Restricted hybridisation in the secondary contact zone of closely related haplodiploid social spider mites

Restricted hybridisation in the secondary contact zone of closely related haplodiploid social spider mites | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Restricted hybridisation in the secondary contact zone of closely related haplodiploid social spider mites Yukie Sato, Shota Konaka, Shun Hirota, Yoshihisa Suyama, Yoshihiko Tsumura This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3947675/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract How frequently hybridisation and gene flow occur in the contact zones of diverging taxa is important for understanding the speciation process. Stigmaeopsis sabelisi and Stigmaeopsis miscanthi HG form are haplodiploid, social spider mites that infest the Chinese silver grass, Miscanthus sinensis . These two species are closely related and parapatrically distributed in Japan. In mountainous areas, S. sabelisi and S. miscanthi HG form are often found in the highlands and lowlands, respectively, suggesting that they are in contact at intermediate altitudes. It is estimated that they diverged from their common ancestors distributed in subtropical regions (south of Japan) during the last glacial period, expanded their distribution into the Japanese Archipelago, and came to have such a parapatric distribution (secondary contact). As their reproductive isolation is strong but incomplete, hybridisation and genetic introgression are expected at their distributional boundaries. In this study, we investigated their spatial distribution patterns along the elevation on Mt. Amagi using male morphological differences and investigated their hybridisation status using single-nucleotide polymorphisms by MIG-seq. We found their contact zone at altitudes of 150–430 m, suggesting that their contact zone is prevalent in the parapatric area, which is in line with a previous study. Interspecific mating was predicted based on the sex ratio in the contact zone. However, no obvious hybrids were found, and genetic introgression was estimated to be extremely low. Here, we discuss why gene flow is extremely restricted to the contact zone. Biological sciences/Evolution/Speciation Biological sciences/Ecology/Behavioural ecology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Closely related species often overlap in their geographical distribution (Mayr, 1963). Contact frequencies and reproductive, ecological, and genetic relationships at their distribution boundaries are important for their coexistence and evolutionary consequences (Coyne and Orr, 2004; Johannesson et al. , 2020). In particular, hybridisation is likely to occur if reproductive barriers are not completely established. In this case, they may fuse into a single species (Coyne and Orr, 2004) or diversify further by character displacement and/or reinforcement of reproductive barriers (Dobzhansky, 1959; Hoskin et al. , 2005; Smadja and Butlin, 2006; Pfennig and Rice, 2014). If the fitness of the hybrids is high, a new species can be generated from hybridisation through developing reproductive isolation from its parental species (Rieseberg, 1997; Seehausen, 2004; Mallet, 2008; Abbott et al. , 2013).Even though this does not result in new species, genetic introgression by hybridisation may bring genetic diversity to the parent species and drive their subsequent evolution (Edelman et al. , 2019). Therefore, to understand the evolutionary relationships and speciation of closely related species with overlapping distributions, it is important to determine the frequencies of contact, reproductive isolation, and genetic introgression at their distribution boundaries. The Stigmaeopsis miscanthi species group (Acari: Tetranychidae) is a haplodiploid spider mite that infests Chinese silver grass, Miscanthus sinensis , in East Asia (Saito et al. , 2018, 2019). The mites construct woven nests on the undersurface of the host plant leaves and live in groups within the nests. They are called social spider mites because there are two to three generations of overlap among nest members, and they show cooperative nest building, nest sanitation, and brood care (Saito, 2009; Schausberger et al. , 2021). Woven nests are protective against their natural enemies (predatory mites, predatory gall midges, ants, etc.); however, some predators, such as the phytoseiid mite Typhlodromus bambusae , can intrude into the woven nests. To protect nestmates and their offspring against predatory intruders, adult males and females counterattack the intruders cooperatively and sometimes kill the intruders if they are immature (Saitō, 1986a, 1986b; Yano et al. , 2011; Saito et al. , 2011). Males are aggressive not only against predatory intruders but also against conspecific males. They kill each other to establish their own harem (Saitō, 1990). The frequency of male killing varies among populations (Saito, 1995; Saito and Sahara, 1999; Sato, Egas, et al. , 2013; Sato et al. , 2019) and is associated with differences in male aggression and also with their reproductive, phylogenetic, and geographic relationships (Sato et al. , 2000a, 2000b, 2015, 2015, 2018; Sato, Egas, et al. , 2013). Five species and two forms have been described in this species group so far (Saito et al. , 2018, 2019). In Japan, Stigmaeopsis sabelisi with lower male aggression, S. miscanthi high-aggression form (hereafter, S. miscanthi HG form) with higher male aggression, and S. miscanthi mild-aggression form (hereafter, S. miscanthi ML form) with intermediate male aggression are distributed (Saito, 1995; Saito and Sahara, 1999; Sato, Egas, et al. , 2013; Sato et al. , 2019). S. miscanthi ML form is distributed in subtropical regions and is geographically isolated from the two other species (Sato, Egas, et al. , 2013; Sato et al. , 2019) (Fig. 1). On the other hand, S. sabelisi and S. miscanthi HG form show overlap in their geographic distribution: S. sabelisi is distributed in colder regions (from Aomori Prefecture to Kyushu Islands), whereas S. miscanthi HG form is distributed in warmer regions (from Shizuoka Prefecture to the main island of Okinawa) (Fig. 1). Japan is mountainous, and in areas where both species are distributed, S. sabelisi and S. miscanthi HG form are found in the highlands and lowlands, respectively (parapatric distribution). A previous study inferred the population history of the species group using mtDNA (cytochrome c oxidase subunit I; COI) and estimated that S. sabelisi and S. miscanthi HG form were derived from an ancestral group with mild male aggression in the subtropical region during the last glacial period (20,000–40,000 years BP for S. sabelisi and 5,494–10,988 years BP for S. miscanthi HG form) (Sato et al. , 2019). Considering their inferred history together with their ecological and reproductive relationships and the migration history of their host plant (Clark et al. , 2014), it is predicted that (1) S. sabelisi was derived south of Japan and migrated into the Japanese archipelago just after the host plant expanded its distribution into the Japanese archipelago; (2) as temperature increased more, the ancestral groupexpanded its distribution northward and migrated into the Ryukyus Islands; (3) S. miscanthi HG form was derived from the ancestral group in and around the Japanese archipelago; and (4) S. miscanthi HG form expanded its distribution in the Japanese archipelago and drove S. sabelisi to the colder region through competition and reproductive interference (Saito et al. , 2013; Sato, Sabelis, et al. , 2013; Sato et al. , 2015), resulting in their present geographic distributions (Sato et al. , 2019). A previous field study at Mt. Unzen, one of the mountains on the Kyushu Islands at the southern end of the parapatric area (Nagasaki Prefecture; Fig. 1), found that the distributions of these two species broadly overlapped at intermediate altitudes (100–400 m), and both species were collected from the same host plant colonies in the contact zone (Sato et al. , 2008). It is known that their reproductive isolation is strong but incomplete; there is a strong post-mating and pre-zygotic reproductive barrier, but a few hybrids are produced from interspecific crosses (proportion of hybrids: 0– 30%) (Sato et al. , 2000a, 2000b, 2015, 2018), and their hybrids are fertile (Sato, 2004). Therefore, hybridisation and gene flow are likely to occur in the contact zones formed on each mountain in the parapatric area. In particular, males of S. miscanthi HG form actively approach the females of S. sabelisi for mating, as they do for conspecific females (Sato et al. , 2015) . In addition, interspecific male fights occur easily between the two species, and males in the S. miscanthi HG form tend to win interspecific male fights (Sato, Sabelis, et al. , 2013). This suggests that S. sabelisi females are at a higher risk of interspecific mating than S. miscanthi HG form females, indicating that genetic introgression is likely asymmetric. However, it has not been confirmed whether hybridisation occurs in the contact zones. Furthermore, their contact zone has been reported only on Mt. Unzen, and it is unclear whether their contact zones are widespread in their parapatric areas. In this study, to address whether the contact zone of S. sabelisi and S. miscanthi HG form is widespread in their parapatric areas, we investigated their distribution patterns along the elevation on and around Mt. Amagi in Shizuoka Prefecture, Japan. Mt. Amagi was selected as the study site because it is located at the northern end of the parapatric area (Fig. 1). The presence of contact zones at both the southern and northern ends of the parapatric areas (Mt. Unzen and Mt. Amagi) supports the hypothesis that contact zones are prevalent in parapatric areas. To determine whether interspecific mating occurred in the contact zone, we analysed the sex ratio of mite colonies in the contact zone. The mites are haplodiploid, in which females develop from fertilised eggs and males develop from unfertilised eggs. Virgin females lay unfertilised eggs, and the number of eggs is significantly lower than that of mated females (Sato et al. , 2000a, 2000b, 2018). Females mated with males of different species lay unfertilised eggs because of the strong post-mating pre-zygotic barrier (reproductive barrier in the egg fertilisation stage); however, the number of eggs was similar to that of females mated with conspecific males, possibly because females control the number of eggs by copulation stimuli (Sato et al. , 2000a, 2000b, 2018). As females that mate with males of different species produce an overabundance of sons (Sato et al. , 2000a, 2000b, 2018), the sex ratio would become relatively male-biased in mite colonies where interspecific mating occurs (Sato et al. , 2008); although, the spider mite species originally showed an extremely female-biased sex ratio (male ratio: 0.10–0.20) (Sato and Saito, 2007). To address whether hybridisation and genetic introgression occurred between the two mite species, we analysed the genetic population structure of the mite colonies in their contact zones. Spider mites are too small to obtain sufficient DNA from a single mite for molecular analysis (body length is less than 0.5 mm). Therefore, in the genetic analyses, we used multiplexed Inter-Simple Sequence Repeat (ISSR) genotyping by sequencing (MIG-seq), which is useful for small amounts of low-quality DNA (Suyama and Matsuki, 2015). Single-nucleotide polymorphisms (SNPs) were detected using MIG-seq for genetic analysis. To confirm whether S. sabelisi and S. miscanthi HG form collected from Mt. Amagi produced hybrids, we performed cross-experiments. Previous cross-experiments have found hybridisation; however, these studies used different populations, so there is a possibility that the two species from Mt. Amagi do not produce hybrids that are different from those reported in previous studies. Finally, based on these findings, we discuss the prevalence of their contact zones, interspecific mating, gene flow, and the effect of the contact zone on their evolution. Materials and Methods Study site and mite collection We collected spider mites of S. miscanthi species group from the eastern area of Mt. Amagi (Shizuoka Prefecture, Japan) at altitudes of 20–680 m in July 2020 (Table 1, Fig. 1). This area is dominated by S. sabelisi ;therefore, to ensure the collection of S. miscanthi HG form, we collected mites from the area northeast of Mt. Amagi at an altitude of 20 m, additionally (Shiofuki in Table 1). We brought host plant leaves with mite nests to the laboratory and recorded the number of males, females, and immatures in each nest using stereoscopic microscopes. Slide specimens of the collected males were prepared and used for species identification based on the male morphology. We stored the collected females in microtubes containing 99% acetone for genetic analysis. We reared the collected immatures on detached M. sinensis leaves under controlled conditions (25℃, 75–100% relative humidity, and 15:9 h light:dark). Since spider mites are haplodiploid, male genes are derived only from their mothers. Therefore, we prepared slide specimens of males developed from reared immatures within several days after mite collection and used them for species identification of females based on male morphology. We established laboratory mite cultures for each population using the remaining collected mites and used them for the cross-experiment. Species identification by male morphology As the relative length of leg I to leg III and body width were greater in males of the S. miscanthi HG form than in those of S. sabelisi , we used morphological differences to identify the species of the collected mites (Saito, 1995; Sato et al. , 2000a, 2019; Sato, Egas, et al. , 2013). One to five males were placed on a drop of Hoyer’s solution on a slide glass and covered with cover glass. We placed a 10-g weight on the cover glass to flatten the mite body evenly for accurate measurement and dried it at 45℃ for more than 3 days. We took photos of the slide specimen with a microscope (Axioskop, Zeiss, Germany) and an eyepiece camera (Dino-Eye AM4023X, Anmo Electronics Corporation, Taiwan), after which we measured the lengths of the first and third legs (the four leg segments of the tarsus, tibia, genu, and femur), body length, and body width of the slide specimen using image-processing software (Image J ver. 1.53a; National Institute of Health, Bethesda, MD, USA) (Schneider et al. , 2012). We calculated the relative length of the first to third legs (leg I to leg III) from the total of the four leg segment lengths and used the log-transformed values in the analysis. The specimens used to construct the linear discriminant function were measured three times, and the average values were used. The specimens used for species identification were measured once, and the value was used for species identification. In a previous study conducted on Mt. Unzen (Nagasaki Prefecture, Japan), a contact zone was found at altitudes of 100–400 m (Sato et al. , 2008). Therefore, we assumed that the mite colonies at altitudes below 100 m and above 500 m were S. miscanthi HG form and S. sabelisi , respectively. We constructed a linear discriminant function using body width ( BW ), body length ( BL ), and log-transformed values of the relative length of the first to third legs ( BLL ) of males from the colonies ( S. miscanthi HG form: 31 males and 41 males developed from immatures collected from Shiofuki and Amagi 1 and 2; S. sabelisi : 28 males and 41 males developed from immatures collected from Amagi 17–20; Table 1). We used the discriminant function for species identification of males and males developed from immatures collected from colonies at an altitude of 100–500 m (Amagi 3–16; Table 1). For discriminant analysis, we used statistic software R ver. 4.0.2 (R Core Team, 2022) and the MASS package (Venables and Ripley, 2002). Interspecific mating inferred from the sex ratio in the field The sex ratio is expected to be relatively male-biased in mite colonies where interspecific mating occurs (Sato et al. , 2008), because females that mate with different species produce an overabundance of sons (Sato et al. , 2000a, 2000b, 2018). We estimated interspecific mating in the field by analysing the calculated sex ratio. Based on the results of species identification using male morphology, we categorised the mite colonies into three types: S. miscanthi HG, S. sabelisi alone, and both species. The male ratio was analysed using a generalised linear model (GLM) with a binomial error distribution and likelihood ratio test (LRT). Multiple comparisons were performed using Tukey’s post-hoc test. For the analysis, we used statistic software R ver. 4.0.2 (R Core Team, 2022) and the multcomp package (Hothorn et al. , 2008). Genetic analysis In the genetic analysis, we used 237 females from 11 colonies at altitudes of 100–500 