). For copulation duration, identical \nsuperscripts indicate non-significant differences at the 5% level among crosses within each test (Italic: \nmale choice test, lowercase: female choice test, uppercase: 1 st mating event of the no -choice test). \nNote that infected females were not used in the no -choice test (hence, black dots are not displayed \nfor Ri and Gi females in panel d). \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n29 \n \nFigure 3. Mating propensity observed in two successive mating events in the no-choice test. For \neach cross category, circles and diamonds  represent mean (± s.e.) proportion of females that mated \nduring the first and the second mating event, respectively. The population of the female is displayed \nat the bottom level of the x -axis and the population of the first male at the top level (the population \nof the second male is always the same as that of the female ). Identical superscripts indicate non -\nsignificant differences at the 5% level among crosses across mating events. \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n30 \n \nFigure 4. Latency to copulation (a, b) and copulation duration (c, d) observed in two successive \nmating events in the no-choice test. In (a) and (c), circles and diamonds represent mean time (± s.e.) \nin seconds for each cross category during the first and the second mating event, respectively. Identical \nor absent superscripts indicate non-significant differences at the 5% level among crosses within each \nmating event. In (b) and (d), squares represent the mean time difference (± s.e.) observed between the \ntwo mating events for each female that mated with both males (i.e. [time spent for the second mating] \n– [time spent for the first mating]). Superscripts indicate significant differences from zero at the 10% \nlevel (‡: p<0.10) and at the 5% level ( *: p≤0.05). In all panels, t he population of the female is \ndisplayed at the bottom of the x-axis and the population of the first male at the top (the population of \nthe second male is always the same as that of the female).  \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n31 \n \nFigure 5. Effect of re-mating on offspring production in crosses affected by Wolbachia-induced \nCI and/or HI. (a) Outcome of egg development from each cross category, with bars representing the \nmean (± s.e.) relative proportions of unhatched eggs ( i.e. embryonic mortality), adult daughters and \nsons. (b) Boxplot of the proportion of males produced in all crosses relative to control crosses \n(MDcorr). (c) Boxplot of the proportion of estimated unhatched female eggs relative to control crosses \n(FMcorr). (d) Boxplot of the proportion of F1 adult females in the brood (FP). The population of the \nfemale (♀) is displayed at the bottom level of the x-axis, that of the first male (1st ♂) at the middle \nlevel, and that of the second male (2nd ♂) at the top level (“-” indicates females that did not mate with \nthe second male). \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint \n\n32 \n \nFigure 6. Contribution of the different reproductive barriers to reducing gene flow within and \nbetween populations. Percent contributions to total reproductive isolation were computed based on \nthe estimated strength of reproductive isolation (RI) caused by a given reproductive barrier. They are \nshown for the six most representative types  of cross in this system  (see Table S11 for all \ncrosses). Mate preference corresponds to male preferences only, as females overall showed no mate \npreferences. ‘ns’ indicates no significant difference to zero at the 5% level.  \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 12, 2024. ; https://doi.org/10.1101/2024.05.09.593295doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}