Yasuoki Takami

Genetic diversity and structure of urban populations of Pieris butterflies assessed using amplified fragment length polymorphism.

Conservation programs in urban ecosystems need to determine the genetic background in populations of urban dwellers. We examined the genetic diversity and structure of Pieris rapae and P. melete using AFLP markers, and compared them between species and between urban and rural environments. As a result: (i) in both species, there was no reduction in genetic diversity within urban populations by direct comparison of diversity measurements, although the analysis of molecular variance suggested significant reductions in the variance within seasonal subpopulations in urban populations; (ii) P. rapae retained greater genetic diversity within species and populations; (iii) populations of both species showed significant genetic differentiation, and P. melete was more strongly subdivided; (iv) in both species, geographically close populations did not cluster with one another in the upgma analysis; (v) there was no genetic isolation due to geographical distance in either species; (vi) the genetic composition of seasonal subpopulations differed in urban populations of both species, and the genetic distances among subpopulations were correlated with seasonal differences in P. rapae and with temporal differences in P. melete. These results indicate that the genetic diversity in urban populations of both species was reduced at times, but was maintained by dispersal from genetically differentiated populations. Differences in the ability and mode of dispersal in the two species may be reflected in the degree of population subdivision and patterns of seasonal change in the genetic composition.

Interspecific body size differentiation in species assemblages of the carabid subgenus Ohomopterus in Japan

Abstract Geographic variation and interspecific differentiation in body size (body length) were analyzed for 15 species of the carabid subgenus Ohomopterus (genus Carabus; Coleoptera, Carabidae) in Japan. Local species assemblages of this subgenus consist of up to 5 species of different size classes. These beetles exhibited sexual dimorphism in body size where females are larger than males, except Carabus uenoi, in which the male and female sizes were equivalent, possibly because of the exaggerated male genitalia. In 9 of 15 species, there was a positive correlation between mean body size and annual mean temperature of habitat, representing the converse of Bergmanns rule. However, in some cases this correlation does not hold over the range of a species because of regional differences. When allopatric and sympatric populations were compared, allopatric populations of Carabus albrechti and C. japonicus had larger bodies than sympatric populations. These intraspecific differences may have resulted from character displacement. In each local assemblage with 2 or more species, there was little interspecific overlap of body size, although the body size ratio between two species with adjacent body sizes seldom showed strict constancy. The mean size ratio between 2 adjacent species in an assemblage was reduced with the number of species, whereas the size ratio of the largest to smallest species in an assemblage increased with the number of species (i.e., the expansion of body size range). These results indicate that the body size of Ohomopterus species may have evolved in response to both climatic conditions and interspecific interactions. Because each species or species group represents the same size class over the distribution range and similar-sized species are parapatric or allopatric, the interspecific segregation in body size in local assemblages may have resulted mainly from a size assortment process during colonization.

Experimental analysis of the effect of genital morphology on insemination success in the ground beetle Carabus insulicola (Coleoptera Carabidae)

The hypothesis that the elaborated genitalia of male insects serve to improve insemination success were tested using the ground beetle Carabus insulicola. To enhance variation in genital size, the genital hooks of experimental males were cut, and these males were then mated with virgin females. Logistic regression showed that the length of the male genital hook affected insemination success. Males with a shorter genital hook tended not to deposit spermatophores at the proper site, and failed to transfer sperm into the spermatheca. Therefore, the male genital hook serves to increase insemination success by depositing a spermatophore at the site where sperm are likely to be transferred. The duration of copulation and post-copulatory guarding may also be explained by these determinants. Stepwise regressions indicated that the occurrence of ejaculation, and the location of the spermatophore determined the duration of copulation and post-copulatory guarding, respectively.

