Phylogenetic studies on some Japanese amphibia by means of immunoelectrophoresis. II. Relationships of some amphibians to Japanese pond frogs (author's transl)

Since the Japanese pond frogs (Rana nigromaculata and R. brevipoda) are known to be very closely allied with each other in morphological, ecological, physiological or immunological characters, the phylogenetical relationships between the Japanese pond frogs and other 10 species of Japanese amphibians were investigated by means of immunoelectrophoretic analysis of liver extract. The results obtained are as follows: (1) There are conservative antigens which are commonly found in all species of Anura. (2) The Japanese pond frogs have specific antigens. (3) R. nigromaculata and R. brevidpoda are very closely allied with each other. (4) Four species of the genus Rana (R. rugosa, R. catesbeiana, R. ornativentris and R. japonica) are closely related to the Japanese pond frogs. (5) Two species of the genus Rhacophorus (Rh. arboreus and Rh. burergeri) are related to the Japanese pond frogs. (6) R. limnocharis is related to the Japanese pond frogs at the same extent as the genus Rhcophorus is. (7) Tow species of the suborder Procoela (Hyla arbored and Bufo bufo) are only partially related to the Japanese pond frogs. (8) Cynops pyrrhogaster pyrrhogaster of Urodera had only a few common antigens with the Japanese pond frogs.     

Diversity of opsin immunoreactivities in the extraretinal tissues of four anuran amphibians

The pineal complex, deep brain, and skin have been known to function as extraretinal photoreceptors in non-mammalian vertebrates. To see the diversity of localization of extraretinal photoreceptors in lower vertebrates having different habitats, we analyzed the opsin-like immunoreactivities in anuran amphibians, Xenopus laevis, Rana catesbeiana, Rana nigromaculata, and Bufo japonicus. An antiserum (toad Rh-AS) was raised against rhodopsin purified from the retinas of Japanese toad, B. japonicus. In the retina of all the anurans examined, the outer segments of rods were immunopositive to toad Rh-AS. The outer segments of most pinealocytes were immunopositive in R. catesbeiana, R. nigromaculata, and B. japonicus. The outer segments of photoreceptor-like cells within the frontal organ of R. nigromaculata were immunostained. Interestingly, toad Rh-AS immunostained many secretory cells of mucous glands in the head skin of B. japonicus, implying the presence of a novel photoreceptive molecule. Within the hypothalamus, toad Rh-AS immunostained many cells in the magnocellular preoptic nucleus of R. catesbeiana and B. japonicus. Toad Rh-AS also labeled cerebrospinal fluid (CSF)-contacting cells in the anterior preoptic nucleus of R. nigromaculata and those adjacent to the lateral ventricle within the septum of R. catesbeiana. Thus the distribution patterns of the rhodopsin-like immunoreactivities among the anurans were highly diverged, and there was no relationship between the distribution patterns and their habitats. J. Exp. Zool. 286:136-142, 2000.     

Intraspecific molecular phylogeny, genetic variation and phylogeography of Reticulitermes speratus (Isoptera: Rhinotermitidae)

Population structure was investigated in Reticulitermes speratus populations in the Korean Peninsula and the Japanese Archipelago. All trees derived from analyses of the combined sequence dataset of two mitochondrial genes, COII and COIII, showed that R. speratus populations cluster into two major clades comprising the Korean/southern Japanese populations and the north-ern Japanese populations. Analysis of population ge-netic structure showed strong genetic partitioning between populations of the two clades. To understand historical migration routes and current distributions, the phylogeographic history of R. speratus was inferred from intra-/interspecific phylogeny and diver-gence times estimated between the clades of the phylogenetic tree. The estimated migration route and divergence time of ancestral R. speratus are congruent with recent paleogeographic hypotheses involving land-bridge connections between the Asian continent and the Japanese Archipelago. We suggest that ancestral R. speratus separated into northern and southern Japanese populations after its migration into the Japanese main islands from East China during the early Pleistocene via the East China Sea basin, which may have been exposed during that period. The Korean populations seem to have diverged recently from southern Japanese populations; this may explain the current distribution of R. speratus in the Japanese Arachipelago, and account for why it is restricted to northern areas of the Tokara Strait.     