m (contact zone), two colonies below 100 m altitude (pure S. miscanthi HG form), and two colonies above 500 m altitude (pure S. sabelisi ). DNA was extracted from a single female mite, which was stored in 99% acetone in a microtube, using PrepMan Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA). To construct the MIG-seq library, we followed the protocol described by Suyama et al. (2022), which is modified from Suyama and Matsuki (2015). Briefly, an MIG-seq library was prepared using a two-step PCR method. In the first PCR, ISSR regions were amplified using MIG-seq primer set 1 (Suyama and Matsuki 2015). In the second PCR, indices and Illumina adapter sequences were added to the first PCR products. The Illumina MiSeq platform and MiSeq Reagent Kit v3 (150 cycles; Illumina) were used for sequencing. We skipped the sequencing of the first 17 bases of reads 1 and 2 (SSR primer region and anchors) using “DarkCycle”. We detected genome-wide SNPs from the sequenced raw reads and filtered SNPs and females, as described in Suyama et al. (2022). After the removal of extremely short reads and low-quality reads using trimmomatic 0.39 (Bolger et al. , 2014), 42,183,313 reads (177,989 ± 4,186 reads per sample) remained from 43,713,012 raw reads (184,443 ± 4,323 reads per sample). De novo SNP genotyping was performed using Stacks 2.55 (Rochette et al. , 2019), utilizing the following parameters: minimum depth of coverage required to create a stack ( m ) of 3, maximum distance allowed between stacks ( M ) of 2, and number of mismatches allowed between sample loci while building the catalogue ( n ) of 2. Using the ‘populations’ program in Stacks, we removed SNP sites exhibiting high heterozygosity ( H o ≥ 0.6) and filtered out SNP sites with fewer than three minor alleles. SNPs that were retained by 50% or more samples were included in the SNP dataset. To avoid linked SNPs, only the first SNP from each locus was considered. Observed ( H O ) and expected ( H E ) heterozygosities as well as nucleotide diversities at SNP sites were calculated using the ‘populations’ program in Stacks. To determine the presence of hybrids and genetic introgression between the species, the selected SNPs were used to analyse the population structure using STRUCTURE Software ver. 2.3.4 (Pritchard et al. , 2000) and Structure Harvester (Earl and von Holdt, 2012). We conducted 30 independent runs with a burn-in of 100,000 steps, followed by an additional 100,000 steps using an admixture model. We estimated the log-likelihood of each cluster ( K = 1–10). A Neighbour-Net network was constructed using SplitsTree4 4.14 (Huson and Bryant, 2006), utilising the uncorrelated P distance matrix and ignoring ambiguous sites. Cross-experiment We used laboratory cultures collected from Amagi 17 and Amagi 1 in the cross-experiment as S. sabelisi and S. miscanthi HG form, respectively (Table 1). We placed a detached M. sinensis leaf (1.0 × 3.0 cm) on water-soaked cotton wool in a Petri dish (5.0 cm diameter, 1.5 cm high; SPL Life Sciences, Gyeonggi-do, Korea). We collected a teleiochrysalis female from the mite culture and placed it onto the prepared leaf arena. One day after mite introduction, we checked the emergence and construction of a woven nest and then introduced a male collected from the mite culture onto the leaf arena. We allowed them to mate and oviposit for 10 days, after which we removed the females and males from the leaf arena and recorded the number of eggs. We checked the development of their offspring daily until they reached the adult phase and recorded their survival and sex. We performed cross-experiments between the populations: Amagi 17 (female) × Amagi 1 (male) and Amagi 1 (female) × Amagi 17 (male), and within species as controls: Amagi 17 (female) × Amagi 17 (male) and Amagi 1 (female) × Amagi 1 (male). As another control, we observed virgin oviposition because spider mite females produce sons without mating, and the number of eggs is significantly fewer in the virgin oviposition than in the mated oviposition in the species group (Sato et al. , 2000a, 2000b, 2018). We carried out the cross-experiments under controlled conditions (25℃, 75–100% relative humidity, and 15:9 h light:dark). To determine the pre-mating barrier, we compared the number of eggs among the cross combinations (interspecific crosses, intrapopulation crosses, and virgin oviposition) of each female species. For comparison, we used a GLM with a negative binomial error distribution because we detected overdispersion in the Poisson GLM. To determine post-mating and pre-zygotic barriers, we compared the offspring sex ratios among cross combinations (interspecific cross, intraspecific cross, and virgin oviposition) using a GLM with a binomial error distribution or quasibinomial error distribution when overdispersion was detected in the binomial GLM. To determine the zygotic barrier, we compared the offspring survival ratios among the cross combinations (interspecific cross, intraspecific cross, and virgin oviposition) using a GLM with a quasibinomial error distribution, as we detected overdispersion in the binomial GLM. For the analysis, we used statistic software R ver. 4.0.2 (R Core Team, 2022). Results Contact zone inferred by species identification by male morphology The discriminant function was obtained as follows: where DW , DL , and RLL are the body width, body length, and relative lengths of the first to third legs, respectively. When the male individuals used for formulating the function were randomly sampled, the function provided 100% correct answers. Using the discriminant function, we identified 137 males for species identification in males and 175 males developed from the immatures for species identification in females, both of which were collected from colonies at altitudes of 100–500 m. For species identification in males, 32 and 105 individuals were identified as S. miscanthi HG form and S. sabelisi , respectively. Both species were found together in six colonies at altitudes of 160–420 m, although S. sabelisi dominated in seven colonies and S. miscanthi HG form in one colony (Fig. 2). In the species identification of females, 34 and 141 individuals were identified as S. miscanthi HG form and S. sabelisi , respectively. Both species were found together in five colonies at altitudes of 150–430 m, although S. sabelisi was dominant in nine colonies (Fig. 2). These results indicate that their contact zone exists at least in the area at altitudes of 150–430 m on Mt. Amagi. We examined the morphology of 312 males in total and found that a male that developed from the immatures collected at an altitude of 350 m (Amagi 10; Table 1; Fig. 2) had a deformity (Fig. 3). The legs usually consist of four segments (tarsus, tibia, genu, and femur); however, in the deformed male, only three segments were present on one of the third legs (Fig. 3). Interspecific mating inferred from the sex ratio in the field The male ratio varied among the mite colonies (male ratio: 0.077–0.355; Fig. 4). The effect of colony type on the male ratio was significant (binomial GLM, df = 2, LRT = 15.123, P < 0.001). The male ratio in the colonies in which both species were found was significantly higher than that in the colonies of pure S. sabelisi or S. miscanthi HG form, although the male ratio was not significantly different between the colonies of pure S. miscanthi HG form and S. sabelisi (Tukey’s test, both species vs. S. miscanthi HG form: z = 3.756, P = 0.001; both species vs. S. sabelisi : z = 2.563, P = 0.027; S. miscanthi HG form vs. S. sabelisi : z = 1.389, P = 0.344). These results indicate that the overproduction of sons by interspecific mating occurred in colonies where both species were found. Hybridization and genetic introgression A total of 111 SNPs from 189 loci in 237 females were used for the genetic analysis. All raw reads of MIG-seq data were submitted to the DDBJ Sequence Read Archive under the BioProject ID PRJDB17427. The S. miscanthi HG form populations had lower heterozygosity and nucleotide diversity than the populations of S. sabelisi (Table S1). Neighbour-Net analysis demonstrated the distinction between S. sabelisi and S. miscanthi HG form as two separate genetic clusters (Fig. 5). None of the samples occupied an intermediate position between S. sabelisi and S. miscanthi HG form. Under the delta K method, the best STRUCTURE model assigned individuals to two genetic clusters (Fig. S1), corresponding to S. sabelisi and S. miscanthi HG form, respectively (Fig. 6). A total of 232 individuals were assigned as pure S. sabelisi or pure S. miscanthi HG form, and five individuals collected from their contact zones were assigned as S. sabelisi with very small fragments of S. miscanthi HG form. These results showed that there were no hybrids and that genetic introgression was very low. Reproductive isolation in laboratory cross-experiment In the cross experiments with S. sabelisi females, the number of eggs in interspecific crosses was significantly higher than that in virgin oviposition (negative-binomial GLM, z = 3.418, P < 0.001; Table 2) and similar to that in intrapopulation crosses (negative-binomial GLM, z = 0.793, P = 0.428; Table 2), indicating that mating occurred in the interspecific cross. A few hybrids were produced in two of the 24 pairs of interspecific crosses; however, the female ratio in the offspring of interspecific crosses was significantly lower than that in intrapopulation crosses (binomial GLM, z = 7.919, P < 0.001; Table 2) and similar to that in virgin oviposition (binomial GLM, z = 0.006, P = 0.995; Table 2), indicating the presence of a strong pre-zygotic barrier. The survival rate of offspring from egg to adult in interspecific crosses was significantly lower than that in intrapopulation crosses (quasibinomial GLM, t = 2.993, P < 0.01; Table 2) but not significantly different from that in virgin oviposition (quasibinomial GLM, t = 1.230, P = 0.223; Table 2). The results of the cross-experiments with S. miscanthi HG form females were similar to those of the cross-experiments with S. sabelisi females. The number of eggs in the interspecific cross was significantly higher than that in the virgin oviposition (negative-binomial GLM, z = 4.870, P < 0.001; Table 2) and was similar to that in the intrapopulation cross (negative-binomial GLM, z = 0.513, P = 0.608; Table 2), indicating that mating occurred in the interspecific cross. A few hybrids were produced in one of the 25 pairs of interspecific crosses; however, the female ratio in the offspring of interspecific crosses was significantly lower than that in intrapopulation crosses (quasibinomial GLM, t = 6.311, P < 0.001; Table 2) and similar to that in virgin oviposition crosses (quasibinomial GLM, t = 0.004, P = 0.997; Table 2), indicating the presence of a strong pre-zygotic barrier. The survival rate of offspring from egg to adult in interspecific crosses was significantly lower than that in intrapopulation crosses (quasibinomial GLM, t = 2.692, P < 0.01; Table 2) but not significantly different from that in virgin oviposition (quasibinomial GLM, t = 1.565, P = 0.122; Table 2). Discussion We found that the geographic distributions of S. sabelisi and S. miscanthi HG form overlapped widely at intermediate altitudes (150–430 m) on Mt. Amagi. We conducted a field survey on Mt. Amagi at the northern end of their parapatric area because a previous study detected a contact zone on Mt. Unzen at the southern end of their parapatric area (Sato et al. , 2008; Fig. 1). As their contact zone was confirmed at both the southern and northern ends of their parapatric area, we concluded that their contact zone would be prevalent in their parapatric area and that their contact zone may have a significant impact on their evolution and ecological relationships. In several host-plant fields in their contact zones, both species were found together for both sexes. This suggests that interspecific mating has occurred in these fields. In fact, the sex ratio of spider mites in the colonies of both species was male-biased compared to that of pure S. sabelisi or S. miscanthi HG form colonies. As for their reproductive barrier, the post-mating pre-zygotic barrier contributes the most (Sato et al. , 2000a, 2000b, 2015, 2018), which causes the overproduction of males owing to their haplodiploid genetic system and regulation of the number of eggs by copulation stimuli. The male-biased sex ratio strongly supports interspecific mating in the contact zone. A previous study conducted on Mt. Unzen detected a male-biased sex ratio at intermediate altitudes (Sato et al. , 2008). Therefore, we conclude that interspecific mating is widespread in their parapatric area. A previous study reported the presence of a pre-mating barrier, but it was very weak and asymmetric (Sato et al. , 2015). The post-mating pre-zygotic barrier contributes most to the reproductive barrier; however, it is also incomplete, and a few hybrids were produced in their cross-experiments in the laboratory (proportion of hybrids: 0–30%) (Sato et al. , 2000a, 2000b, 2015, 2018). In this study, we carried out cross experiments using the populations collected from Mt. Amagi and confirmed that they produce hybrids, although the proportion of hybrids was slightly lower compared to those in previous studies (3–13%). Therefore, we expected that hybrids would exist in the contact zone and that gene flow would occur between them. However, our genetic analysis did not identify obvious hybrids, and it was estimated that genetic introgression was extremely low (Figs. 5 and 6). This suggests that gene flow is strongly restricted by mechanisms that act later than post-mating pre-zygotic isolation. Because their hybrids are fertile in the laboratory (Sato, 2004), they may have problems surviving in nature. For example, adult female spider mites enter diapause and overwinter. Diapause attributes differ between the two species: S. sabelisi takes a much longer time to emerge from diapause (high-intensity diapause), whereas the S. miscanthi HG form emerges from diapause very quickly (low-intensity diapause) (Saito et al. , 2002). The difference in diapause is considered the result of adaptation to colder and warmer regions in each species, and this difference contributes to maintaining their parapatric distribution. The diapause attributes of their hybrids have not yet been investigated; however, if they are not able to enter diapause or emerge from diapause at a suitable time in the local season, they will be selected against from the field, even though they are able to survive in the laboratory. It is also likely that hybrids have reproductive problems in nature. As described before, males of this species kill each other to establish their own harems, and only the victorious male can reproduce (Saitō, 1990). In this study, we identified a deformed male in the contact zone. We do not know if the deformity was caused by hybridisation, but it is worth investigating the influence of hybridisation on the morphology and fighting ability of males. Therefore, to understand why gene flow is extremely restricted in the contact zone, it is necessary to investigate hybrid traits, such as diapause attributes, morphology, and behaviour. This field survey was conducted at the northern end of S. miscanthi HG form distribution. It is common for populations at the edge of the distribution to have extremely low genetic diversity owing to bottlenecks and genetic drift. Because the genetic diversity of S. miscanthi HG form populations has not been investigated in other regions, it is not possible to confirm whether the genetic diversity is lower in this area than in other areas. However, S. miscanthi HG form populations showed lower heterozygosity and nucleotide diversity than S. sabelisi (Table S1) . This may explain the low genetic diversity of S. miscanthi HG form populations in this study. The low genetic diversity and harshness of the environment for S. miscanthi HG form likely make it difficult to produce hybrids. Therefore, before concluding that gene flow is extremely restricted between them in their parapatric area, it is worth conducting this study on other mountains where both species are abundant. Although not as much as expected, some negligible genetic introgression was detected between S. sabelisi and S. miscanthi HG form (Fig. 6). As described previously, these two species have different distribution areas and male aggression levels: S. sabelisi is distributed in colder regions and shows lower male aggression, whereas S. miscanthi HG form is distributed in warmer regions and shows higher male aggression (Saito, 1995; Sato, Egas, et al. , 2013). Male aggression varies among populations in each species, and the same relationship between winter coldness and male aggression was found in each species. S. miscanthi HG form populations distributed in relatively colder regions showed lower male aggression compared to