Flower orientation enhances pollen transfer in bilaterally symmetrical flowers

Zygomorphic flowers are usually more complex than actinomorphic flowers and are more likely to be visited by specialized pollinators. Complex zygomorphic flowers tend to be oriented horizontally. It is hypothesized that a horizontal flower orientation ensures effective pollen transfer by facilitating pollinator recognition (the recognition-facilitation hypothesis) and/or pollinator landing (the landing-control hypothesis). To examine these two hypotheses, we altered the angle of Commelina communis flowers and examined the efficiency of pollen transfer, as well as the behavior of their visitors. We exposed unmanipulated (horizontal-), upward-, and downward-facing flowers to syrphid flies (mostly Episyrphus balteatus), which are natural visitors to C. communis. The frequency of pollinator approaches and landings, as well as the amount of pollen deposited by E. balteatus, decreased for the downward-facing flowers, supporting both hypotheses. The upward-facing flowers received the same numbers of approaches and landings as the unmanipulated flowers, but experienced more illegitimate landings. In addition, the visitors failed to touch the stigmas or anthers on the upward-facing flowers, leading to reduced pollen export and receipt, and supporting the landing-control hypothesis. Collectively, our data suggested that the horizontal orientation of zygomorphic flowers enhances pollen transfer by both facilitating pollinator recognition and controlling pollinator landing position. These findings suggest that zygomorphic flowers which deviate from a horizontal orientation may have lower fitness because of decreased pollen transfer.

Mating Behavior, Insemination and Sperm Transfer in the Ground Beetle Carabus insulicola

Abstract Mating behavior and the processes of insemination and sperm transfer in the ground beetle Carabus insulicola were analyzed. C. insulicola has elaborate genitalia, in which the strongly sclerotized male copulatory piece is inserted into the female vaginal appendix in copula. During mating, I observed pre-copulatory struggles of males and females, as well as delays in ejaculation, suggesting the presence of intersexual conflicts. Insemination was achieved with a spermatophore, which strongly adhered to the openings of the spermatheca, common oviduct, and vaginal appendix. The spermatophore dissolved after copulation, and sperm were transferred into the spermatheca within three hours after copulation. Sperm bundles were contained within the testes and spermatophores, but free spermatozoa were found in the spermatheca.

Resource partitioning or reproductive isolation: the ecological role of body size differences among closely related species in sympatry.

1. Body size differences among coexisting related species are common, but the actual effect of these differences in mitigating interspecific interactions, such as resource competition and reproductive interference, is poorly understood. 2. Local assemblages of the ground beetle genus Carabus (subgenus Ohomopterus) typically consist of two or more species of varying sizes. Through foraging and mating experiments using four Ohomopterus species in parapatry and sympatry, we examined whether interspecific body size differences are effective in partitioning food resources or reducing reproductive interference. 3. Because larval Ohomopterus feed exclusively on earthworms, body size differences may be related to partitioning earthworms of different sizes. However, larvae did not exhibit differences in selectivity or attack success on earthworms of different sizes based on larval body size, indicating little possibility of partitioning food by body size. 4. In contrast, interspecific mating behaviours, such as mate recognition, mounting, and copulation, were hindered when body size differences were large; copulation was frequently accomplished between parapatric species with smaller body size differences. 5. These results suggest that body size differences between species effectively reduce reproductive interference, rather than resource competition. Although body size differences in coexisting closely related species have been considered to function in resource partitioning, they may function primarily in reproductive isolation and thereby facilitate coexistence of species.

Rapid diversification of male genitalia and mating strategies in Ohomopterus ground beetles.

We analysed evolutionary diversification and covariation in male genitalia and four mating traits related to sexual selection, i.e. testis size, spermatophore size, copulation duration and post‐copulatory guarding duration, in Ohomopterus ground beetles using phylogenetically independent contrasts. Male genital size and mating duration have evolved more rapidly than body size and the other traits studied. Male genital size was negatively correlated with copulation duration, suggesting that elongated male genitalia may enable decreased time investment in a single copulation because it is more effective at facilitating spermatophore deposition. Male genital size was positively correlated with spermatophore size, suggesting coevolution between offensive and defensive male mating tactics because the elongated male genitalia may be advantageous in displacement of rivals’ plug‐like spermatophores, and decreased mating duration may intensify sperm competition. Thus, the remarkable diversity of male genitalia in Ohomopterus may have been facilitated by the interplay between inter‐ and intrasexual selection processes.