Evaluation of introgressive hybridization among Cervidae in Japan's Kinki District via two novel genetic markers developed from public NGS data

Hybridization and backcrossing of native populations with introduced species can lead to introgression and genetic alteration. In this study, we evaluated introgression in 43 deer from a potential hybrid zone around Okinoshima Island, Kinki District, Japan. This region witnessed the migration of a hybrid population (cross between the Formosan sika deer [Cervus nippon taiouanus] and other deer species) that could potentially breed with the native Japanese sika deer (C. n. centralis). We used an existing genetic marker for the mitochondrial cytochrome b gene and two novel markers for nuclear DNA, developed using publicly available next‐generation sequencing data. We identified one mainland deer with a mitochondrial haplotype identical to that of the Formosan sika deer as well as nuclear heterozygous sequences identical to those of Formosan and Japanese sika deer. This suggests that the mainland deer is a hybrid offspring of the Okinoshima population and native deer. However, only Japanese sika deer sequences were found in the other 42 samples, indicating limited introgression. Nevertheless, hybridization pre‐ and postintroduction in the Okinoshima population could cause multispecies introgression among Japanese sika deer, negatively affecting genetic integrity. We developed a simple test based on polymerase chain reaction–restriction fragment length polymorphism to detect introgression in natural populations. Our method can accelerate genetic monitoring of Japanese sika deer in Kinki District. In conclusion, to prevent further introgression and maintain genetic integrity of Japanese sika deer, we recommend establishing fences around Okinoshima Island to limit migration, besides a continued genetic monitoring of the native deer.     

Genetic relationships among Korean brown frog species (Anura,Ranidae), with special reference to evolutionary divergences between two allied species Rana dybowskii and R. huanrenensis

Allozyme analysis for 41 populations of brown frog species, Rana dybowskii, R. huanrenensis, and R. amurensis from Korea and three reference species (Chinese R. chensinensis and Japanese R. dybowskii and R. tsushimensis), were performed to clarify taxonomic status of Korean brown frogs. The level of average genetic differentiation (Nei's D) among local populations of each species in Korea was very low (D<0.01 2) and Korean and Japanese R. dybowskii also showed conspecific level of differentiation (D=0.070). Whereas, much larger, discrete genetic differences were detected in the interspecific comparisons (D>0.370). In the genetic relationships among five species examined, the 24 chromosome brown frogs (R. dybowskii, R. huanrenensis, and R. chensinensis) did not form a monophyletic group. Rana dybowskii with the chromosome number of 2n=24 was grouped together with R. amurensis with the chromosome number of 2n=26. The hypothesis of reversal change from 24 to 26 in Korean R. amurensis seems to better explain the phylogenetic relationships of east Asian brown frogs than the assumption of parallel reduction in chromosome number from 2n=26 to 24 in R. dybowskii and in the common ancestor of R. huanrenensis and R. chensinensis. The genetic, morphological, and reproductive divergences between Korean R. dybowskii and R. huanrenensis were compared.     

壬基酚对黑斑蛙的毒性效应

通过研究壬基酚对黑斑蛙(Rana nigromaculata)血浆渗透压以及血细胞的影响,探讨壬基酚对黑斑蛙血液的毒性效应。用200、400和600mg/kg壬基酚分别对黑斑蛙腹部淋巴囊注射染毒,在不同的时间间隔内利用渗透压仪测量各组血浆渗透压,同时制作血涂片观察血细胞的异常现象。结果表明,在相同处理时间内,随着壬基酚浓度的增加,黑斑蛙血浆渗透压值上升,血细胞膨大,血细胞核分裂以及核质不均匀现象明显;在相同浓度处理组中,随着处理时间的延长,黑斑蛙血浆渗透压上升,血细胞膨大,细胞核损害严重。壬基酚可诱发红细胞出现微核现象,随着壬基酚浓度的增加,同一处理时间内黑斑蛙红细胞微核及核异常率呈现先上升后下降的变化规律;随着处理时间的延长,各处理组红细胞微核率及核异常率呈现下降的趋势。     