populations distributed in warmer regions, and the same clinal trend was found in S. sabelisi populations (Saito and Sahara, 1999). Male aggression is genetically determined, as laboratory mite populations maintain similar aggression regardless of rearing temperature. To date, attempts have been made to explain the clinical trends of male aggression, both intra- and interspecies, through the application of kin selection theory (Saito, 1995; Saito and Sahara, 1999; Saito and Mori, 2005; Sato, Egas, et al. , 2013). Severe winter colds can induce inbreeding in the spider mite species. Overwintered virgin females establish their spring nests by mating with the surrounding males; however, if there are no males, they produce haploid sons and mate with them for spring nest establishment. Whether males are around is affected by winter coldness because non-diapause males overwinter by chance if the winter coldness is mild (Saito, 1995; Sato, Egas, et al. , 2013). As predicted by kin selection theory, male aggression is higher in populations where the average kinship in a colony is expected to be low from the spring nest establishment event, and vice versa. However, if genetic introgression affects male aggression, then a clinical trend in each species may be generated by genetic introgression. For example, S. miscanthi HG form populations distributed in relatively colder regions are expected to encounter S. sabelisi much more frequently than those in warmer regions. Milder male aggression was likely caused by genetic introgression from S. sabelisi . To test this hypothesis, it is necessary to investigate the inheritance of male aggression and genetic introgression status in other contact zones. In this study, we revealed that S. sabelisi and S. miscanthi HG form have broad contact zones throughout their parapatric area, and interspecific mating occurs in their contact zones; however, gene flow is strongly restricted between them. The contact zones of closely related species influence their evolution in various ways; they can cause species breakdown, promote diversification by character displacement and/or reinforcement of reproductive barriers, and generate new species by hybridisation (Coyne and Orr, 2004; Johannesson et al. , 2020). In spider mites, contact zones are likely to contribute to diversification. In particular, reinforcement of reproductive isolation can occur when the fitness of a hybrid is low. The traits and fitness of their hybrids have not been well investigated; however, it is quite likely that hybrids have problems surviving and reproducing in nature, considering that we did not find obvious hybrids in their contact zones, despite incomplete reproductive isolation. Reinforcement of reproductive isolation in spider mites was suggested by a previous study, which found that post-mating, a pre-zygotic barrier evolved faster in S. sabelisi females collected from parapatric areas than in allopatric areas (Sato et al. , 2018). This tendency was not found in S. miscanthi HG form females; however, it fits their behavioural differences (Sato et al. , 2018). Specifically, males readily fight with different species of males for nests, S. miscanthi HG form males tend to win interspecific male fights (Sato, Sabelis, et al. , 2013), and S. miscanthi HG form males are active to mate regardless of female species much more than S. sabelisi males (Sato et al. , 2015). These behavioural differences suggest that S. sabelisi females are likely exposed to the risk of reproductive interference by other male species, whereas S. miscanthi HG form females are guarded by the same male species against such risk. We expect genetic analyses to provide insights into this hypothesis. However, in this study, we could not analyse the direction and details of genetic introgression because the gene flow was extremely low. As discussed before, their gene flow may be restricted by mechanisms acting later than post-mating pre-zygotic isolation. However, the amount of gene flow likely changes depending on the power relationships between S. sabelisi and S. miscanthi HG form. In this study, genetic analyses were performed on Mt. Amagi, where S. sabelisi was found to be superior. From this perspective, it would be worthwhile to conduct this study on other mountains. Declarations Acknowledgements We thank Prof. Kentaro Nakano and Prof. Yooichi Kainoh at the University of Tsukuba for providing the microscope for male morphology measurements and the space to cultivate the host plants of the mites. We thank Mr. Gomei Yoda, Ms. Hisaho Kobatyashi, Mr. Taito Sano, Mr. Ryuto Uchiyama, Ms. Sayuka (Nagase) Nitta, Ms. Aina Yokoi, and Ms. Ayana Tanino for their valuable suggestions and support. This research was supported in part by JSPS KAKENHI, Grant Number 20K06810 (Grant-in-Aid for Scientific Research C to Y. Sato) and by The Suzuki Takahisa Memorial Grant, the University of Tsukuba (to Y. Sato). Author contribution statement Y. Sato conceived the study. Y. Sato, S. Konaka, Y. Tsumura, and Y. Suyama designed the study. S. Konaka, Y. Sato, and N. Matsumoto conducted the field surveys. S. Konaka performed the measurements, Y. Sato and S. Konaka analysed of male morphology. S. Hirota, Y. Suyama, S. Konaka, Y. Sato, and Y. Tsumura performed molecular analyses. Y. Sato conducted the cross-experiment and analysed reproductive isolation. Y. Sato and S. Hirota wrote the first draft of the manuscript. All authors read and approved the final manuscript. Conflicts of Interest The authors have no conflicts of interest to declare. Data availability All raw reads of the MIG-seq data are available from the DDBJ Sequence Read Archive under BioProject ID PRJDB17427. The morphology, sex ratio, and cross-experimental data are shown in the Supplementary Materials. Research Ethics Statement Not applicable, as the study was conducted on spider mites. References Abbott R, Albach D, Ansell S, Arntzen JW, Baird SJE, Bierne N, et al. (2013). Hybridization and speciation. Journal of Evolutionary Biology 26 : 229–246. Bolger AM, Lohse M, Usadel B (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30 : 2114–2120. Clark LV, Brummer JE, Głowacka K, Hall MC, Heo K, Peng J, et al. (2014). A footprint of past climate change on the diversity and population structure of Miscanthus sinensis . Ann Bot 114 : 97–107. Coyne JA, Orr HA (2004). Speciation , 1st edition. Sinauer Associates, Inc.: Sunderland, Mass. Dobzhansky T (1959). Genetics and the Origin of Species , Third Edition. Columbia University Press. Earl DA, vonHoldt BM (2012). STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genet Resour 4 : 359–361. Edelman NB, Frandsen PB, Miyagi M, Clavijo B, Davey J, Dikow RB, et al. (2019). Genomic architecture and introgression shape a butterfly radiation. Science 366 : 594–599. Hoskin CJ, Higgie M, McDonald KR, Moritz C (2005). Reinforcement drives rapid allopatric speciation. Nature 437 : 1353–1356. Hothorn T, Bretz F, Westfall P (2008). Simultaneous inference in general parametric models. Biom J 50 : 346–363. Huson DH, Bryant D (2006). Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 23 : 254–267. Johannesson K, Le Moan A, Perini S, André C (2020). A Darwinian Laboratory of Multiple Contact Zones. Trends in Ecology & Evolution 35 : 1021–1036. Mallet J (2008). Hybridization, ecological races and the nature of species: empirical evidence for the ease of speciation. Philosophical Transactions of the Royal Society B: Biological Sciences 363 : 2971–2986. Pfennig KS, Rice AM (2014). Reinforcement generates reproductive isolation between neighbouring conspecific populations of spadefoot toads. Proceedings of the Royal Society of London B: Biological Sciences 281 : 20140949. Pritchard JK, Stephens M, Donnelly P (2000). Inference of population structure using multilocus genotype data. Genetics 155 : 945–959. R Core Team (2022). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria . Rieseberg LH (1997). Hybrid origins of plant species. Annual Review of Ecology and Systematics 28 : 359–389. Rochette NC, Rivera-Colón AG, Catchen JM (2019). Stacks 2: Analytical methods for paired-end sequencing improve RADseq-based population genomics. Molecular Ecology 28 : 4737–4754. Saitō Y (1986a). Prey kills predator: Counter-attack success of a spider mite against its specific phytoseiid predator. Exp Appl Acarol 2 : 47–62. Saitō Y (1986b). Biparental defence in a spider mite (Acari: Tetranychidae) infesting Sasa bamboo. Behav Ecol Sociobiol 18 : 377–386. Saitō Y (1990). ‘Harem’ and ‘non-harem’ type mating systems in two species of subsocial spider mites (Acari, Tetranychidae). Res Popul Ecol 32 : 263–278. Saito Y (1995). Clinal variation in male-to-male antagonism and weaponry in a subsocial mite. Evolution 49 : 413–417. Saito Y (2009). Plant Mites and Sociality: Diversity and Evolution . Springer Science & Business Media. Saito Y, Chittenden AR, Kanazawa M (2011). Counterattack success of a social spider mite against two predominant phytoseiid predator species. Exp Appl Acarol 55 : 249. Saito Y, Kanazawa M, Sato Y (2013). Life history differences between two forms of the social spider mite, Stigmaeopsis miscanthi . Exp Appl Acarol 60 : 313–320. Saito Y, Mori K (2005). Where does male-to-male “aggression” compromise “cooperation”? Popul Ecol 47 : 159–166. Saito Y, Sahara K (1999). Two clinal trends in male-male aggressiveness in a subsocial spider mite ( Schizotetranychus miscanthi ). Behav Ecol Sociobiol 46 : 25–29. Saito Y, Sakagami T, Sahara K (2002). Differences in diapause attributes between two clinal forms distinguished by male-to-male aggression in a subsocial spider mite, Schizotetranychus miscanthi Saito. Ecological Research 17 : 645–653. Saito Y, Sato Y, Chittenden AR, Lin J-Z, Zhang Y-X (2018). Description of two new species of Stigmaeopsis , Banks 1917 (Acari, Tetranychidae) inhabiting Miscanthus grasses (Poaceae). Acarologia 58 : 414–429. Saito Y, Sato Y, Kongchuensin M, Chao J-T, Sahara K (2019). New Stigmaeopsis species on Miscanthus grasses in Taiwan and Thailand (Acari, Tetranychidae). Systematic and Applied Acarology 24 : 675–682. Sato Y (2004). Studies on variation in social structure and the maintenance mechanism associated with diversification in the Stigmaeopsis species group. In: Hokkaido University, Ph. D. thesis ,, p 162. Sato Y, Breeuwer JAJ, Egas M, Sabelis MW (2015). Incomplete premating and postmating reproductive barriers between two parapatric populations of a social spider mite. Exp Appl Acarol 65 : 277–291. Sato Y, Egas M, Sabelis MW, Mochizuki A (2013). Male–male aggression peaks at intermediate relatedness in a social spider mite. Ecol Evol 3 : 2661–2669. Sato Y, Sabelis MW, Mochizuki A (2013). Asymmetry in male lethal fight between parapatric forms of a social spider mite. Exp Appl Acarol 60 : 451–461. Sato Y, Saito Y (2007). Can the extremely female-biased sex ratio of the social spider mites be explained by Hamilton’s local mate competition model? Ecological Entomology 32 : 597–602. Sato Y, Saito Y, Chittenden AR (2008). The parapatric distribution and contact zone of two forms showing different male-to-male aggressiveness in a social spider mite, Stigmaeopsis miscanthi (Acari: Tetranychidae). Exp Appl Acarol 44 : 265–276. Sato Y, Saito Y, Mori K (2000a). Reproductive isolation between populations showing different aggression in a subsocial spider mite, Schizotetranychus miscanthi Saito (Acari: Tetranychidae). Applied Entomology and Zoology 35 : 605–610. Sato Y, Saito Y, Mori K (2000b). Patterns of reproductive isolation between two groups of Schizotetranychus miscanthi Saito (Acari: Tetranychidae) showing different male aggression traits. Applied Entomology and Zoology 35 : 611–618. Sato Y, Sakamoto H, Gotoh T, Saito Y, Chao J-T, Egas M, et al. (2018). Patterns of reproductive isolation in a haplodiploid – strong postmating, prezygotic barriers among three forms of a social spider mite. Journal of Evolutionary Biology 31 : 866–881. Sato Y, Tsuda Y, Sakamoto H, Egas M, Gotoh T, Saito Y, et al. (2019). Phylogeography of lethal male fighting in a social spider mite. Ecology and Evolution 9 : 1590–1602. Schausberger P, Yano S, Sato Y (2021). Cooperative behaviors in group-Living spider mites. Frontiers in Ecology and Evolution 9 : 745036. Schneider CA, Rasband WS, Eliceiri KW (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9 : 671–675. Seehausen O (2004). Hybridization and adaptive radiation. Trends in Ecology & Evolution 19 : 198–207. Smadja C, Butlin R (2006). Speciation: A new role for reinforcement. Heredity 96 : 422–423. Suyama Y, Hirota SK, Matsuo A, Tsunamoto Y, Mitsuyuki C, Shimura A, et al. (2022). Complementary combination of multiplex high-throughput DNA sequencing for molecular phylogeny. Ecological Research 37 : 171–181. Suyama Y, Matsuki Y (2015). MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform. Sci Rep 5 : 16963. Venables WN, Ripley BD (2002). Modern Applied Statistics with S , 4th edn. Springer-Verlag: New York. Yano J, Saito Y, Chittenden AR, Sato Y (2011). Variation in counterattack effect against a phytoseiid predator between two forms of the social spider mite, Stigmaeopsis miscanthi . J Ethol 29 : 337–342. Tables Table 1 Location where spider mites of Stigmaeopsis miscanthi species group were collected and the numbers of nests, females, males immatures, eggs and the male ratio collected from each location. Location Latitude Longitude Altitude (m) No. of Male ratio Nest Female Male Immature Egg Shiofuki 34.96863 139.12685 20 8 48 4 23 133 0.08 Amagi 1 34.89088 139.13757 20 19 70 13 143 267 0.16 Amagi 2 34.84611 139.07361 50 18 74 17 157 331 0.19 Amagi 3 34.84833 139.06611 150 14 35 12 59 53 0.26 Amagi 4 34.88358 139.09616 160 10 13 6 26 79 0.32 Amagi 5 34.85333 139.06028 210 22 20 11 39 52 0.35 Amagi 6 34.88972 139.08833 250 7 15 3 25 61 0.17 Amagi 7 34.85750 139.05889 280 10 18 8 31 81 0.31 Amagi 8 34.88667 139.08917 290 4 6 2 22 20 0.25 Amagi 9 34.90222 139.10972 300 8 17 4 28 66 0.19 Amagi 10 34.88806 139.08306 350 6 7 2 10 25 0.22 Amagi 11 34.85582 139.06557 390 17 42 14 122 127 0.25 Amagi 12 34.90583 139.07472 400 28 80 33 139 293 0.29 Amagi 13 34.88917 139.07889 420 11 26 9 85 112 0.26 Amagi 14 34.85806 139.06444 430 21 40 4 50 172 0.09 Amagi 15 34.88583 139.07694 450 20 27 9 69 132 0.25 Amagi 16 34.90639 139.06500 460 35 103 29 164 228 0.22 Amagi 17 34.88361 139.07139 500 10 25 5 41 92 0.17 Amagi 18 34.86194 139.05944 530 11 20 6 47 45 0.23 Amagi 19 34.88139 139.06639 560 4 7 2 7 28 0.22 Amagi 20 34.87944 139.05972 680 10 19 5 30 94 0.21 Table 2 The number of eggs laid in 10 days after female emergence, the survival rate from egg to adult, and the female ratio in interspecific crosses, intra-population crosses and virgin oviposition in the cross experiments using S. sabelisi females (a) and S. miscanthi HG form females (b). (a) Cross experiment of S. sabelisi female Cross type Cross combination No. of pairs No. of eggs Survival rate from egg to adult Female ratio in offspring Female Male (Mean ± SE) (Mean ± SE) (Mean ± SE) Interspecific Amagi 17 ( S. sabelisi ) Amagi 1 ( S. miscanthi HG form) 24 7.708 ± 0.850 0.797 ± 0.060 0.013 ± 0.009 Intra-population Amagi 17 ( S. sabelisi ) Amagi 17 ( S. sabelisi ) 27 8.444 ± 0.590 0.951 ± 0.017 0.806 ± 0.014 Virgin oviposition Amagi 17 ( S. sabelisi ) - 23 4.870 ± 0.352 0.891 ± 0.037 0.000 ± 0.000 (b) Cross experiment of S. miscanthi HG form female Cross type Cross combination No. of pairs No. of eggs Survival rate from egg to adult Female ratio in offspring Female Male (Mean ± SE) (Mean ± SE) (Mean ± SE) Interspecies Amagi 1 ( S. miscanthi HG form) Amagi 17 ( S. sabelisi ) 25 8.320 ± 0.725 0.773 ± 0.038 0.030 ± 0.030 Intra-population Amagi 1 ( S. miscanthi HG form) Amagi 1 ( S. miscanthi HG form) 22 7.818 ± 0.737 0.932 ± 0.032 0.820 ± 0.023 Virgin oviposition Amagi 1 ( S. miscanthi HG form) - 21 4.143 ± 0.416 0.900 ± 0.044 0.000 ± 0.000 Additional Declarations There is no duality of interest Supplementary Files TableS1.docx Table S1 Fig.S1.pptx Figure S1 SupplementaryMaterials.xlsx Data Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 10 Apr, 2024 Review # 2 received at journal 19 Mar, 2024 Review # 1 received at journal 19 Mar, 2024 Reviewer # 2 agreed at journal 05 Mar, 2024 Reviewer # 1 agreed at journal 28 Feb, 2024 Reviewers invited by journal 22 Feb, 2024 Editor assigned by journal 11 Feb, 2024 First submitted to journal 11 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3947675","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":274293137,"identity":"a349d04d-416b-4b7a-8159-052895be1402","order_by":0,"name":"Yukie Sato","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYFACHoYDIIofRCQUEK0lgYFBsgGkxYBILQwgLQZgu4jRYnD87MEDH3/Y5RmfX5344YEBgzy/2AECWs7kJRyckZBcbHbj7WYJoMMMZ85OIKDlBo/BYZ4E5sRtN85uAGlJMLhNjJY/CfWJm2ec3fyDeC0MCYcTN/D3biPOFskzOQYHe9KOJ864wbvNIsFAgrBf+I6fMf7ww6Y6sb//7OabPyps5PmlCWhBAAmwSglilYMA/wFSVI+CUTAKRsFIAgB3zUnWFbhIIgAAAABJRU5ErkJggg==","orcid":"","institution":"University of Tsukuba","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yukie","middleName":"","lastName":"Sato","suffix":""},{"id":274293138,"identity":"6353f436-d7dc-418b-8282-c62e9a0b9578","order_by":1,"name":"Shota Konaka","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shota","middleName":"","lastName":"Konaka","suffix":""},{"id":274293139,"identity":"05ede002-c6d0-4a90-a14f-f7f106efce2e","order_by":2,"name":"Shun Hirota","email":"","orcid":"https://orcid.org/0000-0002-6104-1119","institution":"Tohoku University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shun","middleName":"","lastName":"Hirota","suffix":""},{"id":274293140,"identity":"9860fd32-707c-402e-8f01-f1024551a50c","order_by":3,"name":"Yoshihisa Suyama","email":"","orcid":"https://orcid.org/0000-0002-3136-5489","institution":"Tohoku University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yoshihisa","middleName":"","lastName":"Suyama","suffix":""},{"id":274293141,"identity":"528c2dad-7668-4782-9105-26c8382e2745","order_by":4,"name":"Yoshihiko Tsumura","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yoshihiko","middleName":"","lastName":"Tsumura","suffix":""}],"badges":[],"createdAt":"2024-02-11 05:55:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3947675/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3947675/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51622216,"identity":"f2d4b535-209a-40cb-886d-926bc1dc00ec","added_by":"auto","created_at":"2024-02-26 06:14:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110607,"visible":true,"origin":"","legend":"\u003cp\u003eThe geographic distribution of the social spider mite\u003cem\u003e Stigmaeopsis miscanthi\u003c/em\u003e species group in and around the Japanese Archipelago (Saito, 1995; Saito \u0026amp; Sahara, 1999; Sato et al., 2013), and the locations of Mt. Unzen and Mt. Amagi, where the distribution of the two species at different altitudes was studied in a previous study (Mt. Unzen; Sato et al., 2008) and in this study (Mt. Amagi). Three species are distributed in Japan, and the geographic distributions of two species, \u003cem\u003eStigmaeopsis sabelisi \u003c/em\u003eand \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, overlap widely. In the overlapping area (parapatric region), \u003cem\u003eS. sabelisi\u003c/em\u003e and\u003cem\u003eS. miscanthi\u003c/em\u003e HG form are distributed in the highlands and lowlands, respectively.