Mitsuaki Ogata

The ZZ/ZW sex-determining mechanism originated twice and independently during evolution of the frog, Rana rugosa

The Japanese frog, Rana rugosa, has two distinct sex chromosome types, XX/XY and ZZ/ZW. These two types are found in localized groups, separated geographically by a boundary area predicted to lie somewhere around Lake Biwa in central Japan. To determine this precise boundary, the heterogametic sex of 18 populations around Lake Biwa was examined by genotyping sex-linked genes. Phylogenetic relationships between the populations were also analyzed using mitochondrial 12S rRNA gene. Results showed that the Suzuka-Kii mountain range located east of Lake Biwa separated the XX/XY populations from the ZZ/ZW populations. Unexpectedly, from a phylogenetic perspective, the ZZ/ZW populations around Lake Biwa belonged not to the main ZW group but to the XY group. The authors propose that the ZZ/ZW populations around Lake Biwa diverged secondarily from the XX/XY group through a change of heterogametic sex, eventually forming a new group. This group was thus named the ‘Neo-ZW group’. As the main ZW group inhabiting northwestern Japan is known to have a different male heterogametic origin, this finding shows that change of heterogametic sex from male to female may have occurred twice, and independently, during the frog speciation.

The prototype of sex chromosomes found in Korean populations of Rana rugosa

The seventh largest chromosome in Japanese populations of the frog Rana rugosa morphologically evolved as a sex chromosome. The sex chromosome is XX/XY type in one geographic form and ZZ/ZW type in another. In contrast, the seventh chromosomes are still homomorphic between the sexes in the other two geographic forms: they are more subtelocentric in the Kanto form and subtelocentric in the western Japanese form. To identify a prototype of the sex chromosomes, we extended our investigation in this study to the Korean form, which is supposed to be close to the phylogenetic origin of this species. The karyotype, a sex-linked gene sequence, and mecha- nisms of sex determination and gonadal differentiation were all examined. In addition, phylogenetic analyses were performed based on mitochondrial gene sequences and the results of crossings between the Korean and Japanese forms. As a consequence, the more subtelocentric seventh chromosome, shared by the Korean and Japanese Kanto forms, was concluded to be the prototype of the sex chromosomes. Starting at the prototype, a whole process of morphological sex chromosome evolution was reconstructed.

Intraspecific Differentiation in the Japanese Brown Frog Rana japonica Inferred from Mitochondrial DNA Sequences of the Cytochrome b Gene

Abstract Intraspecific differentiation of the Japanese brown frog Rana japonica was investigated by analyzing nucleotide sequences of the mitochondrial cytochrome b (cyt b) gene in order to clarify phylogenetic relationships among three population groups known to exist in this species. The nucleotide sequences of 447 base pair (bp) segments were determined by the PCR-direct sequencing method on 31 individuals from 14 populations of R. japonica from Honshu, and phylogenetic trees were constructed by the UPGMA and NJ methods using Rana catesbeiana as an outgroup. A sequence alignment provided 92 variable sites (15 corresponded to the first codon position, three to the second, and 74 to the third), and 19 haplotypes were identified from 31 frogs. The sequence divergences were 0.22∼2.50% (x̄ = 0.65%) within populations, 0.22∼12.02% (x̄ = 7.34%) between populations, and 23.59∼27.89% (x̄ = 26.19%) between R. japonica and R. catesbeiana. Although many nucleotide substitutions were silent mutations, 12 amino acid replacements were found to occur within R. japonica. A high frequency of transitions relative to transversions was observed within R. japonica. The present nucleotide sequence data showed that the eastern and western groups of R. japonica was considerably differentiated to each other, and that the Akita population of the northwestern group was evidently derived from the eastern group, but the Nakajo and Izumozaki populations of the northwestern group diverged considerably from each of the eastern and the western groups.

