Zoological Science

Mate choice is important for successful reproduction, and consequently species have evolved various ways to choose potential high-quality mates. Anuran mate choice and underlying processes have been the subject of several recent investigations, however we are far from a complete understanding of mate choice in this system. In the present study, when given a simultaneous choice between a male and a female of identical size, males did not discriminate between the sexes, and attempted to clasp a male or a female with equal frequency. Test males only released the stimulus toad when a release call was emitted by the stimulus male. When two males with distinct size differences were provided with a male, the male chose the larger one. Moreover, males discriminated between gravid females that differed in body size, choosing larger gravid females over smaller ones. These results suggest that male Bufo gargarizans can discriminate between the sexes, probably based on male release calls, and prefer to mate with larger individual using visual cues.

Pelophylax nigromaculatus, P. porosus porosus, and P. p. brevipoda are three pond frog species distributed in Japan. Their distributions overlap at two basins in central Japan (P. nigromaculatus and P. p. porosus in the Matsumoto basin, and P. nigromaculatus and P. p. brevipoda in the Ina basin), and hybrid descendants have been found in these areas. To clarify the distribution areas and hybrid zones of the frogs, and to understand the mode of introgressive hybridization and its impact on the frog populations, we conducted exhaustive sampling at each basin and performed allozyme and mtDNA analyses of 233 individuals. Analysis using genetic markers clearly detected nine F1 hybrids and 94 hybrid descendants of P. nigromaculatus and P. porosus from the overlapping areas of both basins. Allozyme and mtDNA data suggest directional hybridization between female P. p. porosus and male P. nigromaculatus in the Matsumoto basin. Over the past 30 years, the distribution of P. p. porosus has been narrowed and fragmented by the invasion of P. nigromaculatus, seemingly because of directional hybridization in the Matsumoto basin. In the Ina basin, the “pure” P. p. brevipoda (n = 8) population was extremely reduced by gene introgression from P. nigromaculatus, yet its distribution was barely changed compared to the Matsumoto basin. Consequently, this study shows that P. porosus populations are threatened by interspecific hybridization with P. nigromaculatus, and that introgressive hybridization damaged P. porosus populations by different means in each basin.

Pelophylax nigromaculatus, P. porosus porosus, and P. p. brevipoda are three pond frog species distributed in Japan. Their distributions overlap at two basins in central Japan (P. nigromaculatus and P. p. porosus in the Matsumoto basin, and P. nigromaculatus and P. p. brevipoda in the Ina basin), and hybrid descendants have been found in these areas. To clarify the distribution areas and hybrid zones of the frogs, and to understand the mode of introgressive hybridization and its impact on the frog populations, we conducted exhaustive sampling at each basin and performed allozyme and mtDNA analyses of 233 individuals. Analysis using genetic markers clearly detected nine F1 hybrids and 94 hybrid descendants of P. nigromaculatus and P. porosus from the overlapping areas of both basins. Allozyme and mtDNA data suggest directional hybridization between female P. p. porosus and male P. nigromaculatus in the Matsumoto basin. Over the past 30 years, the distribution of P. p. porosus has been narrowed and fragmented by the invasion of P. nigromaculatus, seemingly because of directional hybridization in the Matsumoto basin. In the Ina basin, the “pure” P. p. brevipoda (n = 8) population was extremely reduced by gene introgression from P. nigromaculatus, yet its distribution was barely changed compared to the Matsumoto basin. Consequently, this study shows that P. porosus populations are threatened by interspecific hybridization with P. nigromaculatus, and that introgressive hybridization damaged P. porosus populations by different means in each basin.