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3947675/v1/740771923ff79d603b533b70.png"},{"id":51622219,"identity":"d4d016ca-c7b9-481d-a2bb-ec00e6d21833","added_by":"auto","created_at":"2024-02-26 06:14:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51449,"visible":true,"origin":"","legend":"\u003cp\u003eDistributional patterns of \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form along altitude in both sexes based on the discriminant scores in the species identification by male morphology. Squares show the field points, and the numbers near the squares correspond to Table 1. The numbers with underlines are the colonies used to construct the discriminant function, and others are the colonies identified by the discriminant function. The colour of the square shows the species composition, and black, white, and grey indicate \u003cem\u003eS. sabelisi\u003c/em\u003eonly, \u003cem\u003eS. miscanthi \u003c/em\u003eHG form only, and the mixture, respectively. The right part of the square shows males and the left part shows females. Lines on the map are contour lines.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3947675/v1/f39eb9bbad24ab75464cef89.png"},{"id":51622223,"identity":"b99df373-b437-48c3-b235-8883fa40a960","added_by":"auto","created_at":"2024-02-26 06:14:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":447733,"visible":true,"origin":"","legend":"\u003cp\u003eThe third leg of a normal \u003cem\u003eS. sabelisi \u003c/em\u003emale specimen (a), a normal \u003cem\u003eS. miscanthi\u003c/em\u003e HG form male specimen (b), and an abnormal \u003cem\u003eS. miscanthi\u003c/em\u003e HG form male specimen (c). The males of (a) and (b) were collected from 680 m (Amagi 20) and 20 m altitudes (Amagi 1), respectively, and the male of (c) was collected from 350 m altitude (Amagi 10) and identified as \u003cem\u003eS. miscanthi\u003c/em\u003e HG form by measuring the other leg. Legs usually consist of four segments (tarsus, tibia, genu, and femur), as shown in (a) and (b); however, in (c), only three segments were present on one of the third legs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3947675/v1/492d32cf7318cc53236e6ff4.png"},{"id":51622217,"identity":"51f4114c-6481-40a2-bd4d-eab786ba2cb2","added_by":"auto","created_at":"2024-02-26 06:14:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7607,"visible":true,"origin":"","legend":"\u003cp\u003eSex ratio (proportion of males) in mite colonies along altitude. Blue squares show the mite colonies of pure \u003cem\u003eS. sabelisi\u003c/em\u003e, red circles show the mite colonies of pure \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, and yellow squares show the mite colonies of a mixture of \u003cem\u003eS. sabelisi \u003c/em\u003eand \u003cem\u003eS. miscanth\u003c/em\u003ei HG form.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3947675/v1/a25de6e84b79bd8c6f31e076.png"},{"id":51622218,"identity":"f2c32749-2e46-49f8-b85a-4141ec4aab69","added_by":"auto","created_at":"2024-02-26 06:14:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5688,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-Net network of \u003cem\u003eStigmaeopsis\u003c/em\u003e reconstructed based on the uncorrected P distance. The filled cycle represents the samples.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3947675/v1/435b5600b78501394d995018.png"},{"id":51622225,"identity":"e6b2ba8b-5466-42b1-9f67-4840f9bf8e39","added_by":"auto","created_at":"2024-02-26 06:14:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":389614,"visible":true,"origin":"","legend":"\u003cp\u003eThe proportion of the genome of every individual originating from each of the inferred clusters, K = 2: red is cluster 1 corresponding to \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, and green is cluster 2 corresponding to \u003cem\u003eS. sabelisi\u003c/em\u003e. Individuals 1 to 61 were from the colonies where \u003cem\u003eS. miscanthi \u003c/em\u003eHG form was found, individuals 62 to 101 were from the colonies where both \u003cem\u003eS. miscanthi\u003c/em\u003e HG form and \u003cem\u003eS. sabelis\u003c/em\u003ewere found, and 102 to 237 were from the colonies where \u003cem\u003eS. sabelis\u003c/em\u003e was found according to the results of identification by male morphology. Individuals 69, 75, 77, 117, and 120 have elements of \u003cem\u003eS. sabelisi\u003c/em\u003e, as well as very few elements of \u003cem\u003eS. miscanthi\u003c/em\u003e HG from.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3947675/v1/e7e46daa4c2089c190677133.png"},{"id":51622817,"identity":"cc822fe1-6971-4149-bfc2-a2a6e49d360d","added_by":"auto","created_at":"2024-02-26 06:22:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1246444,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3947675/v1/895a655b-0348-4b8c-a66c-8ccbed74c0bd.pdf"},{"id":51622221,"identity":"ca52851e-ef2b-4984-808b-a8befcb51f86","added_by":"auto","created_at":"2024-02-26 06:14:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19033,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3947675/v1/b4e2cb94bfbdcd703a789496.docx"},{"id":51622224,"identity":"ba231224-bfc1-4a2c-8f91-f58fc320527d","added_by":"auto","created_at":"2024-02-26 06:14:31","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":111677,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1\u003c/p\u003e","description":"","filename":"Fig.S1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-3947675/v1/6fd5b0215961e0762d7623fc.pptx"},{"id":51622220,"identity":"b4d6b712-e25f-4405-9aaf-9b981645f28e","added_by":"auto","created_at":"2024-02-26 06:14:31","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":38851,"visible":true,"origin":"","legend":"\u003cp\u003eData\u003c/p\u003e","description":"","filename":"SupplementaryMaterials.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3947675/v1/a9b3ea04189210690c94beaa.xlsx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Restricted hybridisation in the secondary contact zone of closely related haplodiploid social spider mites","fulltext":[{"header":"Introduction","content":"\u003cp\u003eClosely related species often overlap in their geographical distribution (Mayr, 1963). Contact frequencies and reproductive, ecological, and genetic relationships at their distribution boundaries are important for their coexistence and evolutionary consequences\u0026nbsp;(Coyne and Orr, 2004; Johannesson \u003cem\u003eet al.\u003c/em\u003e, 2020). In particular, hybridisation is likely to occur if reproductive barriers are not completely established. In this case, they may fuse into a single species\u0026nbsp;(Coyne and Orr, 2004)\u0026nbsp;or diversify further by character displacement\u0026nbsp;and/or\u0026nbsp;reinforcement of reproductive barriers\u0026nbsp;(Dobzhansky, 1959; Hoskin \u003cem\u003eet al.\u003c/em\u003e, 2005; Smadja and Butlin, 2006; Pfennig and Rice, 2014). If the fitness of the hybrids is high, a new species can be generated from hybridisation through developing reproductive isolation from its parental species\u0026nbsp;(Rieseberg, 1997; Seehausen, 2004; Mallet, 2008; Abbott \u003cem\u003eet al.\u003c/em\u003e, 2013).Even though this does not result in new species, genetic introgression by hybridisation may bring genetic diversity to the parent species and drive their subsequent evolution\u0026nbsp;(Edelman \u003cem\u003eet al.\u003c/em\u003e, 2019).\u0026nbsp;Therefore, to understand the evolutionary relationships and speciation of closely related species with overlapping distributions, it is important to\u0026nbsp;determine the frequencies of contact, reproductive isolation, and genetic introgression at their distribution boundaries.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The\u003cem\u003eStigmaeopsis miscanthi\u003c/em\u003e species group (Acari: Tetranychidae)\u0026nbsp;is\u0026nbsp;a\u0026nbsp;haplodiploid spider mite that infests Chinese silver grass,\u0026nbsp;\u003cem\u003eMiscanthus sinensis\u003c/em\u003e, in East Asia\u0026nbsp;(Saito \u003cem\u003eet al.\u003c/em\u003e, 2018, 2019). The mites construct woven nests on the undersurface of the host plant leaves and live in groups within the nests. They are called social spider mites because there are two to three generations of\u0026nbsp;overlap among nest members, and they show cooperative nest building, nest sanitation, and brood care\u0026nbsp;(Saito, 2009; Schausberger \u003cem\u003eet al.\u003c/em\u003e, 2021). Woven nests are protective against their natural enemies (predatory mites, predatory gall midges, ants, etc.); however, some predators, such as\u0026nbsp;the phytoseiid mite \u003cem\u003eTyphlodromus bambusae\u003c/em\u003e, can intrude\u0026nbsp;into the woven nests. To protect nestmates and their offspring against predatory intruders, adult males and females counterattack the intruders cooperatively and sometimes kill the intruders if they are immature\u0026nbsp;(Saitō, 1986a, 1986b; Yano \u003cem\u003eet al.\u003c/em\u003e, 2011; Saito \u003cem\u003eet al.\u003c/em\u003e, 2011). Males are aggressive not only against predatory intruders\u0026nbsp;but also against\u0026nbsp;conspecific males. They kill each other to establish their own harem\u0026nbsp;(Saitō, 1990). The frequency of male killing varies among populations\u0026nbsp;(Saito, 1995; Saito and Sahara, 1999; Sato, Egas, \u003cem\u003eet al.\u003c/em\u003e, 2013; Sato \u003cem\u003eet al.\u003c/em\u003e, 2019)\u0026nbsp;and is associated with differences in male aggression and also with their reproductive, phylogenetic, and geographic relationships\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2000a, 2000b, 2015, 2015, 2018; Sato, Egas, \u003cem\u003eet al.\u003c/em\u003e, 2013). Five species and two forms have been described in this species group so far\u0026nbsp;(Saito \u003cem\u003eet al.\u003c/em\u003e, 2018, 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn Japan, \u003cem\u003eStigmaeopsis sabelisi\u003c/em\u003e with lower male aggression, \u003cem\u003eS. miscanthi\u003c/em\u003e high-aggression form (hereafter, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form) with higher male aggression, and \u003cem\u003eS. miscanthi\u003c/em\u003e mild-aggression form (hereafter, \u003cem\u003eS. miscanthi\u0026nbsp;\u003c/em\u003eML form) with intermediate male aggression are distributed\u0026nbsp;(Saito, 1995; Saito and Sahara, 1999; Sato, Egas, \u003cem\u003eet al.\u003c/em\u003e, 2013; Sato \u003cem\u003eet al.\u003c/em\u003e, 2019). \u003cem\u003eS. miscanthi\u003c/em\u003e ML form is distributed in subtropical regions and\u0026nbsp;is\u0026nbsp;geographically isolated from\u0026nbsp;the\u0026nbsp;two other species\u0026nbsp;(Sato, Egas, \u003cem\u003eet al.\u003c/em\u003e, 2013; Sato \u003cem\u003eet al.\u003c/em\u003e, 2019)\u0026nbsp;(Fig. 1). On the other hand, \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form show overlap in their geographic distribution: \u003cem\u003eS. sabelisi\u003c/em\u003e is distributed in colder regions\u0026nbsp;(from Aomori Prefecture to Kyushu Islands), whereas \u003cem\u003eS. miscanthi\u003c/em\u003e HG form is distributed in warmer regions (from Shizuoka Prefecture to the main island of Okinawa) (Fig. 1). Japan is mountainous, and in areas where both species are distributed,\u0026nbsp;\u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form\u0026nbsp;are found in the highlands and lowlands, respectively (parapatric distribution). A previous study inferred the population history of the species group using mtDNA (cytochrome c oxidase subunit I; COI) and estimated that \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form\u0026nbsp;were derived from an ancestral group with mild male aggression in the subtropical region during the last glacial period (20,000–40,000 years BP for \u003cem\u003eS. sabelisi\u003c/em\u003e and 5,494–10,988 years BP for \u003cem\u003eS. miscanthi\u003c/em\u003e HG form)\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2019). Considering their inferred history together with their ecological and reproductive relationships and the migration history of their host plant\u0026nbsp;(Clark \u003cem\u003eet al.\u003c/em\u003e, 2014), it is predicted that (1) \u003cem\u003eS. sabelisi\u003c/em\u003e was derived south of Japan and migrated into the Japanese archipelago just after the host plant expanded its distribution into the Japanese archipelago; (2) as temperature increased more, the ancestral groupexpanded its distribution northward and migrated into the Ryukyus Islands; (3) \u003cem\u003eS. miscanthi\u003c/em\u003e HG form was derived from the ancestral group in and around the Japanese archipelago; and (4) \u003cem\u003eS. miscanthi\u003c/em\u003e HG form expanded its distribution in the Japanese archipelago and drove \u003cem\u003eS. sabelisi\u003c/em\u003e to the colder region through competition and reproductive interference\u0026nbsp;(Saito \u003cem\u003eet al.\u003c/em\u003e, 2013; Sato, Sabelis, \u003cem\u003eet al.\u003c/em\u003e, 2013; Sato \u003cem\u003eet al.\u003c/em\u003e, 2015), resulting in their present geographic distributions\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA previous field study at Mt. Unzen, one of the mountains on the Kyushu Islands at the southern end of the parapatric area (Nagasaki Prefecture; Fig. 1), found that the distributions of these two species broadly overlapped at intermediate altitudes (100–400 m), and both species were collected from the same host plant colonies in the contact zone\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2008). It is known that their reproductive isolation is strong but incomplete; there is a strong\u0026nbsp;post-mating\u0026nbsp;and\u0026nbsp;pre-zygotic\u0026nbsp;reproductive barrier, but a few hybrids are produced from interspecific crosses (proportion of hybrids: 0– 30%)\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2000a, 2000b, 2015, 2018), and their hybrids are fertile\u0026nbsp;(Sato, 2004). Therefore, hybridisation and gene flow are likely to occur in the contact zones formed\u0026nbsp;on each mountain in the parapatric area. In particular, males of \u003cem\u003eS. miscanthi\u003c/em\u003e HG form actively approach the females of \u003cem\u003eS. sabelisi\u003c/em\u003e for mating, as they do for conspecific females\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2015)\u003cem\u003e.\u003c/em\u003e In addition, interspecific male fights\u0026nbsp;occur easily\u0026nbsp;between the two species, and males in the \u003cem\u003eS. miscanthi\u003c/em\u003e HG form tend to win interspecific male fights\u0026nbsp;(Sato, Sabelis, \u003cem\u003eet al.