Investigating temporal changes in hybridization and introgression in a predominantly bimodal hybridizing population of invasive sika (Cervus nippon) and native red deer (C. elaphus) on the Kintyre Peninsula,Scotland

We investigated temporal changes in hybridization and introgression between native red deer (Cervus elaphus) and invasive Japanese sika (Cervus nippon) on the Kintyre Peninsula, Scotland, over 15 years, through analysis of 1513 samples of deer at 20 microsatellite loci and a mtDNA marker. We found no evidence that either the proportion of recent hybrids, or the levels of introgression had changed over the study period. Nevertheless, in one population where the two species have been in contact since ～1970, 44% of individuals sampled during the study were hybrids. This suggests that hybridization between these species can proceed fairly rapidly. By analysing the number of alleles that have introgressed from polymorphic red deer into the genetically homogenous sika population, we reconstructed the haplotypes of red deer alleles introduced by backcrossing. Five separate hybridization events could account for all the recently hybridized sika‐like individuals found across a large section of the Peninsula. Although we demonstrate that low rates of F1 hybridization can lead to substantial introgression, the progress of hybridization and introgression appears to be unpredictable over the short timescales.     

Amphibian metallothionein. Induction in the frogs Rana japonica, R. nigromaculata and Rhacophorus schlegelii

Cadmium (Cd) was injected intramuscularly into three species of frogs to examine the number of isoforms in the induced metallothionein (MT). The induced MT was shown to consist of a single isoform in the three species of frogs Rana japonica, R. nigromaculata and Rhacophorus schlegelii. Native MT in the livers of R. japonica and R. nigromaculata was copper (Cu)-MT and the induced MT in the three species was Cd, Cu-MT, the Cd/Cu ratio in the induced MT being different from species to species. The effect of Cd injections on the concentrations of 10 elements in the livers and kidneys of the Rana species was not significant except for Cu.     

Chlorophyll-binding proteins revisited - a multigenic family of light-harvesting and stress proteins from a brown algal perspective

Background

Mitochondrial introgression may result in the mitochondrial genome of one species being replaced by that of another species without leaving any trace of past hybridization in its nuclear genome. Such introgression can confuse the species genealogy estimates and lead to absurd inferences of species history. We used a phylogenetic approach to explore the potential mitochondrial genome introgression event(s) between two closely related green pond frog species, Pelophylax nigromaculatus and P. plancyi.

Results

DNA sequence data of one mitochondrial and two nuclear genes from an extensive sampling of the two species were collected, and the genealogies of the three genes were constructed and compared. While the two nuclear genes congruently showed mutual reciprocal monophyly of both species, the mitochondrial phylogeny separated a Korean P. nigromaculatus clade, a paraphyletic central China P. plancyi assemblage, and a large well-supported introgression clade. Within the introgression clade, the mitochondrial haplotypes of the two species were mixed together. This reticulated pattern can be most parsimoniously explained by an ancient mitochondrial introgression event from P. plancyi to P. nigromaculatus that occurred at least 1.36 MYA, followed by multiple recent introgression events from P. nigromaculatus back to P. plancyi within the last 0.63 MY. The re-constitution of previously co-adapted genomes in P. plancyi may be responsible for the recent rampant introgression events. The Korean P. nigromaculatus clade likely represents the only surviving "true" mitochondrial lineage of P. nigromaculatus, and the central China P. plancyi assemblage likely represents the "original" P. plancyi mitochondrial lineage. Refugia in the Korean Peninsula and central China may have played a significant role in preserving these ancient lineages.

Conclusions

The majority of individuals in the two species have either introgressed (P. nigromaculatus) or reclaimed (P. plancyi) mitochondrial genomes while no trace of past hybridization in their nuclear genomes was detected. Asymmetrical reproductive ability of hybrids and continuous backcrossing are likely responsible for the observed mitochondrial introgression. This case is unique in that it includes an ancient "forward" introgression and many recent "backward" introgressions, which re-constitutes the original nuclear and mitochondrial genomes of P. plancyi. This hybrid system provides an excellent opportunity to study cyto-nuclear interaction and co-adaptation.     