The population genetic structure of a reef-dwelling aeolid nudibranch, Pteraeolidia ianthina ( Angas, 1864 ) (Gastropoda, Mollusca), was investigated by analyzing the nuclear ribosomal RNA gene region, including 18S and 5.8S rRNA genes and the ITS1 region (545 bp). Among 235 individuals from 10 localities in the northwestern Pacific, two genetically distinct groups were detected: Groups A and B, which are separated by a minimum sequence difference of 5.3% (p-distance). The marked genetic differentiation of these two groups, even in localities where both co-occur, suggests that they are distinct species, although intergroup hybrids were observed in very low frequency. Within-population genetic variability was low in Group A, but high in Group B. Geographic analysis of between-population genetic distance and “allele” sharing data indicated three subgroups within Group A (temperate Japan, Ryukyus + Philippines, and Guam), whose distribution ranges appear to have some relationship to climate zones (temperate and subtropics + tropics) and ocean currents (Kuroshio and North Equatorial Currents). Within Group B detected only from three subtropic and tropic localities, however, genetic distances between populations were much smaller than those in Group A, and thus no significant geographic pattern was found. These differences in genetic variability and population structure between Groups A and B may reflect differences in biological features between them, such as the duration of the pelagic larval stage. Furthermore, some morphological differences were observed between Groups A and B. These observations provide further support for the suggestion that the two groups are separate species.

The population genetic structure of a reef-dwelling aeolid nudibranch, Pteraeolidia ianthina (Angas, 1864) (Gastropoda, Mollusca), was investigated by analyzing the nuclear ribosomal RNA gene region, including 18S and 5.8S rRNA genes and the ITS1 region (545 bp). Among 235 individuals from 10 localities in the northwestern Pacific, two genetically distinct groups were detected: Groups A and B, which are separated by a minimum sequence difference of 5.3% (p-distance). The marked genetic differentiation of these two groups, even in localities where both co-occur, suggests that they are distinct species, although intergroup hybrids were observed in very low frequency. Within-population genetic variability was low in Group A, but high in Group B. Geographic analysis of between-population genetic distance and “allele” sharing data indicated three subgroups within Group A (temperate Japan, Ryukyus + Philippines, and Guam), whose distribution ranges appear to have some relationship to climate zones (temperate and subtropics + tropics) and ocean currents (Kuroshio and North Equatorial Currents). Within Group B detected only from three subtropic and tropic localities, however, genetic distances between populations were much smaller than those in Group A, and thus no significant geographic pattern was found. These differences in genetic variability and population structure between Groups A and B may reflect differences in biological features between them, such as the duration of the pelagic larval stage. Furthermore, some morphological differences were observed between Groups A and B. These observations provide further support for the suggestion that the two groups are separate species.

Theory predicts that the degree of testes asymmetry should be positively correlated with male body condition in species with directional testis asymmetry. We tested this prediction in Rhacophorus omeimontis, a species in which females mate with more than one male. Our results showed that the treefrogs did not exhibit the absence of directional asymmetry in testis size, but rather the occurrence of fluctuating asymmetry. Moreover, we also tested differences in body size, body mass, testis mass, testis asymmetry, and sperm size among initially paired, jointly paired, and unpaired males. We found that body size and mass, testis mass, testis asymmetry and sperm length did not differ among the three male types. Testis mass showed a positive relationship with soma mass, but the correlations between the extent of fluctuating testis asymmetry and sperm length, and between testis mass and sperm length were not significant. Our data suggest that testes size and sperm length do not play an important role in determining male mating success in the presence of sperm competition.

Temperature data loggers (TDL) are mostly used to monitor avian incubation behavior in bird studies. In this paper we demonstrate how TDL can also be used to determine different breeding stages and nest success of the vulnerable Emei Shan Liocichla (Liocichla omeiensis). All nests that contained at least one egg were divided into two groups. Group I included six nests monitored traditionally by the observers' visits, while Group II included eight nests monitored by TDL. Group I and Group II were visited every 1–4 days and 7 days, respectively, to check nest contents and status (e.g., active vs. inactive, and the breeding process) until fledging or nest failure. The time of each observation was recorded to verify the interpretation of TDL. The data recorded by TDL were converted into line graphs of temperature against time and assessed visually. The results indicated that TDL can reliably identify different breeding stages and estimate daily nest survival rates (DSR) and total nest success. The nest success of Group II (0.3015) was higher than that of Group I (0.2387), suggesting that deployment of TDL did not negatively influence nest survival rate of Emei Shan Liocichla. In contrast to traditional nest visits, TDL minimized disturbance by observers and provided a more precise estimate of nest survival. We suggest that TDL should be used more widely in studies of the breeding ecology of rare and endangered birds.