\u003c/em\u003e, 2013). This suggests that\u0026nbsp;\u003cem\u003eS. sabelisi\u0026nbsp;\u003c/em\u003efemales are at a higher risk of interspecific mating than \u003cem\u003eS. miscanthi\u0026nbsp;\u003c/em\u003eHG form females, indicating that genetic introgression is likely asymmetric. However, it has not been confirmed whether hybridisation occurs in\u0026nbsp;the contact zones. Furthermore, their contact zone\u0026nbsp;has been reported only on Mt. Unzen, and it is unclear whether their contact zones are widespread in their parapatric areas.\u003c/p\u003e\n\u003cp\u003eIn this study, to address whether the contact zone of \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u0026nbsp;\u003c/em\u003eHG form is widespread in their parapatric areas, we investigated their distribution patterns along\u0026nbsp;the elevation\u0026nbsp;on and around Mt. Amagi in Shizuoka Prefecture, Japan. Mt. Amagi was selected as the study site because it is located at the northern end of the parapatric area (Fig. 1). The presence of contact zones at both the southern and northern ends of the parapatric areas (Mt. Unzen and Mt. Amagi) supports\u0026nbsp;the hypothesis that contact zones are prevalent in parapatric areas. To determine whether interspecific mating occurred in the contact zone, we analysed the sex ratio of mite colonies in the contact zone. The mites are haplodiploid, in which females develop from fertilised eggs and males develop from unfertilised eggs. Virgin females lay unfertilised eggs, and the number of eggs is significantly lower than that of mated females\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2000a, 2000b, 2018). Females mated with males of different species lay unfertilised eggs because of the strong post-mating pre-zygotic barrier (reproductive barrier in the egg fertilisation stage); however, the number of eggs was similar to that of females mated with conspecific males, possibly because females control the number of eggs by copulation stimuli\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2000a, 2000b, 2018). As females that mate with males of different species produce\u0026nbsp;an overabundance of sons\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2000a, 2000b, 2018), the sex ratio would become relatively male-biased in mite colonies where interspecific mating occurs\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2008); although, the spider mite species originally showed\u0026nbsp;an extremely female-biased sex ratio (male ratio: 0.10–0.20)\u0026nbsp;(Sato and Saito, 2007). To address whether hybridisation and genetic introgression occurred between the two mite species, we analysed the genetic population structure of the mite colonies in their contact zones. Spider mites are too small to obtain sufficient DNA\u0026nbsp;from a single mite\u0026nbsp;for molecular analysis (body length is less than 0.5 mm). Therefore, in the genetic analyses, we used multiplexed Inter-Simple Sequence Repeat (ISSR) genotyping by sequencing (MIG-seq), which is useful for small amounts of low-quality DNA\u0026nbsp;(Suyama and Matsuki, 2015). Single-nucleotide polymorphisms (SNPs) were detected using MIG-seq for genetic analysis. To confirm whether \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form\u0026nbsp;collected from Mt. Amagi produced hybrids, we\u0026nbsp;performed cross-experiments. Previous cross-experiments have found hybridisation; however, these studies used different populations, so there is a possibility that the two species from Mt. Amagi do not produce hybrids\u0026nbsp;that are different from those reported in previous studies.\u0026nbsp;Finally, based on these findings, we discuss the prevalence of their contact zones, interspecific mating, gene flow, and the effect of the contact zone on their evolution.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eStudy site and mite collection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe collected spider mites of \u003cem\u003eS. miscanthi\u003c/em\u003e species group from the eastern area of Mt. Amagi (Shizuoka Prefecture, Japan) at altitudes of 20–680 m in July 2020 (Table 1, Fig. 1). This area is dominated by \u003cem\u003eS. sabelisi\u003c/em\u003e;therefore, to ensure the collection of \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, we collected mites\u0026nbsp;from the area northeast of Mt. Amagi at an altitude\u0026nbsp;of 20 m, additionally\u0026nbsp;(Shiofuki in Table 1). We brought host plant leaves with mite nests to the laboratory and recorded the number of males, females, and immatures in each nest using stereoscopic microscopes. Slide specimens of the collected males\u0026nbsp;were prepared\u0026nbsp;and used for species identification based on the male morphology. We stored the collected females in microtubes containing 99% acetone for\u0026nbsp;genetic analysis.\u0026nbsp;We reared the collected immatures on detached \u003cem\u003eM. sinensis\u003c/em\u003e leaves under controlled conditions (25℃, 75–100% relative humidity, and 15:9 h light:dark). Since spider mites are haplodiploid, male genes are\u0026nbsp;derived\u0026nbsp;only from their mothers. Therefore, we prepared slide specimens of males developed from reared immatures within several days after mite collection and used them for species identification of females based on male morphology. We established laboratory mite cultures for each population using the remaining collected mites and used them for\u0026nbsp;the cross-experiment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSpecies identification by male morphology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs the relative length of leg I to leg III and body width were greater in males of the \u003cem\u003eS. miscanthi\u003c/em\u003e HG form than\u0026nbsp;in\u0026nbsp;those of \u003cem\u003eS. sabelisi\u003c/em\u003e, we used morphological differences to identify the species of the collected mites\u0026nbsp;(Saito, 1995; Sato \u003cem\u003eet al.\u003c/em\u003e, 2000a, 2019; Sato, Egas, \u003cem\u003eet al.\u003c/em\u003e, 2013). One to five males\u0026nbsp;were placed on a drop of Hoyer’s solution on a slide\u0026nbsp;glass and covered with cover glass. We placed a 10-g weight on the cover glass to flatten the mite body evenly for accurate measurement and dried it at 45℃\u0026nbsp;for more than 3 days. We took photos of the slide specimen with a microscope (Axioskop, Zeiss, Germany) and an eyepiece camera (Dino-Eye AM4023X, Anmo Electronics Corporation, Taiwan), after which we measured the lengths of the first and third legs (the four leg segments of\u0026nbsp;the\u0026nbsp;tarsus,\u0026nbsp;tibia, genu, and femur), body length,\u0026nbsp;and body width of the slide specimen using image-processing software (Image J ver. 1.53a; National Institute of Health, Bethesda, MD, USA)\u0026nbsp;(Schneider \u003cem\u003eet al.\u003c/em\u003e, 2012). We calculated the relative length of the first to third legs (leg I to leg III) from the total of the four leg segment lengths and used the log-transformed values in the analysis. The specimens used to construct the linear discriminant function were measured\u0026nbsp;three times, and the average values\u0026nbsp;were used.\u0026nbsp;The specimens used for species identification were\u0026nbsp;measured once,\u0026nbsp;and the value was used for species identification.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;In a previous study conducted on Mt. Unzen (Nagasaki Prefecture, Japan), a contact zone was found at altitudes of 100–400 m\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2008). Therefore, we assumed that\u0026nbsp;the\u0026nbsp;mite colonies at altitudes below 100 m and above 500 m were \u003cem\u003eS. miscanthi\u003c/em\u003e HG form and \u003cem\u003eS. sabelisi\u003c/em\u003e, respectively. We constructed a linear discriminant function using body width (\u003cem\u003eBW\u003c/em\u003e), body length (\u003cem\u003eBL\u003c/em\u003e), and log-transformed values of the relative length of\u0026nbsp;the\u0026nbsp;first to third legs (\u003cem\u003eBLL\u003c/em\u003e) of males from the colonies (\u003cem\u003eS. miscanthi\u003c/em\u003e HG form: 31 males and 41 males developed from immatures collected from Shiofuki and Amagi 1 and 2;\u003cem\u003e\u0026nbsp;S. sabelisi\u003c/em\u003e: 28 males and 41 males developed from immatures\u0026nbsp;collected\u0026nbsp;from Amagi 17–20; Table 1). We used the discriminant function for species identification of males and males developed from immatures collected from colonies at an altitude\u0026nbsp;of 100–500 m\u0026nbsp;(Amagi 3–16; Table 1). For discriminant analysis, we used\u0026nbsp;statistic software R\u0026nbsp;ver. 4.0.2\u0026nbsp;(R Core Team, 2022)\u0026nbsp;and the \u003cem\u003eMASS\u003c/em\u003e package\u0026nbsp;(Venables and Ripley, 2002).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInterspecific mating inferred from the sex ratio in the field\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe sex ratio is expected to be relatively male-biased in mite colonies where interspecific mating occurs\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2008), because females that mate with different species produce\u0026nbsp;an overabundance\u0026nbsp;of sons\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2000a, 2000b, 2018). We estimated interspecific mating in the field by analysing the calculated\u0026nbsp;sex ratio. Based on the results of species identification using male morphology, we categorised the mite colonies into three types:\u003cem\u003e\u0026nbsp;S. miscanthi\u003c/em\u003e HG, \u003cem\u003eS. sabelisi\u003c/em\u003e alone, and both species. The male ratio was analysed using a generalised linear model (GLM) with\u0026nbsp;a binomial error distribution\u0026nbsp;and likelihood ratio test (LRT). Multiple comparisons were performed using Tukey’s post-hoc\u0026nbsp;test. For the analysis, we used\u0026nbsp;statistic software R\u0026nbsp;ver. 4.0.2\u0026nbsp;(R Core Team, 2022)\u0026nbsp;and the \u003cem\u003emultcomp\u003c/em\u003e package\u0026nbsp;(Hothorn \u003cem\u003eet al.\u003c/em\u003e, 2008).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGenetic analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the genetic analysis, we used 237 females from 11 colonies at altitudes of 100–500 m (contact zone), two colonies below 100 m altitude (pure \u003cem\u003eS. miscanthi\u003c/em\u003e HG form), and two colonies above 500 m altitude (pure \u003cem\u003eS. sabelisi\u003c/em\u003e). DNA was extracted from a single female mite, which was stored in 99% acetone in a microtube, using PrepMan Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo construct the MIG-seq library, we followed the protocol described by Suyama et al. (2022),\u0026nbsp;which is modified from Suyama and Matsuki (2015).\u0026nbsp;Briefly, an MIG-seq library was prepared using a two-step PCR method. In the first PCR, ISSR regions were amplified using MIG-seq primer set 1 (Suyama and Matsuki 2015). In the second PCR, indices and Illumina adapter sequences were added to\u0026nbsp;the first PCR\u0026nbsp;products. The Illumina MiSeq platform and MiSeq Reagent Kit v3 (150 cycles; Illumina) were used for sequencing. We skipped the sequencing of the first 17 bases of reads 1 and 2 (SSR primer region and anchors) using “DarkCycle”. We detected genome-wide SNPs from the sequenced raw reads and filtered SNPs and females, as described in Suyama \u003cem\u003eet al.\u003c/em\u003e (2022). After the removal of extremely short reads and low-quality reads using trimmomatic 0.39\u0026nbsp;(Bolger \u003cem\u003eet al.\u003c/em\u003e, 2014), 42,183,313 reads (177,989 ± 4,186 reads per sample) remained from 43,713,012 raw reads (184,443 ± 4,323 reads per sample). De novo SNP genotyping was performed using Stacks 2.55\u0026nbsp;(Rochette \u003cem\u003eet al.\u003c/em\u003e, 2019), utilizing the following parameters: minimum depth of coverage required to create a stack (\u003cem\u003em\u003c/em\u003e) of 3, maximum distance allowed between stacks (\u003cem\u003eM\u003c/em\u003e) of 2, and number of mismatches allowed between sample loci while building the catalogue (\u003cem\u003en\u003c/em\u003e) of 2. Using the ‘populations’ program in Stacks, we removed SNP sites exhibiting high heterozygosity (\u003cem\u003eH\u003c/em\u003eo ≥ 0.6) and filtered out SNP sites with fewer than three minor alleles. SNPs that were retained by 50% or more samples were included in the SNP dataset. To avoid linked SNPs, only the first SNP from each locus\u0026nbsp;was considered. Observed (\u003cem\u003eH\u003c/em\u003e\u003csub\u003eO\u003c/sub\u003e) and expected (\u003cem\u003eH\u003c/em\u003e\u003csub\u003eE\u003c/sub\u003e) heterozygosities as well as nucleotide diversities at SNP sites were calculated using the ‘populations’ program in Stacks. To determine the presence of hybrids and genetic introgression between the species, the selected SNPs were used to analyse the population structure using\u0026nbsp;STRUCTURE Software ver. 2.3.4\u0026nbsp;(Pritchard \u003cem\u003eet al.\u003c/em\u003e, 2000)\u0026nbsp;and Structure Harvester\u0026nbsp;(Earl and von\u0026nbsp;Holdt, 2012). We conducted 30 independent runs with a burn-in of 100,000 steps, followed by an additional 100,000 steps using an admixture model. We estimated the log-likelihood of each cluster (\u003cem\u003eK\u003c/em\u003e = 1–10). A Neighbour-Net network was constructed using SplitsTree4 4.14\u0026nbsp;(Huson and Bryant, 2006), utilising the uncorrelated P distance matrix and ignoring ambiguous sites.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCross-experiment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe used laboratory cultures collected from Amagi 17 and Amagi 1 in the cross-experiment as \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, respectively (Table 1). We placed\u0026nbsp;a detached \u003cem\u003eM. sinensis\u003c/em\u003e leaf (1.0 × 3.0 cm) on water-soaked cotton wool in a Petri dish (5.0 cm diameter, 1.5 cm high; SPL Life Sciences, Gyeonggi-do, Korea). We collected a teleiochrysalis female from the mite culture and placed it onto the prepared leaf arena. One day after mite introduction, we checked the emergence and construction of a woven nest\u0026nbsp;and then\u0026nbsp;introduced a male collected from the mite culture onto the leaf arena. We allowed them to mate and oviposit for 10 days, after which we removed the females and\u0026nbsp;males from the leaf arena\u0026nbsp;and recorded the number of eggs. We checked the development of their offspring daily until they\u0026nbsp;reached the adult phase and recorded their survival\u0026nbsp;and sex. We performed cross-experiments between the populations: Amagi 17 (female) × Amagi 1 (male) and Amagi 1 (female) × Amagi 17 (male), and within species as controls: Amagi 17 (female) × Amagi 17 (male) and Amagi 1 (female) × Amagi 1 (male). As another control, we observed virgin oviposition because spider mite females produce sons without mating, and the number of eggs is significantly fewer in the virgin oviposition\u0026nbsp;than in the mated oviposition in the species group\u0026nbsp;(Sato \u003cem\u003eet al.\u003c/em\u003e, 2000a, 2000b, 2018). We carried out the cross-experiments under controlled conditions (25℃, 75–100% relative humidity, and 15:9 h light:dark).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; To determine the pre-mating barrier, we compared the number of eggs among the cross combinations (interspecific crosses, intrapopulation crosses, and virgin oviposition) of each female species. For comparison, we used a GLM with a negative binomial error distribution because we detected overdispersion in the Poisson GLM. To determine post-mating and pre-zygotic barriers, we compared the offspring sex ratios among cross combinations (interspecific cross, intraspecific cross, and virgin oviposition) using a GLM with a binomial error distribution or quasibinomial error distribution when overdispersion was detected in the binomial GLM. To determine the zygotic barrier, we compared the offspring survival ratios among the cross combinations (interspecific cross, intraspecific cross, and virgin oviposition) using a GLM with a quasibinomial error distribution, as we detected overdispersion in the binomial GLM. For the analysis, we used statistic software R ver. 4.0.2 (R Core Team, 2022).