The Epilachna vigintioctomaculata complex

The Epilachna vigintioctomaculata complex differs greatly in external morphology and food plants in and around the Japanese Archipelago. This species complex has been the subject of intensive studies since 1940's by researchers of diverse fields of evolutionary biology, in particular those who have a specific interest in speciation (Watanabe and Sakagami, 1948; Ehara, 1952, 1953; Yasutomi, 1954; Iwao, 1959; Watanabe and Suzuki, 1965; Kuboki, 1978; Katakura, 1981a, 1988; Katakura et al., 1981, 1989; Nakano, 1981, 1985; Hoshikawa, 1983a, b, 1984; Tsurusaki et al., 1993; Katakura and Hosogai, 1994, 1997).

Based on various sorts of information covering morphology, food habits and their geographic variations, Katakura (1981a) classified the E. vigintioctomaculata complex into two groups and four species (Table 1, Fig. 1): group A, E. vigintioctomaculata Motschulsky; group B, E. pustulosa Kôno, E. niponica Lewis, E. yasutomii (Katakura). The separation of the species complex into two groups is based primarily on morphological evidence and the difference of host plants (Katakura, 1981a). Adults are discriminated by subtle but relatively stable differences in a number of characteristics such as elytral spot patterns, antennal segments and the genitalia of both sexes. Furthermore, group A occurs on solanaceous and cucurbitaceous plants while the members of group B usually occur on thistle (Cirsium spp.) and/or blue cohosh (Caulophyllum robustum). The dichotomy has recently been supported by karyological evidence (Tsurusaki et al., 1993) and analyses of mitochondrial DNA (N. Kobayashi et al., unpubl. data).

Table 1

Composition of the Epilachna vigintioctomaculata species complex, with geographic distribution and variation of component species

Fig. 1

Geographic distribution (A - C) and variation of elytral shape (D) (dorsal view for a, c, d - j; lateral view for d - i; markings on pronotum and elytra are omitted except the dorsal view of g) of the members of the Epilachna vigintioctomaculata complex (based on Katakura, 1988). (A) E. vigintioctomaculata (a - c); (B) E. pustulosa (d - g) and E. niponica (h - j); (C) E. yasutomii (the range of the so-called “Western Tokyo form” is shaded), a - c, V-I to V-III; e - g, P-I to III; d, P-III′; h - j, N-I to III.

On the other hand, recognition of the three species of group B is operational, and is mainly based on biological evidence and distributional information (Katakura, 1981a). Recent analyses of the cytochrome oxidaze I gene of mitochondrial DNA clarified that the three species are very closely related genetically (N. Kobayashi et al., unpubl. data). The difference between the populations of group B was 0.002 to 0.005 /bp, nearly equivalent to that found among the individuals of the same population. This result implies that the morphological and biological divergence of group B occurred rapidly over a very short geological time.

Life history and modes of reproduction

Various factors can act as intrinsic barriers to gene flow between sympatric taxa (Mayr, 1963; Dobzhansky, 1970; Littlejohn, 1981). These barriers are classified into those that operate before mating (=premating) and after mating (postmating). Since life history traits and modes of reproduction often function as important premating barriers, those of the members of the E. vigintioctomaculata complex are summarized here before describing the reproductive isolation of selected pairs.

1) Life cycle: All members of the E. vigintioctomaculata complex are univoltine and hibernate as adults (Katakura, 1981a). New adults emerge in mid summer to early fall, and enter hibernation by late fall in a litter in or near their habitats. Most overwintered adults die by summer but some enter a second hibernation (Nakamura and Ohgushi, 1979).

2) Oviposition and mating activities: Usually only overwintered females oviposit (Katakura, 1976). On the other hand, both males and females mate repeatedly during their lives (Nakano, 1987), from the summer of emergence to just before death (in most cases by the mid summer of the next year) with the intervention of winter dormancy. They usually mate on their host plants. More than half of newly emerging females of E. vigintioctomaculata and E. pustulosa mate before entering hibernation (Katakura, 1982).

3) Sperm storage and sperm longevity: After copulation, females keep spermatozoa in a pair of lateral swellings located at the common oviduct, a structure thus far known only in epilachnine beetles (Dobzhansky, 1924; Katakura, 1981b; Katakura et al., 1994). Spermatozoa kept by females are viable and fertile for a few months in laboratory conditions. In natural conditions, too, stored spermatozoa seem long-lived (Katakura, 1982).