Temperature data loggers (TDL) are mostly used to monitor avian incubation behavior in bird studies. In this paper we demonstrate how TDL can also be used to determine different breeding stages and nest success of the vulnerable Emei Shan Liocichla (Liocichla omeiensis). All nests that contained at least one egg were divided into two groups. Group I included six nests monitored traditionally by the observers' visits, while Group II included eight nests monitored by TDL. Group I and Group II were visited every 1–4 days and 7 days, respectively, to check nest contents and status (e.g., active vs. inactive, and the breeding process) until fledging or nest failure. The time of each observation was recorded to verify the interpretation of TDL. The data recorded by TDL were converted into line graphs of temperature against time and assessed visually. The results indicated that TDL can reliably identify different breeding stages and estimate daily nest survival rates (DSR) and total nest success. The nest success of Group II (0.3015) was higher than that of Group I (0.2387), suggesting that deployment of TDL did not negatively influence nest survival rate of Emei Shan Liocichla. In contrast to traditional nest visits, TDL minimized disturbance by observers and provided a more precise estimate of nest survival. We suggest that TDL should be used more widely in studies of the breeding ecology of rare and endangered birds.

Transforming growth factor-α (TGFα) is thought to be involved in the regulation of endometrial cells. We investigated Tgfa mRNA expression, and the effects of TGFα on DNA-synthesis and gene expression of insulin-like growth factor 1 (IGF1), IGF binding protein-3 (IGFBP3) and IGF1 receptor in the mouse endometrial cells, because IGF1 is involved in estrogen-induced growth of endometrial cells. We also investigated the role of TGFα on matrix metalloproteinase (MMP) expression, as MMPs are involved both in tissue remodeling during cell proliferation and in enhancement of IGF1 signaling through the degradation of IGFBP3. Tgfa mRNA expression was detected in endometrial luminal and glandular epithelial cells, and stromal cells. Tgfa mRNA signals did not appear to change in endometrial luminal epithelial cells, but signals in glandular epithelial cells were higher at diestrus 1, 2 and proestrus, and the number of stromal cells showing strong signals appeared to increase at diestrus 1 and 2. Endometrial epithelial and stromal cells were treated with estradiol-17β (E2) or progesterone (P4). E2 or P4 stimulated Tgfa mRNA expression in stromal cells. TGFα stimulated DNA synthesis in endometrial epithelial and stromal cells, while E2 and P4 stimulated DNA synthesis in stromal cells. In stromal cells, TGFα, at as low as 1 ng/ml, decreased Igfbp3 and Mmp9 mRNA levels, while high dose (10 ng/ml) of TGFα decreased Igf1 mRNA level and increased Mmp3 mRNA level. These results imply that TGFα stimulates proliferation of endometrial stromal cells through multiple mechanisms, including its regulation of Igfbp3 and Mmp3 transcription.

Transforming growth factor-α (TGFα) is thought to be involved in the regulation of endometrial cells. We investigated Tgfa mRNA expression, and the effects of TGFα on DNA-synthesis and gene expression of insulin-like growth factor 1 (IGF1), IGF binding protein-3 (IGFBP3) and IGF1 receptor in the mouse endometrial cells, because IGF1 is involved in estrogen-induced growth of endometrial cells. We also investigated the role of TGFα on matrix metalloproteinase (MMP) expression, as MMPs are involved both in tissue remodeling during cell proliferation and in enhancement of IGF1 signaling through the degradation of IGFBP3. Tgfa mRNA expression was detected in endometrial luminal and glandular epithelial cells, and stromal cells. Tgfa mRNA signals did not appear to change in endometrial luminal epithelial cells, but signals in glandular epithelial cells were higher at diestrus 1, 2 and proestrus, and the number of stromal cells showing strong signals appeared to increase at diestrus 1 and 2. Endometrial epithelial and stromal cells were treated with estradiol-17β (E2) or progesterone (P4). E2 or P4 stimulated Tgfa mRNA expression in stromal cells. TGFα stimulated DNA synthesis in endometrial epithelial and stromal cells, while E2 and P4 stimulated DNA synthesis in stromal cells. In stromal cells, TGFα, at as low as 1 ng/ml, decreased Igfbp3 and Mmp9 mRNA levels, while high dose (10 ng/ml) of TGFα decreased Igf1 mRNA level and increased Mmp3 mRNA level. These results imply that TGFα stimulates proliferation of endometrial stromal cells through multiple mechanisms, including its regulation of Igfbp3 and Mmp3 transcription.