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eContact zone inferred by species identification by male morphology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe discriminant function was obtained as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003eDW\u003c/em\u003e, \u003cem\u003eDL\u003c/em\u003e, and \u003cem\u003eRLL\u003c/em\u003e are the body width, body length, and relative lengths of the first to third legs, respectively. When the male individuals used for formulating the function were randomly sampled, the function provided 100% correct answers. Using the discriminant function, we identified 137 males for species identification in males and 175 males developed from the immatures for species identification in females, both of which were collected from colonies at altitudes of 100\u0026ndash;500 m. For species identification in males, 32 and 105 individuals were identified as \u003cem\u003eS. miscanthi\u003c/em\u003e HG form and \u003cem\u003eS. sabelisi\u003c/em\u003e, respectively. Both species were found together in six colonies at altitudes of 160\u0026ndash;420 m, although \u003cem\u003eS. sabelisi\u003c/em\u003e dominated in seven colonies and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form in one colony (Fig. 2). In the species identification of females, 34 and 141 individuals were identified as \u003cem\u003eS. miscanthi\u003c/em\u003e HG form and \u003cem\u003eS. sabelisi\u003c/em\u003e, respectively. Both species were found together in five colonies at altitudes of 150\u0026ndash;430 m, although \u003cem\u003eS. sabelisi\u003c/em\u003e was dominant in nine colonies (Fig. 2). These results indicate that their contact zone exists at least in the area at altitudes of 150\u0026ndash;430 m on Mt. Amagi.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe examined the morphology of 312 males in total and found that a male that developed from the immatures collected at an altitude of 350 m (Amagi 10; Table 1; Fig. 2) had a deformity (Fig. 3). The legs usually consist of four segments (tarsus, tibia, genu, and femur); however, in the deformed male, only three segments were present on one of the third legs (Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInterspecific mating inferred from the sex ratio in the field\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe male ratio varied among the mite colonies (male ratio: 0.077\u0026ndash;0.355; Fig. 4). The effect of colony type on the male ratio was significant (binomial GLM, \u003cem\u003edf\u003c/em\u003e = 2, LRT = 15.123, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). The male ratio in the colonies in which both species were found was significantly higher than that in the colonies of pure \u003cem\u003eS. sabelisi\u003c/em\u003e or \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, although the male ratio was not significantly different between the colonies of pure\u003cem\u003e\u0026nbsp;S. miscanthi\u003c/em\u003e HG form and \u003cem\u003eS. sabelisi\u003c/em\u003e (Tukey\u0026rsquo;s test, both species vs. \u003cem\u003eS. miscanthi\u003c/em\u003e HG form: \u003cem\u003ez\u003c/em\u003e = 3.756, \u003cem\u003eP\u003c/em\u003e = 0.001; both species vs. \u003cem\u003eS. sabelisi\u003c/em\u003e: \u003cem\u003ez\u003c/em\u003e = 2.563, P = 0.027; \u003cem\u003eS. miscanthi\u003c/em\u003e HG form vs. \u003cem\u003eS. sabelisi\u003c/em\u003e: \u003cem\u003ez\u003c/em\u003e = 1.389, P = 0.344). These results indicate that the overproduction of sons by interspecific mating occurred in colonies where both species were found.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHybridization and genetic introgression\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 111 SNPs from 189 loci in 237 females were used for the genetic analysis. All raw reads of MIG-seq data were submitted to the DDBJ Sequence Read Archive under the BioProject ID PRJDB17427. The \u003cem\u003eS.\u003c/em\u003e \u003cem\u003emiscanthi\u003c/em\u003e HG form populations had lower heterozygosity and nucleotide diversity than the populations of \u003cem\u003eS. sabelisi\u0026nbsp;\u003c/em\u003e(Table S1). Neighbour-Net analysis demonstrated the distinction between \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form as two separate genetic clusters (Fig. 5). None of the samples occupied an intermediate position between \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form. Under the delta \u003cem\u003eK\u003c/em\u003e method, the best STRUCTURE model assigned individuals to two genetic clusters (Fig. S1), corresponding to \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, respectively (Fig. 6). A total of 232 individuals were assigned as pure \u003cem\u003eS. sabelisi\u003c/em\u003e or pure \u003cem\u003eS. miscanthi\u003c/em\u003e HG form, and five individuals collected from their contact zones were assigned as \u003cem\u003eS. sabelisi\u003c/em\u003e with very small fragments of \u003cem\u003eS. miscanthi\u003c/em\u003e HG form. These results showed that there were no hybrids and that genetic introgression was very low.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eReproductive isolation in laboratory cross-experiment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the cross experiments with \u003cem\u003eS. sabelisi\u003c/em\u003e females, the number of eggs in interspecific crosses was significantly higher than that in virgin oviposition (negative-binomial GLM, \u003cem\u003ez\u003c/em\u003e = 3.418, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; Table 2) and similar to that in intrapopulation crosses (negative-binomial GLM, \u003cem\u003ez\u003c/em\u003e = 0.793, \u003cem\u003eP\u003c/em\u003e = 0.428; Table 2), indicating that mating occurred in the interspecific cross. A few hybrids were produced in two of the 24 pairs of interspecific crosses; however, the female ratio in the offspring of interspecific crosses was significantly lower than that in intrapopulation crosses (binomial GLM, \u003cem\u003ez\u003c/em\u003e = 7.919, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; Table 2) and similar to that in virgin oviposition (binomial GLM, \u003cem\u003ez\u003c/em\u003e = 0.006, \u003cem\u003eP\u003c/em\u003e = 0.995; Table 2), indicating the presence of a strong pre-zygotic barrier. The survival rate of offspring from egg to adult in interspecific crosses was significantly lower than that in intrapopulation crosses (quasibinomial GLM, \u003cem\u003et\u003c/em\u003e = 2.993, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; Table 2) but not significantly different from that in virgin oviposition (quasibinomial GLM, \u003cem\u003et\u003c/em\u003e = 1.230, \u003cem\u003eP\u003c/em\u003e = 0.223; Table 2).\u003c/p\u003e\n\u003cp\u003eThe results of the cross-experiments with \u003cem\u003eS. miscanthi\u003c/em\u003e HG form females were similar to those of the cross-experiments with \u003cem\u003eS. sabelisi\u003c/em\u003e females. The number of eggs in the interspecific cross was significantly higher than that in the virgin oviposition (negative-binomial GLM, \u003cem\u003ez\u003c/em\u003e = 4.870, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; Table 2) and was similar to that in the intrapopulation cross (negative-binomial GLM, \u003cem\u003ez\u003c/em\u003e = 0.513, \u003cem\u003eP\u003c/em\u003e = 0.608; Table 2), indicating that mating occurred in the interspecific cross. A few hybrids were produced in one of the 25 pairs of interspecific crosses; however, the female ratio in the offspring of interspecific crosses was significantly lower than that in intrapopulation crosses (quasibinomial GLM, \u003cem\u003et\u003c/em\u003e = 6.311, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; Table 2) and similar to that in virgin oviposition crosses (quasibinomial GLM, \u003cem\u003et\u003c/em\u003e = 0.004, \u003cem\u003eP\u003c/em\u003e = 0.997; Table 2), indicating the presence of a strong pre-zygotic barrier. The survival rate of offspring from egg to adult in interspecific crosses was significantly lower than that in intrapopulation crosses (quasibinomial GLM, \u003cem\u003et\u003c/em\u003e = 2.692, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; Table 2) but not significantly different from that in virgin oviposition (quasibinomial GLM, \u003cem\u003et\u003c/em\u003e = 1.565, \u003cem\u003eP\u003c/em\u003e = 0.122; Table 2).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe found that the geographic distributions of \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form overlapped widely at intermediate altitudes (150\u0026ndash;430 m) on Mt. Amagi. We conducted a field survey on Mt. Amagi at the northern end of their parapatric area because a previous study detected a contact zone on Mt. Unzen at the southern end of their parapatric area (Sato \u003cem\u003eet al.\u003c/em\u003e, 2008; Fig. 1). As their contact zone was confirmed at both\u0026nbsp;the southern and northern ends of their\u0026nbsp;parapatric area, we concluded that their contact zone would be prevalent in their parapatric area and that their contact zone may have a significant impact on their evolution and ecological relationships.\u003c/p\u003e\n\u003cp\u003eIn several host-plant fields in their contact zones, both species were found together for both sexes. This suggests that interspecific mating has occurred in these fields. In fact, the sex ratio of spider mites in the colonies of both species was male-biased compared to that of pure \u003cem\u003eS. sabelisi\u003c/em\u003e or \u003cem\u003eS. miscanthi\u003c/em\u003e HG form colonies. As for their reproductive barrier, the post-mating pre-zygotic barrier contributes the most (Sato \u003cem\u003eet al.\u003c/em\u003e, 2000a, 2000b, 2015, 2018), which causes the overproduction of males owing to their haplodiploid genetic system and regulation of the number of eggs by copulation stimuli. The male-biased sex ratio strongly supports interspecific mating in the contact zone. A previous study conducted on Mt. Unzen detected a male-biased sex ratio at intermediate altitudes (Sato \u003cem\u003eet al.\u003c/em\u003e, 2008). Therefore, we conclude that interspecific mating is widespread in their parapatric area.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA previous study reported the presence of a pre-mating barrier, but it was very weak and asymmetric (Sato \u003cem\u003eet al.\u003c/em\u003e, 2015). The post-mating pre-zygotic barrier contributes most to the reproductive barrier; however, it is also incomplete, and a few hybrids were produced in their cross-experiments in the laboratory (proportion of hybrids: 0\u0026ndash;30%) (Sato \u003cem\u003eet al.\u003c/em\u003e, 2000a, 2000b, 2015, 2018). In this study, we carried out cross experiments using the populations collected from Mt. Amagi and confirmed that they produce hybrids, although the proportion of hybrids was slightly lower compared to those in previous studies (3\u0026ndash;13%). Therefore, we expected that hybrids would exist in the contact zone and that gene flow would occur between them. However, our genetic analysis did not identify obvious hybrids, and it was estimated that genetic introgression was extremely low (Figs. 5 and 6). This suggests that gene flow is strongly restricted by mechanisms that act later than post-mating pre-zygotic isolation. Because their hybrids are fertile in the laboratory (Sato, 2004), they may have problems surviving in nature. For example, adult female spider mites enter diapause and overwinter. Diapause attributes differ between the two species: \u003cem\u003eS. sabelisi\u003c/em\u003e takes a much longer time to emerge from diapause (high-intensity diapause), whereas the \u003cem\u003eS. miscanthi\u003c/em\u003e HG form emerges from diapause very quickly (low-intensity diapause) (Saito \u003cem\u003eet al.\u003c/em\u003e, 2002). The difference in diapause is considered the result of adaptation to colder and warmer regions in each species, and this difference contributes to maintaining\u0026nbsp;their parapatric distribution. The diapause attributes of their hybrids\u0026nbsp;have not\u0026nbsp;yet been investigated; however, if they are not able to enter diapause or emerge from diapause at a suitable time in the local season, they will be selected against from the field, even\u0026nbsp;though they are able to survive in the laboratory. It is also likely that hybrids have reproductive problems in nature. As described before, males of this species kill each other to establish their own harems, and only the victorious male can reproduce\u0026nbsp;(Saitō, 1990). In this study, we identified a deformed male in the contact zone. We do not know if the deformity was caused by hybridisation, but it is worth investigating the influence of hybridisation on\u0026nbsp;the morphology and fighting ability\u0026nbsp;of males. Therefore, to understand why gene flow is extremely restricted in the contact zone, it is necessary to investigate hybrid traits, such as diapause attributes, morphology, and behaviour.\u003c/p\u003e\n\u003cp\u003eThis field survey was conducted at the northern end of \u003cem\u003eS. miscanthi\u003c/em\u003e HG form distribution. It is common for populations at the edge of the distribution to have extremely low genetic diversity owing to bottlenecks and genetic drift. Because the genetic diversity of \u003cem\u003eS. miscanthi\u003c/em\u003e HG form populations has not been investigated in other regions, it is not possible to confirm whether the genetic diversity is lower in this area than in other areas. However, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form populations showed lower heterozygosity and nucleotide diversity than \u003cem\u003eS. sabelisi\u003c/em\u003e (Table S1)\u003cem\u003e.\u003c/em\u003e This may explain the low genetic diversity of \u003cem\u003eS. miscanthi\u003c/em\u003e HG form populations in this study. The low genetic diversity and harshness of the environment for \u003cem\u003eS. miscanthi\u003c/em\u003e HG form likely make it difficult to produce hybrids. Therefore, before concluding that gene flow is extremely restricted between them in their parapatric area, it is worth conducting this study on other mountains where both species are abundant.\u003c/p\u003e\n\u003cp\u003eAlthough not as much as expected, some negligible genetic introgression was detected between \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form (Fig. 6). As described previously, these two species have different distribution areas and male aggression levels: \u003cem\u003eS. sabelisi\u003c/em\u003e is distributed in colder regions and shows lower male aggression, whereas \u003cem\u003eS. miscanthi\u003c/em\u003e HG form is distributed in warmer regions and shows higher male aggression (Saito, 1995; Sato, Egas, \u003cem\u003eet al.\u003c/em\u003e, 2013). Male aggression varies among populations in each species, and the same relationship between winter coldness and male aggression was found in each species. \u003cem\u003eS. miscanthi\u003c/em\u003e HG form populations distributed in relatively colder regions showed lower male aggression compared to populations distributed in warmer regions, and the same clinal trend was found in \u003cem\u003eS. sabelisi\u003c/em\u003e populations (Saito and Sahara, 1999). Male aggression is genetically determined, as laboratory mite populations maintain similar aggression regardless of rearing temperature. To date, attempts have been made to explain the clinical trends of male aggression, both intra- and interspecies, through the application of kin selection theory (Saito, 1995; Saito and Sahara, 1999; Saito and Mori, 2005; Sato, Egas, \u003cem\u003eet al.\u003c/em\u003e, 2013). Severe winter colds can induce inbreeding in the spider mite species. Overwintered virgin females establish their spring nests by mating with the surrounding males; however, if there are no males, they produce haploid sons and mate with them for spring nest establishment. Whether males are around is affected by winter coldness because non-diapause males overwinter by chance if the winter coldness is mild (Saito, 1995; Sato, Egas, \u003cem\u003eet al.\u003c/em\u003e, 2013). As predicted by kin selection theory, male aggression is higher in populations where the average kinship in a colony is expected to be low from the spring nest establishment event, and vice versa. However, if genetic introgression affects male aggression, then a clinical trend in each species may be generated by genetic introgression. For example, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form populations distributed in relatively colder regions are expected to encounter \u003cem\u003eS. sabelisi\u003c/em\u003e much more frequently than those in warmer regions. Milder male aggression was likely caused by genetic introgression from \u003cem\u003eS. sabelisi\u003c/em\u003e. To test this hypothesis, it is necessary to investigate the inheritance of male aggression and genetic introgression status in other contact zones.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In this study, we revealed that \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form have broad contact zones throughout their parapatric area, and interspecific mating occurs in their contact zones; however, gene flow is strongly restricted between them. The contact zones of closely related species influence their evolution in various ways; they can cause species breakdown, promote diversification by character displacement and/or reinforcement of reproductive barriers, and generate new species by hybridisation (Coyne and Orr, 2004; Johannesson \u003cem\u003eet al.