Reproductive isolation between the two groups of the E. vigintioctomaculata complex

The two groups co-occur in most parts of Hokkaido and Honshu, usually on different host plants. Reproductive isolation has been studied in detail between E. vigintioctomaculata and E. pustulosa in Sapporo and the vicinity as a representative case of the sympatry of the two groups (Katakura, 1976, 1981a, 1982,1986a, b; Katakura and Nakano, 1979; Katakura and Sobu, 1986; Nakano, 1981, 1985, 1987; Tsurusaki et al., 1993). Various factors function as barriers to gene flow between the two species (Table 2).

Table 2

Effects of various factors (isolating mechanisms) on the reproductive isolation between E. vigintioctomaculata (Ev) and E pustulosa (Ep)

Table 3

A list of host plants (plants on which larvae complete the growth under natural conditions) and edible plants (plants occasionally eaten by adults or larvae) of E. vigintioctomaculata (Ev) and E. pustulosa (Ep) confirmed under natural conditions in the Sapporo area (modified from Katakura, 1981a)

1) Ecological and seasonal isolation: Epilachna vigintioctomaculata is known as a pest of potato plants. In Hokkaido, it also occurs on a wild cucurbit, Schizopepon bryoniaefolius (Katakura, 1975). On the other hand, E. pustulosa in the Sapporo area occurs mainly on thistle (mostly Cirsium kamtschaticum) and blue cohosh. This difference in the main “host plants”, here defined as the plants on which larvae complete their growth under natural conditions, reduces the gene flow between the two species to some extent. Furthermore, the difference of host plants seems to result in the phenological difference of the two ladybird beetle species (Katakura, 1976): posthibernating adults of E. pustulosa appear in late April to early May whereas those of E. vigintioctomaculata in late May to early June, coinciding with the sprouting of the respective host plants.

However, isolation by host specificity is not complete (Table 3; Katakura, 1981a). The main host plant of one species may be a subsidiary host plant or an edible plant (a plant occasionally eaten by adults or larvae but not used as a host) of the other species. Moreover, there are many plants, mostly cultivated plants and weeds, used as edible plants by both species. The coexistence of E. vigintioctomaculata and E. pustulosa is occasionally found on these plants.

2) Sexual isolation: When given choices between the two species of females under laboratory conditions, males tend to choose conspecifics (Katakura and Nakano, 1979). However, interspecific matings are not rare under such conditions. In natural conditions as well, occasional interspecific matings are known. In a mixed population of the two species which was found occurring on S. megacarpum in the vicinity of Sapporo, interspecific matings were rather common, totally 38.7% of the matings observed during 1978-1980 (N=223) (Nakano, 1987).

3) Gametic isolation: The slight difference in genitalia between the two species (Katakura, 1981a) does not function as a barrier to gene flow. Insemination is successful and normal in interspecific matings. However, approximately 75 - 90% of spermatozoa inseminated in heterospecific females are lost in the course of sperm migration from the bursa copulatrix to the sperm storage organ (Katakura, 1986a), possibly owing to the incompatibility between the spermatozoa and genital tract of the females.

The spermatozoa kept in the sperm reservoir of heterospecific females are motile, although they often appear to be somewhat anomalous compared with those in conspecific females (Katakura, 1986a).

4) Low hatching rates of eggs produced by hybridization: Hatching rates of eggs produced by interspecific matings are much lower than those by intraspecific matings, though results are variable according to experiments (Katakura and Nakano, 1979; Nakano, 1985; Katakura, 1986a; Katakura and Sobu, 1986). Some examples are given in Table 4. This low hatchability is caused by the failure of fertilization and death of hybrid embryos (Fig. 2; Katakura and Sobu, 1986).

Table 4

Hatching rates of eggs (%, mean ± SD, the number of replicates in parentheses) produced by various combinations of matings involving the two groups of the E vigintioctomaculata complex. Group A: V, E. vigintioctomaculata. Group B: P, E. pustulosa; N, E. niponica; Y, E yasutomii; WT, “Western Tokyo form Epilachna” (= E. yasutomii).