Black-spotted and red-spotted tokay geckos are distributed in different regions and have significant differences in morphological appearance, but have been regarded as the same species, Gekko gecko, in taxonomy. To determine whether black-spotted and red-spotted tokay geckos are genetically differentiated, we sequenced the entire mitochondrial cytochrome b gene (1147 bp) from 110 individuals of Gekko gecko collected in 11 areas including Guangxi China, Yunnan China, Vietnam, and Laos. In addition, we performed karyotypic analyses of black-spotted tokay geckos from Guangxi China and red-spotted tokay geckos from Laos. These phylogenetic analyses showed that black-spotted and red-spotted tokay geckos are divided into two branches in molecular phylogenetic trees. The average genetic distances are as follows: 0.12–0.47% among six haplotypes in the black-spotted tokay gecko group, 0.12–1.66% among five haplotypes in the red-spotted tokay gecko group, and 8.76–9.18% between the black-spotted and red-spotted tokay geckos, respectively. The karyotypic analyses showed that the karyotype formula is 2n = 38 = 8m + 2sm + 2st + 26t in red-spotted tokay geckos from Laos compared with 2n = 38 = 8m + 2sm + 28t in black-spotted tokay geckos from Guangxi China. The differences in these two kinds of karyotypes were detected on the 15th chromosome. The clear differences in genetic levels between black-spotted and red-spotted tokay geckos suggest a significant level of genetic differentiation between the two.

Black-spotted and red-spotted tokay geckos are distributed in different regions and have significant differences in morphological appearance, but have been regarded as the same species, Gekko gecko, in taxonomy. To determine whether black-spotted and red-spotted tokay geckos are genetically differentiated, we sequenced the entire mitochondrial cytochrome b gene (1147 bp) from 110 individuals of Gekko gecko collected in 11 areas including Guangxi China, Yunnan China, Vietnam, and Laos. In addition, we performed karyotypic analyses of black-spotted tokay geckos from Guangxi China and red-spotted tokay geckos from Laos. These phylogenetic analyses showed that black-spotted and red-spotted tokay geckos are divided into two branches in molecular phylogenetic trees. The average genetic distances are as follows: 0.12–0.47% among six haplotypes in the black-spotted tokay gecko group, 0.12–1.66% among five haplotypes in the red-spotted tokay gecko group, and 8.76–9.18% between the black-spotted and red-spotted tokay geckos, respectively. The karyotypic analyses showed that the karyotype formula is 2n = 38 = 8m + 2sm + 2st + 26t in red-spotted tokay geckos from Laos compared with 2n = 38 = 8m + 2sm + 28t in black-spotted tokay geckos from Guangxi China. The differences in these two kinds of karyotypes were detected on the 15th chromosome. The clear differences in genetic levels between black-spotted and red-spotted tokay geckos suggest a significant level of genetic differentiation between the two.