Species status and phylogeography of two closely related Coptolabrus species (Coleoptera: Carabidae) in South Korea inferred from mitochondrial and nuclear gene sequences

We investigated the species status and intraspecific phylogeography in South Korea of two ground beetle species, Coptolabrus jankowskii and Coptolabrus smaragdinus (Coleoptera: Carabidae), using statistical parsimony networks and nested clade analyses based on sequences from the mitochondrial cytochrome oxidase subunit I (COI) and nuclear phosphoenolpyruvate carboxykinase (PepCK) and wingless (Wg) genes. Although traditional parsimony tree construction generally failed to resolve interspecific relationships and construct biologically meaningful genealogies, analysis using statistical parsimony networks yielded statistically significant inter‐ and intraspecific genealogical structures. We found that although these two species represent a notable case of trans‐species polymorphisms in both mitochondrial and nuclear gene sequences, their status as separate species was evidenced by the nonrandom association between species and nested clades at various nesting levels. The exceptional occurrence of shared identical or very similar COI sequences was considered to be the result of introgressive hybridization. In addition, range expansion and fragmentation events across the Korean Peninsula and adjacent islands were inferred from nested clade phylogeographical analyses. The COI gene revealed the geographical divergence of major eastern and western clades and historical biogeographical events within each major clade, whereas the nuclear PepCK gene, which did not reveal corresponding east–west clades, indicated past fragmentation and range expansion across wide areas that may have been the result of older biogeographical events. Thus, phylogeographical inferences drawn from analyses of mitochondrial and nuclear genes can reveal different and potentially complementary information about phylogeographical processes.

QTL for the species‐specific male and female genital morphologies in Ohomopterus ground beetles

Animals with internal fertilization often exhibit marked diversification in genital morphology among closely related species. However, our knowledge of the genetic architecture underlying genital evolution is still limited. We constructed genetic linkage maps and analysed quantitative trait loci (QTL) for F2 hybrids of two closely related species of the carabid beetles Carabus (Ohomopterus) iwawakianus and C. (O.) maiyasanus, which show matching male and female genital shapes within species, but marked differences in genital morphologies between species. The linkage maps comprised both amplified fragment length polymorphism and microsatellite markers. Composite interval mapping to detect QTL for three traits of male copulatory piece (length, width, weight) and two traits for female vaginal appendix (length, width) resulted in the detection of one to five significant QTL for each trait. The QTL explained large proportions of phenotypic variance. Thus, the interspecific difference in the genital morphologies appeared to be determined by relatively small numbers of genes with large genetic effects. QTL of different traits for the same or different sexes co‐occurred on five of eight linkage groups with significant QTL; in particular, three QTL for different male and female genital traits occurred almost at the same position. Each of the male genital traits showed uniform signs of additive genetic effects, suggesting that directional selection has led to species‐specific morphologies. However, the signs of additive genetic effects in each female genital trait were not uniform, suggesting that coevolution between sexes is not necessarily concerted. This result requires further assessment because the sample size of F2 females was small.

Genetic differences and phenotypic plasticity in body size between high‐ and low‐altitude populations of the ground beetle Carabus tosanus

The body size of a univoltine carabid beetle Carabus tosanus on Shikoku Island, Japan, was clearly smaller in higher‐altitude populations (subspecies), which possibly represents incipient speciation. To explore the determinants of altitudinal differences in body size in this species, we studied the degree of phenotypic plasticity by conducting rearing experiments at two constant temperatures and examined genetic differences through interpopulation crosses. At 15 °C, C. tosanus had a longer developmental period and a shorter adult body than at 20 °C. Nevertheless, variation in body size due to temperature effects (phenotypic plasticity) was small compared to the interpopulation differences, which suggests substantial genetic differences between populations (subspecies) at different altitudes. In F1 offspring from crosses between a low‐altitude (subspecies tosanus) and a high‐altitude population (subspecies ishizuchianus), adult body length was affected by the genotypes of both parents, with an interaction effect of parental genotype and offspring sex. Further analyses revealed that adult body length was affected by sex‐linked factors in addition to autosomal factors. These genetic differences in body size may have resulted from adaptations to different altitudes and may be important for the process of incipient speciation because body size differences could contribute to premating reproductive isolation.