Yasuyuki Kondo

Inter- and intraspecific evolutionary relationships of the rice frog Rana limnocharis and the allied species R. cancrivora inferred from crossing experiments and mitochondrial DNA sequences of the 12S and 16S rRNA genes

The rice frog Rana limnocharis is widely distributed in Southeast Asia and the rest of the Asian region extending from India to Japan. In Japan, the Sakishima-island populations of this species were regarded as a distinct species based on morphological and genetic divergences. The main purposes of this study were to confirm the presence of intraspecific reproductively isolating mechanisms in the Sakishima-island populations of R. limnocharis, and to clarify molecular inter- and intraspecific relationships of R. limnocharis and an allied species, Rana cancrivora. The hybridization experiments revealed that there were no reproductively isolating mechanisms between the Sakishima-island populations and other populations of R. limnocharis. The molecular evolutionary relationships were investigated by analyzing nucleotide sequences of the mitochondrial 12S and 16S rRNA genes using 12 populations of R. limnocharis from Japan and Taiwan, and two populations of R. cancrivora from Thailand and the Philippines. The phylogenetic trees constructed by the NJ method showed that the two populations of R. cancrivora were clearly separated from the 12 populations of R. limnocharis, and that the 12 populations of R. limnocharis were broadly divided into three clades; the first comprising eight populations from the main islands of Japan, the second comprising the Sakishima-island populations, and the third comprising the Okinawa-island and Taiwan populations. Interestingly, the Okinawa-island and Taiwan populations of R. limnocharis showed a close relationship that possibly reflected a secondary contact between the two populations. Based on the present crossing experiments and molecular data, it seems reasonable to regard the Sakishima-island populations as a single subspecies of R. limnocharis.

Evolutionary Relationships and Reproductive Isolating Mechanisms in the Rice Frog (Fejervarya limnocharis) Species Complex from Sri Lanka, Thailand, Taiwan and Japan, Inferred from mtDNA Gene Sequences, Allozymes, and Crossing Experiments

Abstract The rice frog (Fejervarya limnocharis) species complex is widely distributed, from India to Japan, and most prevalently in Southeast Asia. Conspicuous morphological variation has been reported for this species complex throughout its distribution range. In the present study, we used mtDNA gene sequence and allozyme analyses to infer evolutionary affinities within this species complex using eight populations (Sri Lanka; Bangkok and Ranong in Thailand; Taiwan; and Hiroshima, Okinawa, Ishigaki and Iriomote in Japan). We also conducted crossing experiments among four populations from Japan, Thailand, and Sri Lanka in order to find out more about the reproductive isolating mechanisms that might exist among the East, Southeast, and South Asian populations of this species complex. The crossing experiments revealed that the Sri Lanka population is reproductively isolated from the Hiroshima, Bangkok, and Ranong populations by complete hybrid inviability, and that the Bangkok population may be reproductively isolated from the Hiroshima population by partial hybrid inviability. Thus, it is not unreasonable to regard the Sri Lanka population as a species separated from F. limnocharis. The mtDNA and allozyme data showed that the Ranong population is most closely related to the Bangkok population in nuclear genome, but more similar to the Okinawa and Taiwan populations in mtDNA genome. The present, preliminary survey may raise questions about the species status of these particular populations and also about the nature of the biological species concept.

Strong expression of the calreticulin gene in the liver of Rana rugosa tadpoles, but not adult frogs.

In the present paper we report the purification of calreticulin (CLT) from livers of the frog, Rana rugosa, the cloning and sequencing of its cDNA, and the CLT gene expression. CLT with M(r) = 52 kDa, estimated by SDS-PAGE, was purified from frog livers. Using rat CLT cDNA as a probe, a 2.4-kilobase frog cDNA clone was isolated from a frog liver cDNA library. The cDNA encoded 419 amino acids including an 18-residue NH2-terminal signal sequence that was 76% homologous to the rat CLT sequence and was 84% homologous to the partial sequence of Xenopus laevis CLT (Treves et al. [1992] Biochem. J. 287:579-581). Phylogenetic relationships estimated from the amino acid sequence of CLTs showed no pronounced variation between the two frog species, R. rugosa and X. laevis. Northern blot analysis indicated that the CLT mRNA level was very high in the liver of tadpoles, but extremely low in adult frogs. Expression levels were also very high in the premature ovary, while moderate expression was observed in the testis and brain of adult frogs. However, there was little histological change in the liver of tadpoles during development. Furthermore, CLT was recognized by Western blot analysis of total proteins in the liver of adult frogs. Immunostaining showed that CLT was distributed in the cytoplasm of liver cells. These results suggest that the expression of the CLT gene is tissue-dependent in the frog, R. rugosa, and that CLT probably functions biochemically in liver cells even when its gene expression is low.