The smooth (Lissotriton vulgaris) and Carpathian (L. montandoni) newts are sister species. These are separated by a moderate genetic distance, but exhibit striking morphological differences, especially in male epigamic traits. In the areas where they co-occur, they readily mate with each other and produce viable hybrids. However, a high level of pre-zygotic isolation with an unknown behavioral basis has been reported. The complex courtship of newts consists of at least three types of modality: chemical, visual, and tactile. The relative significance of these in mate choice is unclear, but it is commonly accepted that pheromones are an important communication channel. The goal of this study was to determine whether the females of L. vulgaris and L. montandoni exhibit preferences for conspecific extracts from the pheromone-producing abdominal (dorsal) glands. Females of both species spent more time in proximity to the source of the abdominal gland extracts of their own species when a liver extract was presented as an alternative. In a second trial, females were simultaneously confronted with conspecific and heterospecific abdominal gland extracts. Asymmetric preferences were found. Lissotriton vulgaris females were not selective, whereas L. montandoni females preferred the conspecific abdominal gland extract. This finding is consistent with the results of earlier experiments on mate choice in these species. The results strongly indicate that pheromones play a crucial role in courtship and species recognition in this pair of closely related, hybridizing species.

Based on 203 specimens belonging to the Rhinolophus “pusillus group” (Mammalia: Chiroptera: Rhinolophidae), univariate and multivariate morphometric analyses using 19 characters were performed to assess the confused species taxonomy. The results indicated that R. pusillus (including calidus, parcus, and szechuanus) in the continental region and Hainan Island of China and “R. cornutus” in Japan are morphologically divergent species. Rhinolophus cornutus should be further split into R. cornutus (including orii, pumilus, and miyakonis) in the main islands of Japan, the Amami and Okinawa Group of the central Ryukyu Archipelago, and Miyako Group of the southern Ryukyus; and R. perditus and R. imaizumii from the Yaeyama Group in the southern Ryukyus. Rhinolophus monoceros from Taiwan is morphologically more similar to species in Japan than to R. pusillus. In addition to R. pusillus, another form that is morphologically similar to species in Japan was recognized from Langzhong in Sichuan Province; this may represent an undescribed species, and further examination is necessary to determine its taxonomic status. Specimens from Guang'an in Sichuan Province, China, are also different from the others, and are characterized by the smallest skull size. Although further studies are required, these specimens were tentatively identified as R. subbadius.

Based on 203 specimens belonging to the Rhinolophus “pusillus group” (Mammalia: Chiroptera: Rhinolophidae), univariate and multivariate morphometric analyses using 19 characters were performed to assess the confused species taxonomy. The results indicated that R. pusillus (including calidus, parcus, and szechuanus) in the continental region and Hainan Island of China and “R. cornutus” in Japan are morphologically divergent species. Rhinolophus cornutus should be further split into R. cornutus (including orii, pumilus, and miyakonis) in the main islands of Japan, the Amami and Okinawa Group of the central Ryukyu Archipelago, and Miyako Group of the southern Ryukyus; and R. perditus and R. imaizumii from the Yaeyama Group in the southern Ryukyus. Rhinolophus monoceros from Taiwan is morphologically more similar to species in Japan than to R. pusillus. In addition to R. pusillus, another form that is morphologically similar to species in Japan was recognized from Langzhong in Sichuan Province; this may represent an undescribed species, and further examination is necessary to determine its taxonomic status. Specimens from Guang'an in Sichuan Province, China, are also different from the others, and are characterized by the smallest skull size. Although further studies are required, these specimens were tentatively identified as R. subbadius.

Three new species of Pristionchus (P. exspectatus, P. arcanus, and P. japonicus) are described from Japan. They are morphologically similar, with P. exspectatus and P. arcanus being almost indistinguishable from the model organism P. pacificus. Reproductive isolation, namely the inability to produce interfertile F1 hybrids, separates all species pairs in the species complex. Additionally, all three new species are distinguished from P. pacificus Sommer, Carta, Kim, and Sternberg, 1996 by having a gonochoristic instead of hermaphroditic mode of reproduction. In addition to its reproductive isolation, P. japonicus is distinct from other Pristionchus species by its arrangement of genital papillae. All species in the complex are separated from each other by molecular sequence divergence, as indicated by analysis of 27 nuclear protein-coding genes and unique sequences of the small subunit ribosomal RNA gene. The identification of a species complex that includes P. pacificus is invaluable for studies of population genetics, speciation, and macroevolution, particularly the evolution of hermaphroditism in the genus.