Characterization of the constituent fatty acids in phosphatidylinositol during early development in Rana nigromaculata

1. Qualitative and quantitative changes in phosphatidylinositol (PI) were analyzed in the eggs, embryos and tadpoles of the Japanese pond frog, Rana nigromaculata, at various stages of development. 2. The weight percentage of PI to total phospholipid and lipid was about 8.4-15.2% and 1.4-2.6%, respectively, during embryonic life. 3. At the early stages of the unfertilized egg and the two-cell embryo, the predominant fatty acids are palmitic, stearic, oleic and linoleic acid. From the dorsal lip, early gastrula stage and beyond, the percentage of linoleic acid declines and there is an increase in palmitoleic acid. A relatively large amount of arachidonic acid was noted at the unfertilized egg stage at the 1-position. 4. A large amount of arachidonic acid was also observed at the 2-position of PI in the unfertilized egg, hatching embryo and post-hatching tadpole stages, relative to palmitic and stearic acid. 5. Palmitic and stearic acid were increased at the 2-position of PI in the other embryo and the feeding tadpole stages, relative to arachidonic acid, indicating a shift in these molecular species. 6. Thus, there were marked changes in the positional distribution of the constituent fatty acids in PI during early development of R. nigromaculata.     

Origin and evolution of invasive naturalized material of Rhododendron ponticum L. in the British isles

Information concerning the area of origin, genetic diversity and possible acquisition of germplasm through hybridization is fundamental to understanding the evolution, ecology and possible control measures for an introduced invasive plant species. Rhododendron ponticum is extensively naturalized in the British Isles, but it is not known whether native material in Turkey, Spain or Portugal gave rise to the naturalized material, or to what extent introgression has affected this material. Chloroplast (cp) and nuclear ribosomal DNA (rDNA) restriction fragment length polymorphisms (RFLPs) were sought which could distinguish between native material of R. ponticum, and between 15 other Rhododendron species including R. ponticum's closest relatives. Thereafter, a total of 260 naturalized accessions of R. ponticum from throughout the British Isles was examined with respect to informative polymorphisms. It was found that 89% of these accessions possessed a cpDNA haplotype that occurred in native material of R. ponticum derived almost entirely from Spain, while 10% of accessions had a haplotype unique to Portuguese material. These results therefore indicated an Iberian origin for British material. rDNA or cpDNA evidence of introgression from R. catawbiense was found in 27 British accessions of R. ponticum, and such accessions were significantly more abundant in Britain's coldest region, eastern Scotland, than elsewhere. This could indicate that introgression from R. catawbiense confers improved cold tolerance. Introgression from R. maximum and an unidentified species was also detected.     

虎纹蛙及其它几种蛙科动物线粒体16S rRNA的空间结构分析

测定了虎纹蛙16S rRNA基因全序列,并从GeneBank上下载了5种蛙科动物同源序列,利用CLUSTAL与MEGA3.1软件比较了种间的序列差异,用RNAdraw软件推测分析二级结构的空间构象,并根据空间结构对这6种蛙科动物的分类地位进行讨论分析。结果表明：虎纹蛙与黑斑蛙的二级结构最为相似,与泽蛙结构大体一致,而与黑斑蛙同属的金线蛙则表现出较大的差异;凹耳蛙与上述几种差别较大;福建大头娃与这几种蛙科动物差距最为明显。空间结构分析与序列比较结果存在一定的差异。     

Volatile profiles in cold-pressed peel oil from Korean and Japanese Shiranui (Citrus unshiu Marcov. x C. sinensis Osbeck x C. reticulata Blanco)

A comparison of the volatile profiles between Korean and Japanese Shiranui cold-pressed peel oil was performed by GC and GC-MS. Limonene was the most abundant in the Japanese (91.8%) and Korean (86.4%) oil. Alcohols accounted for 1.8% in the Korean oil, and 0.2% in the Japanese oil, in which the respective linalool levels were 1.2% and 0.1%. The level of aldehydes was also higher in the Korean oil (1.6%) than in the Japanese oil (0.7%).     