Developmental Capacity and Chromosome Number in the Offspring of Artificially Produced Autotetraploids of Rana nigromaculata

Abstract A large number of autotetraploids of Rana nigromaculata were produced in order to assess their developmental capacity and chromosome number in their offspring. An original autotetraploid male was first produced by transplanting a nucleus from an embryo (triploid) into an unfertilized egg. Next, eggs were inseminated with sperm of the autotetraploid male, then cold-treated to obtain first-, second-, and third-generation offspring. According to an investigation of the chromosome numbers of the tadpoles by the tail-tip squash method, the three generations of offspring included many tetraploids (50–80%), as well as some diploids, triploids, hexaploids and mosaics at the early tadpole stage. In addition, several percent of the second- and third-generation offspring were found to be aneuploids. Evidently, a complete set of diploid chromosomes was not precisely transferred to all of the next-generation offspring from the sperm of the artificially produced autotetraploid males. These observations suggest that there were some abnormalities in the course of spermatogenesis in the male autotetraploid frogs.

Amphidiploidy recovers the viability of hybrids of European and east Asian water frogs.

Reciprocal diploid hybrids, artificially produced from crosses between Rana nigromaculata and Rana lessonae, die at the stage from neurula to tailbud. We found in a previous study that triploid hybrids having two R. nigromaculata genomes and one R. lessonae genome in R. nigromaculata cytoplasm grow into mature frogs, whereas triploid hybrids composed of the other combinations of genome and cytoplasm arrest before hatching. In this study, we made amphidiploid hybrids with two R. nigromaculata and two R. lessonae genomes in the cytoplasm of either species and examined their viability to discover the interaction between parental genomes in each cytoplasm. The amphidiploids with R. nigromaculata cytoplasm developed into mature frogs, whereas ones with R. lessonae cytoplasm arrested at almost the same embryo stage as the lethal triploid hybrids. These findings suggest that the arrest of the reciprocal diploid hybrids is not caused only by the incompatibility between the R. nigromaculata genome and the R. lessonae genome. Taking these together with the results of the triploid hybrids, we suppose that the lethality of the hybrids is mainly due to the incompatibility between the egg cytoplasm and foreign genome(s). With the R. nigromaculata cytoplasm, doubling the maternal genome can ease this incompatibility, but it cannot with the R. lessonae cytoplasm.

Experimentally Induced Autotetraploidy and Allotetraploidy in Two Japanese Pond Frogs

Abstract We examined cytogenetic features and reproductive capacity in newly arisen tetraploid amphibians. Autotetraploids were produced by suppressing second polar body formation in haploid eggs of diploid females of Rana nigromaculata inseminated with diploid sperm of autotetraploid R. nigromaculata. Allotetraploids were also produced by suppressing second polar body formation in haploid eggs of diploid females of Rana porosa brevipoda inseminated with diploid sperm of autotetraploid R. nigromaculata. In male R. nigromaculata autotetraploid offspring, mostly quadri- and bivalents were found in Metaphase I, and 26 dyads were seen in Metaphase II. Autotetraploid males were then mated with autotetraploid females, and most of the resultant offspring were tetraploids possessing four chromosome sets of diploid R. nigromaculata. Male allotetraploid offspring showed 26 bivalents in Metaphase I and 26 dyads in Metaphase II. A large number of the offspring resulting from crosses between allotetraploid males and females were tetraploids having chromosome sets of diploid R. nigromaculata and diploid R. p. brevipoda. These results seem to indicate that, if such tetraploids develop a distinguishing premating mechanism, they should continue as a sexual fertile species in one generation and coexist with their diploid parental species.

Metal ion-responsive transgenic Xenopus laevis as an environmental monitoring animal

We generated germ line-transgenic Xenopus laevis that monitors environmental heavy metal ions. Sperm nuclei were transduced with cDNA of enhanced green fluorescent protein (EGFP) driven by murine metallothionein-1 gene promoters and were microinjected into unfertilized eggs. The eggs developed to sexually matured adults. The transgenic tadpoles at the premetamorphic stage were reared in water containing Zn(2+) and Cd(2+) separately at the concentrations of 0.38-1.52 and 0.09-0.44 μM, respectively. These animals responded to Zn(2+) at as low as 0.38 μM and Cd(2+) at as low as 0.44 μM. The level of EGFP fluorescence emitted by tadpoles increased as the concentration increased up to 1.52 μM and the exposure time prolonged up to 120 h. The fluorescent response was much more sensitive to Cd(2+) than to Zn(2+). We concluded that these transgenic tadpoles are useful as an animal indicator of environmental heavy metal ions.