Phyletic Diversity in the Frog Rana rugosa (Anura: Ranidae) with Special Reference to a Unique Morphotype Found from Sado Island, Japan

Abstract: We investigated a sample of apparently unique frogs from Sado Island, Japan. While being tentatively identified to Rana rugosa on the basis of general morphological resemblance, this form (yellow type) differs from the typical Japanese R. rugosa by showing deep yellow or orange coloration in abdomen and the ventral surfaces of legs, and by having much smoother skin on the entire body. For the Japanese populations of Rana rugosa, following four genetic forms are currently recognized: Kanto form (around Tokyo and northeastern Japan), Western Japan form (western Japan), XY form (central Japan), and ZW form (northwestern Japan). Of these, the latter two forms bear differentiated sex chromosomes of XX/XY and ZZ/ZW types, respectively, and are assumed to have originated through hybridizations between the former two forms. In order to infer the phylogenetic affinity of the yellow type from Sado Island, we examined the karyotype and also analyzed the mitochondrial gene sequences. Our results strongly suggested that the yellow type had originated from the ancestral lineage of the Kanto form early in the divergence of R. rugosa in Japan, achieving genetic characteristics of its own. Also, the results suggested distant affinity of the ZW form to the yellow type, despite their geographic proximity.

An X-Linked Body Color Gene of the Frog Rana rugosa and Its Application to the Molecular Analysis of Gonadal Sex Differentiation

We identified a sex-linked, recessive body color gene, presently designated w (whitish-yellow), in the frog Rana rugosa from the Iwakuni population in Western Japan. This is the first time a sex-linked body color gene was found in amphibians so far. In this population of R. rugosa, males are the heterogametic sex, but the sex chromosomes are still homomorphic. When heterozygous males (Ww), which were produced by crossing a whitish-yellow female (ww) found in the field and a wild-type male (WW) of the same population, were backcrossed to the homozygous whitish-yellow female (ww), the resultant male offspring were all wild-type, whereas the females were all whitish-yellow. This result definitely indicates that w is recessive and X-linked, and its wild-type allele W is located on the Y chromosome. Using this strain (XwXw female and XwYW male), we found that expression of Dmrt1 and Rspo1, which are involved in testicular and ovarian differentiation in vertebrates, was higher in males and females, respectively, prior to the onset of the sexually dimorphic expression of Cyp17 and Cyp19, which are involved in biosynthesis of sex steroids and are critical markers of gonadal sex differentiation.

Evolutionary Changes in Sensitivity to Hormonally Induced Gonadal Sex Reversal in a Frog Species

The Japanese frog Glandirana rugosa is unique in that it shows geographic variation in sex chromosome differentiation and heterogametic sex determination. To elucidate the cause of interpopulation differences in gonadal sex differentiation, we investigated hormonally induced sex reversal and the expression patterns of genes associated with sex determination during early tadpole development. We found that sex reversal was easily induced in XX females and XY males of 2 forms (West-Japan and East-Japan) of G. rugosa with the ancestral homomorphic sex chromosomes under male heterogametic sex determination. During sex reversal, expression of CYP19 and/or FOXL2 was dependent on the phenotypic sex of the gonad. In contrast, sex reversal was not induced in ZW females of a population with a heteromorphic ZW sex chromosome system or in XX females or XY males in a population with a heteromorphic XY sex chromosome system. The latter 2 populations are evolutionarily derived forms. These results indicate an evolutionary direction for the gonadal sex differentiation mechanism. The original system was highly sensitive to sex hormones and allowed almost complete sex reversal. From this ancestral form, a new system evolved that was resistant to hormones and showed a change in the heterogametic sex and the sex chromosome differentiation mechanism.

Meiotic recombination counteracts male-biased mutation (male-driven evolution).

Meiotic recombination is believed to produce greater genetic variation despite the fact that deoxyribonucleic acid (DNA)-replication errors are a major source of mutations. In some vertebrates, mutation rates are higher in males than in females, which developed the theory of male-driven evolution (male-biased mutation). However, there is little molecular evidence regarding the relationships between meiotic recombination and male-biased mutation. Here we tested the theory using the frog Rana rugosa, which has both XX/XY- and ZZ/ZW-type sex-determining systems within the species. The male-to-female mutation-rate ratio (α) was calculated from homologous sequences on the X/Y or Z/W sex chromosomes, which supported male-driven evolution. Surprisingly, each α value was notably higher in the XX/XY-type group than in the ZZ/ZW-type group, although α should have similar values within a species. Interestingly, meiotic recombination between homologous chromosomes did not occur except at terminal regions in males of this species. Then, by subdividing α into two new factors, a replication-based male-to-female mutation-rate ratio (β) and a meiotic recombination-based XX-to-XY/ZZ-to-ZW mutation-rate ratio (γ), we constructed a formula describing the relationship among a nucleotide-substitution rate and the two factors, β and γ. Intriguingly, the β- and γ-values were larger and smaller than 1, respectively, indicating that meiotic recombination might reduce male-biased mutations.

