Zoological Science

The Drosophila ananassae species cluster includes D. ananassae, D. pallidosa, D. parapallidosa, and the cryptic species “pallidosa-like”, “pallidosa-like Wau” and “papuensis-like” Some of the taxa are sympatric in the South Pacific, Papua New Guinea, and Southeast Asia, and gene flow between different taxa has been suspected for a handful of genes. In the present analysis, we examined DNA sequences of introns in four loci: alpha actinin (Actn) on XL, white (w) on XR, CG7785 on 2L, and zinc ion transmembrane transporter 63C (ZnT63C) on 2R. Phylogenetic trees (neighbor-joining and haplotype network) were inconsistent among these loci. Some haplotypes shared between taxa were found for w, CG7785, and ZnT63C, suggesting recent gene flow. However, no haplotypes were shared, for example, between D. ananassae and D. pallidosa for CG7785, which is close to the proximal breakpoint of In(2L)D. This suggests that taxon-specific inversions prevent gene flow, as predicted by the chromosomal speciation hypothesis.

Population genetic analyses were conducted for the flightless beetle Silpha perforata in northern Japan to evaluate effects of straits as dispersal barriers. In all, 19 populations on the Hokkaido mainland and one population on each of seven adjacent islands were included in the study. Based on partial sequences (853 bp) of the mitochondrial ND2 gene from 365 individuals, we calculated haplotype diversity (h), nucleotide diversity (π), Tajima's D, and Fu's F _s statistics for each population. The genetic distance d _A and fixation index F _ST were then estimated between populations, excluding two mainland and two island populations with small sample sizes. The results showed that four island populations (Rebun, Rishiri, Yagishiri, and Teuri) were genetically diverged and isolated from mainland populations, including the closest ones, consistent with the assumption that the straits separating these islands from the mainland have functioned as strong barriers against beetle dispersal. However, some factors other than sea barriers may have resulted in the observed divergence of these island populations, since the straits might be too young to permit such divergence (all are estimated to have been finally established approximately 11,000 to 13,000 years BP). On the other hand, the population on Okushiri Island showed little genetic divergence from the nearest mainland population, suggesting current overseas dispersal across the strait (estimated to have been formed more than 200,000 years BP). The present study thus demonstrated that the effects of straits on dispersal can be diverse, even for species of terrestrial invertebrates with limited dispersal ability.

The Ezo wolf (Canis lupus hattai Kishida, 1931) is an extinct subspecies that inhabited Hokkaido in Japan until the middle of the Meiji Period. Because there are very few preserved skeletons, no osteological and/or genetic analyses of the Ezo wolf have been conducted. In this study, 20 cranial and eight mandibular characters were measured on Ezo wolf skeletons, and mitochondrial DNA (mtDNA) was analyzed to assess genetic relationships between the Ezo wolf and other wolf lineages, including the Japanese wolf on Honshu. The morphological study showed that the Ezo wolf is larger than the Japanese wolf and similar in size to the grey wolf of the Asian and American Continents. MtDNA control sequences (751 bp) from two Ezo wolves were identical to those from the Canadian grey wolf. The morphological and genetic characters indicate that the ancestor of the Ezo wolf was genetically related to that of the grey wolf in Canada.

Females of coenagrionid damselflies exhibit color dimorphism, consisting of an andromorph and a gynomorph. This study compared reproductive traits between the female morphs in both field-captured and laboratory-reared females of the female-dimorphic damselfly Ischnura senegalensis. No difference was found in the onset of egg development between the morphs. The andromorphs developed significantly smaller mature eggs and had significantly more immature eggs than the gynomorphs. These results suggest that the andromorphs are r-strategists (high fecundity with small eggs), whereas the gynomorphs are K-strategists (low fecundity with large eggs). Fecundity and egg size might determine the quantity and quality of the offspring, respectively, indicating that morph-specific reproductive traits would contribute to the overall fitness of each female morph, and consequently be key factors affecting morph frequency in a population.

This study examined polymorphism in the resting egg size, hatching pattern, morphotype, and starvation tolerance of stem females in a local population of the freshwater rotifer Brachionus calyciflorus. Fifteen rotifer families were cultured under laboratory conditions to induce the production of inbred, homozygous resting eggs. Each family of resting eggs was measured for size and aliquots were then incubated at 10°C or 20°C to test the phenotypic plasticity of hatching dynamics in this population. The distribution of resting egg size was approximately normal in each family, and the sizes of the resting egg shell and embyro showed significant differences among families, ranging from 6.4–8.9 (× 10⁵ µm³) and 3.7–5.2 (× 10⁵ µm³), respectively. Hatching rates also varied greatly among families at each temperature, with average values ranging from 10.0–51.5% at 10°C and 3.1– 67.2% at 20°C. Hatching temperature clearly affected the hatching pattern. Resting eggs of most families hatched synchronously at 20°C, whereas they were likely to hatch more sporadically at 10°C. We observed no correlation between size of resting egg and subsequent hatching variability. This was the first time to find a large diversity in morphotype of B. calyciflorus stem females among different families. In some families, most stem females lacked posterolateral spines, but in other families, they had two short posterolateral spines. There was a considerable variation in survival time of stem females that had undergone starvation, and survival time was positively correlated with resting egg size. The divergence in these life history traits may have important implications for understanding the rotifer's adaptive strategy and life history evolution.