Fig. 2

The proportion of eggs that hatched, died during embryonic stages, or remained unfertilized in four combinations of crosses between E. vigintioctomaculata and E. pustulosa (based on Katakura and Sobu, 1986). Combinations of mating: VV, vigintioctomaculata females and vigintioctomaculata males; PP, pustulosa females and pustulosa males; VP, vigintioctomaculata females and pustulosa males; PV, pustulosa females and vigintioctomaculata males. Single: females were kept separate and alone after one mating. Pair: females remained paired with males, permitting repeated matings.

The failure of fertilization can be explained by the gametic isilation mentioned above. Spermatozoa kept by heterospecific females may be too few to fertilize every egg, or they may have suffered a reduction of fertility in the genital tract of the females.

On the other hand, at least three explanations are possible for the death of hybrid embryos. First, the substantially high mortality of hybrid embryos may involve the death due to direct interaction of incompatible gene systems coexisted in the hybrid individual, a set of genes from the mother and the other from the father (Dobzhansky, 1970). Secondly, the death of hybrid embryos may be a delayed effect of gametic isolation (Katakura, 1986a; Katakura and Sobu, 1986). It is obvious that the spermatozoa kept in the sperm reservoir of heterospecific females retain fertility to some extent. However, if they are physiologically or genetically anomalous due to the incompatibility with the genital tract of the females, this may result in the death of embryos. Thirdly, the lethality in hybrid embryos may be attributable to a discordance between the rate of cell division and that of chromosome replication during early embryogenesis (Tsurusaki et al., 1993). This idea is derived from the fact that there is a quantitative difference in karyotypes between groups A and B (Tsurusaki et al., 1993). The group B karyotype is characterized by a larger amount of heterochromatic segments on short arms of certain chromosomes than the group A karyotype.

Meanwhile, there is no effective isolating mechanism that acts after F₁ hybrids hatch. The F₁ larvae grow normally to become fully viable and fertile adults under laboratory conditions (Katakura and Nakano, 1979). Furthermore, the hatching rates of eggs laid by back crossings or crossings between F₁ hybrids are on the average higher than those by the interspecific matings (Nakano, 1981). Thus, reproductive isolation between E. vigintioctomaculata and E. pustulosa is attained by a combination of several kinds of barriers acting before hatching.

This pattern seems to be true for reproductive isolation between group A and group B in general. Except for “Western Tokyo form Epilachna” (=E. yasutomii), a pest of potato plants, host plants of the members of group B are different from those of group A. Furthermore, the hatching rates of eggs produced by crossings between groups A and B are consistently low (Table 4; Nakano, 1987; Tomioka, 1988; Katakura, unpubl.).

Putative hybrids between the two groups, identified on a morphological basis, are occasionally documented in natural conditions where the partial overlap of food plants violates ecological and phenological isolations (Nakano, 1987; Katakura, 1988). Furthermore, a contact zone is known in the Kanto district, middle Honshu, where E. vigintioctomaculata meets the “Western Tokyo form”. The contact zone is narrow and hybrids are very rare, although the occurrence of F₁ and later filial hybrids was ascertained by analyzing allozymes (MDH and Malic enzymes) (Nakano, 1987; Takita and Katakura, unpubl.; Fig. 3). This fact indicates that host specificity is not a crucial mating barrier between group A and group B, and that the postmating barriers are more responsible for maintaining the morphological and biological integrity of the two groups in sympatry. This suggests an allopatric origin of the two groups, since postmating barriers are supposed to evolve as byproducts of genetic differentiation during isolation by extrinsic barriers (Coyne, 1992).

Fig. 3

The contact zone of E. vigintioctomaculata (hatched) and “Western Tokyo form Epilachna” (open) in the western part of the Kanto district, central Honshu (drawn based on unpublished data by H. Takita and Katakura). Occurrence of hybrids, confirmed by allozyme analyses, is designated by arrows (three populations).

Conspecific sperm precedence and reproductive isolation

One noteworthy aspect derived from the study of reproductive isolation between the two groups of the Epilachna vigintioctomaculata complex is the role of conspecific sperm precedence as a reproductive barrier.

As already shown, the hatching rates of eggs produced by the interspecific matings of E. vigintioctomaculata and E. pustulosa are extremely low (Fig. 2). However, a low hatching rate due to an interspecific mating rose close to that of a conspecific mating immediately after a subsequent mating with a conspecific, and a high hatching rate by a preceding conspecific mating did not significantly drop after a succeeding interspecific mating (Table 5; Nakano, 1985). Moreover, almost all the progeny obtained from these doubly mated females were fathered by conspecific males (Table 6; Nakano, 1985). A subsequent study also confirmed very strong conspecific sperm precedence in this species pair (Katakura, 1986b).