The Postzygotic Isolation of a Unique Morphotype of Frog Rana rugosa (Ranidae) Found on Sado Island, Japan

Abstract On a small Japanese island, Sado-ga-shima, we identified a frog that differs from Rana rugosa by having a deep yellow coloration of the abdomen and ventral surfaces of the legs. We tentatively refer to it as “yellow morphotype.” To identify any mechanisms of reproductive isolation from R. rugosa, allopatrically distributed on the same island, we artificially crossed the two types and examined the developmental capacity, sex-ratio, and inner-structure of gonads and fertility in the F1 hybrid offspring. Almost all of the reciprocal hybrids became males with a very low abundance of sperm in the testes and show an extremely low fertility when backcrossed to the females of either type. We conclude that the yellow morphotype is reproductively isolated from R. rugosa through all hybrid maleness with scarce fertility and, therefore, presents a species new to science.

Reconstruction of female heterogamety from admixture of XX-XY and ZZ-ZW sex-chromosome systems within a frog species

Sex‐determining mechanisms change repeatedly throughout evolution, and it is difficult to track this continual process. The Japanese soil‐frog Glandirana rugosa is a remarkable evolutionary witness to the ongoing process of the evolution of sex‐determining modes. The two geographic groups, designated XY and Neo‐ZW, have homologous sex chromosomes, yet display opposite types of sex chromosomes, XX‐XY and ZZ‐ZW, respectively. These two groups are sympatric at the edges of their respective ranges in Central Japan. In this study, we discovered molecular evidence that the eastern part of the Neo‐ZW group (Neo‐ZW2 subgroup), which is found near the sympatric area, shares mitochondrial haplotypes with the XY group. By analysing single nucleotide polymorphism (SNP) loci, we have also discovered that the representative nuclear genome of the Neo‐ZW2 subgroup shares allele clusters with both the XY group and another part of the Neo‐ZW group (Neo‐ZW1 subgroup), indicating a hybrid origin of the Neo‐ZW2. Further analysis of sex‐linked SNP loci revealed that the alleles on the W chromosomes of the Neo‐ZW2 were derived mostly from X chromosomes, while alleles on the Z chromosomes originated from the Z chromosomes of the Neo‐ZW1 subgroup and partly from the Y chromosomes of the XY group. Our study revealed that admixture of the two opposite sex‐chromosome systems reconstructed a female heterogametic system by recycling the X chromosomes into new W chromosomes. This work offers an illustrative example of how de novo sex‐chromosome systems can arise by recycling material from ancestral sex chromosomes.

The Distributions and Boundary of Two Distinct, Local Forms of Japanese Pond Frog, Pelophylax porosus brevipodus, Inferred From Sequences of Mitochondrial DNA

The Nagoya Daruma pond frog Pelophylax porosus brevipodus is distributed in western Japan and is traditionally divided into two local forms: the Okayama form in the west and the Nagoya form in the east. These two forms are genetically differentiated, but have never been defined taxonomically because their distributions are unclear to date. To complete the distributions and identify the boundary of the two forms, we genetically investigated 16 populations including eight populations located within the unexamined area. We found that the distributional boundary is located within a small area of Hyogo Prefecture where haplotypes of mitochondrial cytochrome b (cytb) and D-loop region corresponding to the two forms co-existed. On the other hand, the polymorphic site of the nuclear gene SOX3 revealed introgression over the boundary into Okayama cytb clade. These results suggest that the two forms were geographically isolated from each other in the past, and secondarily contacted and then accepted one-way introgression. As a next step of the research, taxonomic approach is expected to define the two forms.