\u003c/em\u003e, 2020). In spider mites, contact zones are likely to contribute to diversification. In particular, reinforcement of reproductive isolation can occur when the fitness of a hybrid is low. The traits and fitness of their hybrids have not been well investigated; however, it is quite likely that hybrids have problems surviving and reproducing in nature, considering that we did not find obvious hybrids in their contact zones, despite incomplete reproductive isolation. Reinforcement of reproductive isolation in spider mites was suggested by a previous study, which found that post-mating, a pre-zygotic barrier evolved faster in \u003cem\u003eS. sabelisi\u003c/em\u003e females collected from parapatric areas than in allopatric areas (Sato \u003cem\u003eet al.\u003c/em\u003e, 2018). This tendency was not found in \u003cem\u003eS. miscanthi\u003c/em\u003e HG form females; however, it fits their behavioural differences (Sato \u003cem\u003eet al.\u003c/em\u003e, 2018). Specifically, males readily fight with different species of males for nests, \u003cem\u003eS. miscanthi\u003c/em\u003e HG form males tend to win interspecific male fights (Sato, Sabelis, \u003cem\u003eet al.\u003c/em\u003e, 2013), and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form males are active to mate regardless of female species much more than \u003cem\u003eS. sabelisi\u003c/em\u003e males (Sato \u003cem\u003eet al.\u003c/em\u003e, 2015). These behavioural differences suggest that \u003cem\u003eS. sabelisi\u003c/em\u003e females are likely exposed to the risk of reproductive interference by other male species, whereas \u003cem\u003eS. miscanthi\u003c/em\u003e HG form females are guarded by the same male species against such risk. We expect genetic analyses to provide insights into this hypothesis. However, in this study, we could not analyse the direction and details of genetic introgression because the gene flow was extremely low. As discussed before, their gene flow may be restricted by mechanisms acting later than post-mating pre-zygotic isolation. However, the amount of gene flow likely changes depending on the power relationships between \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form. In this study, genetic analyses were performed on Mt. Amagi, where \u003cem\u003eS. sabelisi\u003c/em\u003e was found to be superior. From this perspective, it would be worthwhile to conduct this study on other mountains.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Kentaro Nakano and Prof. Yooichi Kainoh at\u0026nbsp;the University of Tsukuba for providing the microscope for male morphology measurements and the space to cultivate the host plants of the mites. We thank Mr. Gomei Yoda, Ms.\u0026nbsp;Hisaho Kobatyashi, Mr. Taito Sano, Mr. Ryuto Uchiyama, Ms. Sayuka (Nagase) Nitta, Ms. Aina Yokoi,\u0026nbsp;and Ms. Ayana Tanino for their valuable suggestions and support. This research was supported in part by JSPS KAKENHI, Grant Number 20K06810 (Grant-in-Aid for Scientific Research C to Y. Sato) and by The Suzuki Takahisa Memorial Grant, the University of Tsukuba (to Y. Sato).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY. Sato conceived the study. Y. Sato, S. Konaka, Y. Tsumura, and Y. Suyama designed the study. S. Konaka, Y. Sato, and N. Matsumoto conducted the field surveys. S. Konaka\u0026nbsp;performed the measurements, Y. Sato and S. Konaka analysed of male morphology. S. Hirota, Y. Suyama, S. Konaka, Y. Sato, and Y. Tsumura performed molecular analyses. Y. Sato conducted the cross-experiment and analysed reproductive isolation. Y. Sato and S. Hirota wrote the first draft of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw reads of\u0026nbsp;the MIG-seq data\u0026nbsp;are available from the DDBJ Sequence Read Archive under BioProject ID PRJDB17427. The morphology, sex ratio, and cross-experimental\u0026nbsp;data are shown in\u0026nbsp;the\u0026nbsp;Supplementary Materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch Ethics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable, as the study was conducted on spider mites.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbbott R, Albach D, Ansell S, Arntzen JW, Baird SJE, Bierne N, \u003cem\u003eet al.\u003c/em\u003e (2013). Hybridization and speciation. \u003cem\u003eJournal of Evolutionary Biology\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e: 229\u0026ndash;246.\u003c/li\u003e\n \u003cli\u003eBolger AM, Lohse M, Usadel B (2014). Trimmomatic: a flexible trimmer for Illumina sequence data.\u0026nbsp;\u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e: 2114\u0026ndash;2120.\u003c/li\u003e\n \u003cli\u003eClark LV, Brummer JE, Głowacka K, Hall MC, Heo K, Peng J, \u003cem\u003eet al.\u003c/em\u003e (2014). A footprint of past climate change on the diversity and population structure of \u003cem\u003eMiscanthus sinensis\u003c/em\u003e. \u003cem\u003eAnn Bot\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e: 97\u0026ndash;107.\u003c/li\u003e\n \u003cli\u003eCoyne JA, Orr HA (2004). \u003cem\u003eSpeciation\u003c/em\u003e, 1st edition. Sinauer Associates, Inc.: Sunderland, Mass.\u003c/li\u003e\n \u003cli\u003eDobzhansky T (1959). \u003cem\u003eGenetics and the Origin of Species\u003c/em\u003e, Third Edition. Columbia University Press.\u003c/li\u003e\n \u003cli\u003eEarl DA, vonHoldt BM (2012). STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. \u003cem\u003eConservation Genet Resour\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e: 359\u0026ndash;361.\u003c/li\u003e\n \u003cli\u003eEdelman NB, Frandsen PB, Miyagi M, Clavijo B, Davey J, Dikow RB, \u003cem\u003eet al.\u003c/em\u003e (2019). Genomic architecture and introgression shape a butterfly radiation. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e366\u003c/strong\u003e: 594\u0026ndash;599.\u003c/li\u003e\n \u003cli\u003eHoskin CJ, Higgie M, McDonald KR, Moritz C (2005). Reinforcement drives rapid allopatric speciation. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e437\u003c/strong\u003e: 1353\u0026ndash;1356.\u003c/li\u003e\n \u003cli\u003eHothorn T, Bretz F, Westfall P (2008). Simultaneous inference in general parametric models. \u003cem\u003eBiom J\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e: 346\u0026ndash;363.\u003c/li\u003e\n \u003cli\u003eHuson DH, Bryant D (2006). Application of phylogenetic networks in evolutionary studies. \u003cem\u003eMol Biol Evol\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e: 254\u0026ndash;267.\u003c/li\u003e\n \u003cli\u003eJohannesson K, Le Moan A, Perini S, Andr\u0026eacute; C (2020). A Darwinian Laboratory of Multiple Contact Zones. \u003cem\u003eTrends in Ecology \u0026amp; Evolution\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e: 1021\u0026ndash;1036.\u003c/li\u003e\n \u003cli\u003eMallet J (2008). Hybridization, ecological races and the nature of species: empirical evidence for the ease of speciation. \u003cem\u003ePhilosophical Transactions of the Royal Society B: Biological Sciences\u003c/em\u003e \u003cstrong\u003e363\u003c/strong\u003e: 2971\u0026ndash;2986.\u003c/li\u003e\n \u003cli\u003ePfennig KS, Rice AM (2014). Reinforcement generates reproductive isolation between neighbouring conspecific populations of spadefoot toads. \u003cem\u003eProceedings of the Royal Society of London B: Biological Sciences\u003c/em\u003e \u003cstrong\u003e281\u003c/strong\u003e: 20140949.\u003c/li\u003e\n \u003cli\u003ePritchard JK, Stephens M, Donnelly P (2000). Inference of population structure using multilocus genotype data. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e155\u003c/strong\u003e: 945\u0026ndash;959.\u003c/li\u003e\n \u003cli\u003eR Core Team (2022). R: A language and environment for statistical computing. \u003cem\u003eR Foundation for \u0026nbsp; \u0026nbsp;Statistical Computing, Vienna, Austria\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eRieseberg LH (1997). Hybrid origins of plant species. \u003cem\u003eAnnual Review of Ecology and Systematics\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e: 359\u0026ndash;389.\u003c/li\u003e\n \u003cli\u003eRochette NC, Rivera-Col\u0026oacute;n AG, Catchen JM (2019). Stacks 2: Analytical methods for paired-end sequencing improve RADseq-based population genomics. \u003cem\u003eMolecular Ecology\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e: 4737\u0026ndash;4754.\u003c/li\u003e\n \u003cli\u003eSaitō Y (1986a). Prey kills predator: Counter-attack success of a spider mite against its specific phytoseiid predator. \u003cem\u003eExp Appl Acarol\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e: 47\u0026ndash;62.\u003c/li\u003e\n \u003cli\u003eSaitō Y (1986b). Biparental defence in a spider mite (Acari: Tetranychidae) infesting Sasa bamboo. \u003cem\u003eBehav Ecol Sociobiol\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e: 377\u0026ndash;386.\u003c/li\u003e\n \u003cli\u003eSaitō Y (1990). \u0026lsquo;Harem\u0026rsquo; and \u0026lsquo;non-harem\u0026rsquo; type mating systems in two species of subsocial spider mites (Acari, Tetranychidae). \u003cem\u003eRes Popul Ecol\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e: 263\u0026ndash;278.\u003c/li\u003e\n \u003cli\u003eSaito Y (1995). Clinal variation in male-to-male antagonism and weaponry in a subsocial mite. \u003cem\u003eEvolution\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e: 413\u0026ndash;417.\u003c/li\u003e\n \u003cli\u003eSaito Y (2009). \u003cem\u003ePlant Mites and Sociality: Diversity and Evolution\u003c/em\u003e. Springer Science \u0026amp; Business Media.\u003c/li\u003e\n \u003cli\u003eSaito Y, Chittenden AR, Kanazawa M (2011). Counterattack success of a social spider mite against two predominant phytoseiid predator species. \u003cem\u003eExp Appl Acarol\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e: 249.\u003c/li\u003e\n \u003cli\u003eSaito Y, Kanazawa M, Sato Y (2013). Life history differences between two forms of the social spider mite, \u003cem\u003eStigmaeopsis miscanthi\u003c/em\u003e. \u003cem\u003eExp Appl Acarol\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e: 313\u0026ndash;320.\u003c/li\u003e\n \u003cli\u003eSaito Y, Mori K (2005). Where does male-to-male \u0026ldquo;aggression\u0026rdquo; compromise \u0026ldquo;cooperation\u0026rdquo;? \u003cem\u003ePopul Ecol\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e: 159\u0026ndash;166.\u003c/li\u003e\n \u003cli\u003eSaito Y, Sahara K (1999). Two clinal trends in male-male aggressiveness in a subsocial spider mite (\u003cem\u003eSchizotetranychus miscanthi\u003c/em\u003e). \u003cem\u003eBehav Ecol Sociobiol\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e: 25\u0026ndash;29.\u003c/li\u003e\n \u003cli\u003eSaito Y, Sakagami T, Sahara K (2002). Differences in diapause attributes between two clinal forms distinguished by male-to-male aggression in a subsocial spider mite, \u003cem\u003eSchizotetranychus miscanthi\u003c/em\u003e Saito. \u003cem\u003eEcological Research\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e: 645\u0026ndash;653.\u003c/li\u003e\n \u003cli\u003eSaito Y, Sato Y, Chittenden AR, Lin J-Z, Zhang Y-X (2018). Description of two new species of \u003cem\u003eStigmaeopsis\u003c/em\u003e, Banks 1917 (Acari, Tetranychidae) inhabiting \u003cem\u003eMiscanthus\u003c/em\u003e grasses (Poaceae). \u003cem\u003eAcarologia\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e: 414\u0026ndash;429.\u003c/li\u003e\n \u003cli\u003eSaito Y, Sato Y, Kongchuensin M, Chao J-T, Sahara K (2019). New \u003cem\u003eStigmaeopsis\u003c/em\u003e species on \u003cem\u003eMiscanthus\u003c/em\u003e grasses in Taiwan and Thailand (Acari, Tetranychidae). \u003cem\u003eSystematic and Applied Acarology\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e: 675\u0026ndash;682.\u003c/li\u003e\n \u003cli\u003eSato Y (2004). Studies on variation in social structure and the maintenance mechanism associated with diversification in the \u003cem\u003eStigmaeopsis\u003c/em\u003e species group. In: \u003cem\u003eHokkaido University, Ph. D. thesis\u003c/em\u003e,, p 162.\u003c/li\u003e\n \u003cli\u003eSato Y, Breeuwer JAJ, Egas M, Sabelis MW (2015). Incomplete premating and postmating reproductive barriers between two parapatric populations of a social spider mite. \u003cem\u003eExp Appl Acarol\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e: 277\u0026ndash;291.\u003c/li\u003e\n \u003cli\u003eSato Y, Egas M, Sabelis MW, Mochizuki A (2013). Male\u0026ndash;male aggression peaks at intermediate relatedness in a social spider mite. \u003cem\u003eEcol Evol\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e: 2661\u0026ndash;2669.\u003c/li\u003e\n \u003cli\u003eSato Y, Sabelis MW, Mochizuki A (2013). Asymmetry in male lethal fight between parapatric forms of a social spider mite. \u003cem\u003eExp Appl Acarol\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e: 451\u0026ndash;461.\u003c/li\u003e\n \u003cli\u003eSato Y, Saito Y (2007). Can the extremely female-biased sex ratio of the social spider mites be explained by Hamilton\u0026rsquo;s local mate competition model? \u003cem\u003eEcological Entomology\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e: 597\u0026ndash;602.\u003c/li\u003e\n \u003cli\u003eSato Y, Saito Y, Chittenden AR (2008). The parapatric distribution and contact zone of two forms showing different male-to-male aggressiveness in a social spider mite, \u003cem\u003eStigmaeopsis miscanthi\u003c/em\u003e (Acari: Tetranychidae). \u003cem\u003eExp Appl Acarol\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e: 265\u0026ndash;276.\u003c/li\u003e\n \u003cli\u003eSato Y, Saito Y, Mori K (2000a). Reproductive isolation between populations showing different aggression in a subsocial spider mite, \u003cem\u003eSchizotetranychus miscanthi\u003c/em\u003e Saito (Acari: Tetranychidae). \u003cem\u003eApplied Entomology and Zoology\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e: 605\u0026ndash;610.\u003c/li\u003e\n \u003cli\u003eSato Y, Saito Y, Mori K (2000b). Patterns of reproductive isolation between two groups of Schizotetranychus miscanthi Saito (Acari: Tetranychidae) showing different male aggression traits. \u003cem\u003eApplied Entomology and Zoology\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e: 611\u0026ndash;618.\u003c/li\u003e\n \u003cli\u003eSato Y, Sakamoto H, Gotoh T, Saito Y, Chao J-T, Egas M, \u003cem\u003eet al.\u003c/em\u003e (2018). Patterns of reproductive isolation in a haplodiploid \u0026ndash; strong postmating, prezygotic barriers among three forms of a social spider mite. \u003cem\u003eJournal of Evolutionary Biology\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e: 866\u0026ndash;881.\u003c/li\u003e\n \u003cli\u003eSato Y, Tsuda Y, Sakamoto H, Egas M, Gotoh T, Saito Y, \u003cem\u003eet al.\u003c/em\u003e (2019). Phylogeography of lethal male fighting in a social spider mite. \u003cem\u003eEcology and Evolution\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e: 1590\u0026ndash;1602.\u003c/li\u003e\n \u003cli\u003eSchausberger P, Yano S, Sato Y (2021). Cooperative behaviors in group-Living spider mites. \u003cem\u003eFrontiers in Ecology and Evolution\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e: 745036.\u003c/li\u003e\n \u003cli\u003eSchneider CA, Rasband WS, Eliceiri KW (2012). NIH Image to ImageJ: 25 years of image analysis. \u003cem\u003eNat Methods\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e: 671\u0026ndash;675.\u003c/li\u003e\n \u003cli\u003eSeehausen O (2004). Hybridization and adaptive radiation. \u003cem\u003eTrends in Ecology \u0026amp; Evolution\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e: 198\u0026ndash;207.\u003c/li\u003e\n \u003cli\u003eSmadja C, Butlin R (2006). Speciation: A new role for reinforcement. \u003cem\u003eHeredity\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e: 422\u0026ndash;423.\u003c/li\u003e\n \u003cli\u003eSuyama Y, Hirota SK, Matsuo A, Tsunamoto Y, Mitsuyuki C, Shimura A, \u003cem\u003eet al.\u003c/em\u003e (2022). Complementary combination of multiplex high-throughput DNA sequencing for molecular phylogeny. \u003cem\u003eEcological Research\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e: 171\u0026ndash;181.\u003c/li\u003e\n \u003cli\u003eSuyama Y, Matsuki Y (2015). MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e: 16963.\u003c/li\u003e\n \u003cli\u003eVenables WN, Ripley BD (2002). \u003cem\u003eModern Applied Statistics with S\u003c/em\u003e, 4th edn. Springer-Verlag: New York.\u003c/li\u003e\n \u003cli\u003eYano J, Saito Y, Chittenden AR, Sato Y (2011). Variation in counterattack effect against a phytoseiid predator between two forms of the social spider mite, \u003cem\u003eStigmaeopsis miscanthi\u003c/em\u003e. \u003cem\u003eJ Ethol\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e: 337\u0026ndash;342.