Table 5

Average hatching rates of eggs laid by doubly-mated females of E. vigintioctomaculata and E. pustulosa before and after the second mating (M2). Each female mates successively with a conspecific male (C) and a heterospecific male (H) (Nakano, 1985).

Table 6

Frequency of hybrids produced by doubly mated females of E vigintioctomaculata and E. pustulosa. Each female mated successively with a conspecific male (C) and a heterospecific male (H) (Nakano, 1985).

Under such conditions, the number of offspring of interspecifically mated females must be greatly affected by their mating experience before and after interspecific matings, and females would suffer little reduction of the number of offspring from interspecific matings when they mate at least once with conspecifics before oviposition (Nakano, 1985). Indeed, the hatchability of eggs laid by females which had mated with heterospecifics in the field was not always low, and most of the offspring showed the mother's phenotypes (Nakano, 1987). Thus, available evidence strongly suggests that conspecific sperm precedence functions as a post insemination barrier between the two groups of the E. vigintioctomaculata complex.

Furthermore, conspecific sperm precedence may prevent reinforcement of premating isolation (Nakano, 1985, 1987; Katakura, 1986b). Theoretically, premating reproductive isolation can evolve at the zone of the contact of two incipient species at the expense of less fitted hybrid progeny (Dobzhansky, 1940). However, how common the reinforcement is in nature is still controversial (Butlin, 1987,1989; Coyne and Orr, 1989; Howard, 1993). Studies of Epilachna beetles suggest that individuals may not suffer a reduction of fitness from interspecific matings owing to the strong conspecific sperm precedence (Nakano, 1985; Katakura, 1986b). This means that under certain conditions, selection for the reinforcement of premating isolation may be more relaxed than expected from the unfitness of hybrids.

Apparently, however, the effect of conspecific sperm precedence as a reproductive barrier is restrictive. Strong conspecific sperm precedence would serve most effective as a reproductive barrier when two species are equally abundant and females mate often (Gregory and Howard, 1994; Katakura, 1986b). Under such circumstances, a female is likely to mate at least once with a conspecific and therefore her eggs will show a normal level of hatchability. The female would suffer little reduction of fitness from interspecific mating. On the other hand, as the abundance of a species decreases, so too does the chance of a female of a species encountering and mating with a conspecific. The degree of conspecific sperm precedence itself also restricts its effectiveness as a reproductive barrier (Katakura, 1986b).

Thus, the general significance of conspecific sperm precedence in reproductive isolation is not yet fully understood. However, accumulating evidence suggests that reproductive isolation at the gametic level may be a common phenomenon in closely related animal taxa (Howard and Gregory, 1993; Gregory and Howard, 1994; Albuquerque et al., 1996).

Reproductive isolation between E. niponica and E. yasutomii

Among the three species of group B, E. yasutomii and E. niponica are sympatric from the southern part of Hokkaido to middle Honshu, occurring on different plants and having different morphology (Fig. 1, Table 1). Epilachna niponica is consistently and considerably larger (Fig. 4) and occurs on thistle, whereas E. yasutomii is smaller and occurs on blue cohosh and some subsidiary host plants. Furthermore, there are distinct differences in the elytral shape between the two sympatric species (Katakura, 1981a), and the differences are not attributable to allometry due to size difference.

Fig. 4

Epilachna niponica (left) and E. yasutomii (right) from Ohnuma, southern Hokkaido (same scale). Both individuals are female. Note the difference in body size and elytral shape.

Reproductive isolation between these two species has been most intensively studied using populations from Ohnuma, southern Hokkaido (Katakura et al., 1981, 1989; Katakura and Hosogai, 1994, 1997). Unlike E. vigintioctomaculata and E. pustulosa mentioned above, E. niponica and E. yasutomii in Ohnuma are isolated from each other by a strong premating barrier, their fidelity to different host plants (Table 7).