To assess the effects of endocrine-disrupting chemicals on the expression and activity of aromatase in the gonads of Bombina orientalis, a common amphibian, we intraperitoneally injected nonylphenol or bisphenol-A and then examined aromatase mRNA levels by RT-PCR as well as aromatase enzymatic activity by tritiated water release assays. To design primers for the RT-PCR, we cloned the B. orientalis aromatase gene using RT-PCR and degenerate primers. The full-length cDNA was obtained by 5′- and 3′-RACE PCR. The complete sequence of the B. orientalis aromatase gene revealed an open reading frame of 1500 bp encoding a deduced protein of 500 amino acids. Semi-quantitative RT-PCR indicated that nonylphenol or bisphenol-A injection did not significantly affect the expression of B. orientalis aromatase mRNA. However, a 48-hr treatment with nonyphenol or bisphenol A reduced aromatase activity to 47% and 32% of the control, respectively. These results suggest that endocrine disrupters can effectively modulate the activity of B. orientalis aromatase without affecting its mRNA levels.

On the basis of allozyme and mtDNA sequence variation, we elucidated genetic relationships between two sympatric genetic types of Onychodactylus japonicus in Kinki and adjacent districts, and investigated their phylogeography. Allozymic analysis revealed the presence of two distinct genetic types (the SW-Honshu and Kinki groups) in this area, and their sympatric occurrence in three of 10 sampling sites. Fixed or nearly fixed allele differences in several loci strongly suggested reproductive isolation between the two types, although one hybrid specimen was found in a locality. Analyses of mtDNA using 194 specimens from 22 localities also demonstrated two genetic types. From phylogeographic and population genetic analyses, it was surmised that these two types diverged allopatrically, and secondarily contacted to become sympatric by the Pleistocene uplift of mountains. Our results indicate different specific status for these two types and separation of the Kinki group from O. japonicus, to which the SW-Honshu group belongs.

Antennal podomere homology has not been well documented in podocopid ostracods. Difficulties associated with describing this homology are compounded by the occurrence of specialised podomeres in both cytheroids and bairdioids. Our research establishes the existence of two kinds of aesthetascs shared among multiple higher taxa. Overgrowth “t-setae” are present in males in Cytheroidea, Cypridocopina and Darwinuloidea, and “aesthetasc yc” is found in both sexes in Cytheroidea and Bairdioidea. Homology of the antennal podomeres among all podocopid superfamilies was determined by using the chaetotaxy of these aesthetascs, leading to a description of evolutionary modifications of the podocopid antenna, which suggests that changes in function of the articulation were prompted by the temporal demands of copulatory behavior in each lineage.

Dorcus koreanus Jang and Kawai, 2008 was recently described as a valid species in Haenam, South Korea, based on morphology. However, the taxonomic position and relationships of this new species with the related species Dorcus japonicus and Dorcus carinulatus were not examined in detail. To address this issue, we evaluated the phylogenetic relationships of D. koreanus to its related species based on molecular analyses of mitochondrial 16S rRNA gene sequences. The molecular evidence suggested that D. koreanus and D. carinulatus are more closely related to each other than either is to D. Japonicus. The genetic divergence between D. koreanus and D. carinulatus ranged from 1.2 to 1.6%, whereas that between D. koreanus and D. japonicus ranged from 9.0 to 9.2%. By comparing the range of nucleotide substitutions within Lucanidae, we determined that the sequence distance between D. koreanus and D. carinulatus is smaller than that for required for species-level distinction. Therefore, we reduce D. koreanus to subspecies rank, as Dorcus carinulatus koreanus stat. nov.

Amphibians are capable of smelling both volatile and water-soluble (e.g., amino acids) odorants. Adult Japanese newts, Cynops pyrrhogaster, live mostly in water, except during hibernation, but sometimes on land. To examine olfactory responses of the newts to adaptation to a short-term stay on land (land adaptation), we measured the magnitude of the olfactory response at five different time points (land adaptation time: 0, 30, 54, 90, and 114 h after transfer from an aquatic to a terrestrial habitat by using electro-olfactogram (EOG) recordings. Statistical analysis by the weighted linear model (P < 0.05) indicated that the time to land adaptation had a significant effect on the magnitude of the EOG induced by 1 µM and 10 µM amino acid mixtures. Further, the slope estimates of the weighted linear model were significantly positive (P < 0.05). These results indicate that the magnitude of the EOG response to amino acid mixtures (arginine, alanine, proline, and glutamic acid) significantly increases with land adaptation time. On the other hand, we observed no significant relationship between the magnitude of the EOG response induced by an 0.05% volatile odorant mixture (isoamyl acetate, n-amyl acetate, cineole, and limonene) and land adaptation time. Our results indicate that olfactory sensitivity to amino acids significantly increases with land adaptation time in adult Japanese newts.