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.
TheStigmaeopsis 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.