Table 7

Effects of various factors (isolating mechanisms) on the reproductive isolation between E niponica (En) and E yasutomii (Ey)

In Ohnuma, E. niponica occurs on thistle (most cases Cirsium alpicola) growing in forest-edge habitats and around marshes, whereas E. yasutomii occurs on blue cohosh on shaded forest floors. Epilachna yasutomii is also found feeding on S. bryoniaefolius in forest-edge habitats in the late summer to fall when blue cohosh withers. Coexistence of the two species on the same host species has not been documented. When given choices under laboratory conditions, adult beetles of either species prefer their own host plant to the host plant of other species (Table 8; Katakura et al., 1989; Katakura and Hosogai, 1997). Furthermore, larvae are difficult to grow on the host plant of the other species (Fig. 5; Katakura et al., 1989; Katakura and Hosogai, 1994). Since Epilachna beetles usually mate on the host plants, the observed strict host specificity must function as a very strong premating barrier for gene exchange between E. niponica and E. yasutomii.

Table 8

Food preference of newly emerged adults of Epilachna niponica, E. yasutomii and their F₁ hybrids for three plant species: blue cohosh (BC, host plant of E. yasutomii), thistle (TH, host plant of E. niponica) and Solanum japonense (NS, a plant preferred by both species in the laboratory)

Fig. 5

Rate of survival for Epilachna niponica, E. yasutomii and their hybrids on thistle (host of E. niponica) and blue cohosh (host of E. yasutomii) (based on Katakura and Hosogai, 1994). Percentage survival to the second instar (left) and that to adulthood (right) are given separately. Offspring of a single female were divided into two groups and reared on different plants. Each dot represents the survival rates of offspring from a single female.

On the other hand, no evidence for seasonal isolation, sexual isolation, gametic isolation, or reduced hybrid viability or fertility is known (Katakura et al., 1981, 1989). Currently available data suggest that the active periods of adults of the two species overlap from early May through early September at Ohnuma. Furthermore, in spite of their difference in body size, E. niponica and E. yasutomii readily mate with each other under laboratory conditions without showing a particular preference for conspecifics, and produce normally viable and fertile hybrid offspring (Katakura et al., 1981). The F₁ hybrid larvae can grow on both thistle and blue cohosh, and the F₁ adults easily accept both plants (Katakura and Hosogai, 1994, 1997).

At a glance, such reproductive isolation which is totally dependent on host fidelity appears to be very fragile. Although habitats of thistle and blue cohosh are different, the two host plant species occasionally grow side by side in ecotones. Given such circumstances, there is always the potential for interspecific mating, should errors in host choice occur. As there are no effective postmating barriers, occasional hybridization, if it occurs, should obscure the distinctness between the species by subsequent gene flow. However, this is apparently not the case. The two species are sympatric and keep their morphological and biological distinctness from southern Hokkaido to central Honshu (Katakura, 1981a). One explanation for this is that host selection is sufficiently strict and completely prevents hybridization of the two species under natural conditions. Another explanation is that hybrids are produced but, because they are considerably less adaptive than their parents in some ecological properties, for example, in the phenological correspondence with host plants, resistance to natural enemies, or competitive ability to exploit host plants, they are selected against.

Thus, although host preference seems to act as a very strong and probably unique premating barrier to gene flow between E. niponica and E. yasutomii, it is still uncertain whether occasional hybridization occurs or not under natural conditions. For a thorough understanding of reproductive isolation between the two species, we need to know much more about the various aspects of the life histories of the beetles and their host plants, such as demography, phenological relationship with the host plants, and the density, patchiness and distribution pattern of the host plants.

The speciation process of E. niponica and E. yasutomii is also not clear. Some authors believe that speciation in phytophagous insects can occur without geographic isolation (Bush, 1969, 1975, 1994; Tauber and Tauber, 1989), and the members of the E. vigintioctomaculata complex are among such candidates for sympatric speciation (Diehl and Bush, 1984; Bush and Howard, 1986; Tauber and Tauber, 1989). The above-mentioned mode of reproductive isolation between E. niponica and E. yasutomii satisfies certain conditions necessary for some models of sympatric speciation via a host shift (e.g. Bush, 1974; Diehl and Bush, 1989). Again, however, the critical point, a reduction of hybrid fitness has not yet been demonstrated. Furthermore, little is known about the genetic basis of host specificity in these beetles (but see Ueno et al., 1997).