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u0026nbsp; Location where spider mites of \u003cem\u003eStigmaeopsis miscanthi\u003c/em\u003e species group were collected and the numbers of nests, females, males immatures, eggs and the male ratio collected from each location.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\" rowspan=\"2\"\u003e\n \u003cp\u003eLocation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" rowspan=\"2\"\u003e\n \u003cp\u003eLatitude\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" rowspan=\"2\"\u003e\n \u003cp\u003eLongitude\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" rowspan=\"2\"\u003e\n \u003cp\u003eAltitude (m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.053003533568905%\" colspan=\"5\"\u003e\n \u003cp\u003eNo. of\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\" rowspan=\"2\"\u003e\n \u003cp\u003eMale ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eNest\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eImmature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eEgg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eShiofuki\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.96863\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.12685\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e133\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.89088\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.13757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.84611\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.07361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e331\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.84833\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.06611\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.88358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.09616\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.85333\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.06028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.88972\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.08833\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.85750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.05889\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.88667\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.08917\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e290\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.90222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.10972\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.88806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.08306\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.85582\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.06557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e390\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e127\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.90583\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.07472\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.88917\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.07889\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e420\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.85806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.06444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e430\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.88583\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.07694\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.90639\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.06500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e460\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.88361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.07139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.86194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.05944\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e530\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.88139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.06639\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e560\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003eAmagi 20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e34.87944\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\" valign=\"top\"\u003e\n \u003cp\u003e139.05972\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.66077738515901%\"\u003e\n \u003cp\u003e680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.010600706713781%\"\u003e\n \u003cp\u003e94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.303886925795053%\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e The number of eggs laid in 10 days after female emergence, the survival rate from egg to adult, and the female ratio in interspecific crosses, intra-population crosses and virgin oviposition in the cross experiments using \u003cem\u003eS. sabelisi\u003c/em\u003e females (a) and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form females (b).\u003c/p\u003e\n\u003cp\u003e(a) Cross experiment of \u003cem\u003eS. sabelisi\u003c/em\u003e female\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.836448598130842%\" rowspan=\"2\"\u003e\n \u003cp\u003eCross type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.33177570093458%\" colspan=\"2\"\u003e\n \u003cp\u003eCross combination\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.957943925233645%\" rowspan=\"2\"\u003e\n \u003cp\u003eNo. of pairs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.485981308411215%\" colspan=\"3\"\u003e\n \u003cp\u003eNo. of eggs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.485981308411215%\" colspan=\"3\"\u003e\n \u003cp\u003eSurvival rate from egg to adult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.901869158878505%\" colspan=\"3\"\u003e\n \u003cp\u003eFemale ratio in offspring\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.90748898678414%\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.90748898678414%\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.195301027900147%\"\u003e\n \u003cp\u003e(Mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.258443465491924%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.901615271659325%\"\u003e\n \u003cp\u003eSE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.195301027900147%\"\u003e\n \u003cp\u003e(Mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.258443465491924%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.901615271659325%\"\u003e\n \u003cp\u003eSE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.195301027900147%\"\u003e\n \u003cp\u003e(Mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3773861967694567%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.901615271659325%\"\u003e\n \u003cp\u003eSE)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.784633294528522%\"\u003e\n \u003cp\u003eInterspecific\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003eAmagi 17\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS. sabelisi\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003eAmagi 1\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS. miscanthi\u003c/em\u003e HG form)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9371362048894065%\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e7.708\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.850\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.797\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.060\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.6775320139697323%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.784633294528522%\"\u003e\n \u003cp\u003eIntra-population\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003eAmagi 17\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS. sabelisi\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003eAmagi 17\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS. sabelisi\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9371362048894065%\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e8.444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.590\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.951\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.6775320139697323%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.784633294528522%\"\u003e\n \u003cp\u003eVirgin oviposition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003eAmagi 17\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS. sabelisi\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9371362048894065%\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e4.870\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.352\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.891\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.6775320139697323%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e(b) Cross experiment of \u003cem\u003eS. miscanthi\u003c/em\u003e HG form female\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.819136522753793%\" rowspan=\"2\"\u003e\n \u003cp\u003eCross type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.203033838973163%\"\u003e\n \u003cp\u003eCross combination\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.203033838973163%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.950991831971995%\" rowspan=\"2\"\u003e\n \u003cp\u003eNo. of pairs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.469078179696616%\" colspan=\"3\"\u003e\n \u003cp\u003eNo. of eggs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.469078179696616%\" colspan=\"3\"\u003e\n \u003cp\u003eSurvival rate from egg to adult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.885647607934656%\" colspan=\"3\"\u003e\n \u003cp\u003eFemale ratio in offspring\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.90748898678414%\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.90748898678414%\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.195301027900147%\"\u003e\n \u003cp\u003e(Mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.258443465491924%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.901615271659325%\"\u003e\n \u003cp\u003eSE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.195301027900147%\"\u003e\n \u003cp\u003e(Mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.258443465491924%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.901615271659325%\"\u003e\n \u003cp\u003eSE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.195301027900147%\"\u003e\n \u003cp\u003e(Mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3773861967694567%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.901615271659325%\"\u003e\n \u003cp\u003eSE)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.784633294528522%\"\u003e\n \u003cp\u003eInterspecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003eAmagi 1\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS. miscanthi\u0026nbsp;\u003c/em\u003eHG form)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003eAmagi 17\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS. sabelisi\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9371362048894065%\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e8.320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.725\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.773\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.6775320139697323%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.030\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.784633294528522%\"\u003e\n \u003cp\u003eIntra-population\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003eAmagi 1\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS. miscanthi\u0026nbsp;\u003c/em\u003eHG form)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003eAmagi 1\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS. miscanthi\u0026nbsp;\u003c/em\u003eHG form)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9371362048894065%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e7.818\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.737\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.820\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.6775320139697323%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.784633294528522%\"\u003e\n \u003cp\u003eVirgin oviposition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003eAmagi 1\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS. miscanthi\u0026nbsp;\u003c/em\u003eHG form)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.160651920838184%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9371362048894065%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e4.143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.416\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.3760186263096625%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.70430733410943%\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.6775320139697323%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.471478463329452%\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"heredity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"hdy","sideBox":"Learn more about [Heredity](http://www.nature.com/hdy/)","snPcode":"41437","submissionUrl":"https://mts-hdy.nature.com/cgi-bin/main.plex","title":"Heredity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3947675/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3947675/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHow frequently hybridisation and gene flow occur in the contact zones of diverging taxa is important for understanding the speciation process.\u003cem\u003eStigmaeopsis sabelisi \u003c/em\u003eand \u003cem\u003eStigmaeopsis miscanthi\u003c/em\u003e HG form are haplodiploid, social spider mites that infest the Chinese silver grass, \u003cem\u003eMiscanthus sinensis\u003c/em\u003e. These two species are closely related and parapatrically distributed in Japan. In mountainous areas, \u003cem\u003eS. sabelisi\u003c/em\u003e and \u003cem\u003eS. miscanthi\u003c/em\u003e HG form are often found in the highlands and lowlands, respectively, suggesting that they are in contact at intermediate altitudes. It is estimated that they diverged from their common ancestors distributed in subtropical regions (south of Japan) during the last glacial period, expanded their distribution into the Japanese Archipelago, and came to have such a parapatric distribution (secondary contact). As their reproductive isolation is strong but incomplete, hybridisation and genetic introgression are expected at their distributional boundaries. In this study, we investigated their spatial distribution patterns along the elevation on Mt. Amagi using male morphological differences and investigated their hybridisation status using single-nucleotide polymorphisms by MIG-seq. We found their contact zone at altitudes of 150–430 m, suggesting that their contact zone is prevalent in the parapatric area, which is in line with a previous study. Interspecific mating was predicted based on the sex ratio in the contact zone. However, no obvious hybrids were found, and genetic introgression was estimated to be extremely low. Here, we discuss why gene flow is extremely restricted to the contact zone.\u003c/p\u003e","manuscriptTitle":"Restricted hybridisation in the secondary contact zone of closely related haplodiploid social spider mites","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-26 06:14:25","doi":"10.21203/rs.3.rs-3947675/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-04-10T09:13:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-19T12:14:13+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-19T07:46:19+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-03-05T13:29:35+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-02-29T04:14:54+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-02-22T12:06:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-11T05:53:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Heredity","date":"2024-02-11T05:53:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"heredity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"hdy","sideBox":"Learn more about [Heredity](http://www.nature.com/hdy/)","snPcode":"41437","submissionUrl":"https://mts-hdy.nature.com/cgi-bin/main.plex","title":"Heredity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0e769f05-c5e1-4adf-b207-83214e44262b","owner":[],"postedDate":"February 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":28913856,"name":"Biological sciences/Evolution/Speciation"},{"id":28913857,"name":"Biological sciences/Ecology/Behavioural ecology"}],"tags":[],"updatedAt":"2024-07-16T11:56:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-26 06:14:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3947675","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3947675","identity":"rs-3947675","version":["v1"]},"buildId":"cTy_lsJlmDsVRNrSptgXS","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}