The relationship among the three species of group B

As mentioned above, E. niponica and E. yasutomii are distinct biological entities that warrant treatment as different biological species, even though their reproductive isolation is attained by host fidelity alone. However, the relationship between either of the two species and E. pustulosa, their northern counterpart, is enigmatic. Epitachna pustulosa is distributed in Hokkaido except for the southern and eastern parts, and is allopatric with both E. niponica and E. yasutomii (Fig. 1). In the Oshima Peninsula, southern Hokkaido, E. pustulosa and the two southern species (E. niponica and E. yasutomii) are separated by a narrow zone not occupied by any members of group B (Fig. 1). Body size of E. pustulosa is, on average, intermediate between E. niponica and E. yasutomii (Katakura, 1981a). Furthermore, the elytral shape cannot be used as a reliable character that separates the northerly distributed E. pustulosa from southerly distributed E. niponica and E. yasutomii, since there is considerable geographic variation in the elytral shape of both E. niponica and E. pustulosa (Fig. 1).

Crossing experiments revealed that E. pustulosa and the other two species produced fully viable and fertile hybrids (Katakura et al., 1981). Moreover, there seems to be no effective premating barrier between E. niponica and E. pustulosa or between E. yasutomii and E. pustulosa, because the so-called Sapporo form of E. pustulosa (form P-lll in Fig. 1) distributed just north of E. niponica and E. yasutomii occurs on both thistle and blue cohosh (Katakura, 1976; Kimura and Katakura, 1986). Indeed, host preference and the performance of the Sapporo form of E. pustulosa under laboratory conditions are very similar to those of the F₁ hybrids of E. niponica and E. yasutomii (Table 9; Fig. 6; Katakura, unpubl.). These lines of evidence indicate that E. pustulosa cannot be discriminated from either E. niponica or E. yasutomii. Epilachna pustulosa is “conspecific” with both E. niponica and E. yasutomii, nevertheless the latter two are “heterospecific”. In other words, group B behaves as one species in the north but as two separate species in the south. The situation is further complicated by the fact that the preference of E. pustulosa for blue cohosh decreases in the northern parts of the distribution range (Hoshikawa, 1984). The recognition of the three species in group B is therefore operational, since such a complex situation cannot be adequately treated by conventional taxonomy.

Table 9

Food preference of posthibernating adults of E. pustulosa (Sapporo form) collected on thistle and blue cohosh

Fig. 6

Rate of survival for Epilachna pustulosa (Sapporo form) on thistle and blue cohosh. Offspring of females collected on thistle and blue cohosh are shown by open and solid circles, respectively. For further explanation, see Fig. 5 (Katakura, unpubl. data).

At the present, it is not obvious how such a situation evolved. Group B might be in the process of gradual splitting, assuming that the Sapporo form of E. pustulosa is an ongoing sympatrically speciating population. Alternatively, it might be in the process of convergence owing to hybridization at the zone of secondary overlap following allopatric divergence. On the other hand, there might be a very complex situation involving partial divergence and partial hybridization.

Conclusion

The mode of reproductive isolation is diverse among the members of the Epilachna vigintioctomaculata complex as summarized above. Several factors function as barriers to gene flow between group A and group B. Postmating-prezygotic barriers, thus far little studied in animals, are considered to be of primary importance to maintaining the distinctness of members of the two groups in sympathy.

On the other hand, E. niponica and E. yasutomii, two sympatric species of group B, are reproductively isolated from each other by a single premating barrier, fidelity to different host plants. Their relationship is, however, complicated by the occurrence of an allopatric third “species”, E. pustulosa, that shows an intermediate host specificity and presumably lacks any potential barriers to gene exchange with either E. niponica or E. yasutomii.

These findings suggest that the mode of speciation may also be diverse among the E. vigintioctomaculata complex. For groups A and B, an allopatric origin is more likely, but for the members within group B, an origin without geographic isolation is also plausible.

Attempts are being made to clarify the phylogenetic relationships of the members of the E. vigintioctomaculata complex (N. Kobayashi et al., unpubl.), extent of gene flow between sympatric taxa, genetic basis of host specificity (Ueno et al., 1997; Ueno, unpubl.), and extrinsic and intrinsic factors responsible for the process of host selection in natural conditions (Koizumi et al., 1997, unpubl.; Fujiyama and Katakura, 1997; Fujiyama, unpubl.). These studies will greatly enrich our knowledge of the evolutionary history of the E. vigintioctomaculata complex, and will contribute to better understanding of animal species and their origin.