Geographic variation and diversity of the cytochrome b gene in wild populations of medaka (Oryzias latipes) from Korea and China

We analyzed the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) of the mitochondrial cytochrome b (cyt b) gene in wild populations of medaka from Korea and China. We surveyed 258 wild specimens from 75 different sites, and identified 17 mitotypes. Sequencing analysis of the complete cyt b gene (1141-bp) was subsequently carried out to infer the phylogenetic relationships among these mitotypes. Phylogenetic trees indicated two major clades, D and E, which were different from the Japanese clades (A, B and C). These two clades were completely identical to two clusters previously identified by RFLP analysis of entire mitochondrial DNAs. The geographic distribution of the mitotypes in clades D and E was consistent with the China-West Korean Population and the East Korean Population as defined by allozymic and karyological analyses. This agreement among different analyses suggests long-term isolation between the two groups. In the region where the distributions of two major clades overlapped, a limited extent of gene flow was observed. These results suggested the existence of some reproductive isolation mechanisms between the two clades, or the introgression between them followed by a random drift in each local population. Furthermore, clade D was subdivided into three subclades (D-I to D-III). The phylogenetic relationship and distribution pattern of subclade D-II suggested a dispersal event of medaka from China to southwest Korea. Our results also showed that the East Korean Population has recently expanded its distribution area because little diversity was observed in clade E.     

SNP identification, linkage disequilibrium, and haplotype analysis for a 200-kb genomic region in a Korean population

Understanding patterns of linkage disequilibrium (LD) across genomes may facilitate association mapping studies to localize genetic variants influencing complex diseases, a recognition that led to the International Haplotype Mapping Project (HapMap). Divergent patterns of haplotype frequency and LD across global populations require that the HapMap database be supplemented with haplotype and LD data from additional populations. We conducted a pilot study of the LD and haplotype structure of a genomic region in a Korean population. A total of 165 SNPs were identified in a 200-kb region of 22q13.2 by direct sequencing. Unphased genotype data were generated for 76 SNPs in 90 unrelated Korean individuals. LD, haplotype diversity, and recombination rates were assessed in this region and compared with the HapMap database. The pattern of LD and haplotype frequencies of Korean samples showed a high degree of similarity with Japanese data. There was a strong correlation between high LD and low recombination frequency in this region. We found considerable similarities in local LD patterns between three Asian populations (Han Chinese, Japanese, and Korean) and the CEPH population. Haplotype frequencies were, however, significantly different between them. Our results should further the understanding of distinctive Korean genomic features and assist in designing appropriate association studies.     

3种无尾两栖类肾形态结构及其对环境的适应性

从显微和亚显微结构以及酸性磷酸酶组织化学等方面,比较研究了史氏蟾蜍(Bufo stejnegeri)、黑斑侧褶蛙(Pelophylax nigrontaculata)和中国林蛙(Rana chensinensis)肾实质,尤其是肾单位的结构特点.3种动物在非繁殖期活动于不同的生境类型,黑斑褶摺蛙属于水生类型,而史氏蟾蜍...     

黑斑蛙肾的组织结构

应用组织学和组织化学方法研究了黑斑蛙(Rana nigromaculata)肾的显微结构。结果表明,黑斑蛙的肾除具有肾单位和集合小管外,还见有淋巴样组织分散于肾实质中。在肾腹侧发现有与真骨鱼类斯坦尼斯小体相似的结构,其中聚集有较多的肥大细胞。说明黑斑蛙的肾具有多种生理功能。     

Variation of BM224 microsatellite in green frogs of the genus Rana

The variable microsatellite repeat BM224 has been discovered in the genomes of eight species of green frogs (Rana ridibunda, R. cf bedriagae, R. cretensis, R. esculenta, R. lessonae, R. shquiperica, R. saharica, R. nigromaculata). Earlier, this repeat had been observed in members of the genus Bufo. In this paper, a possibility of usage of this genetic marker for species identification is discussed.     

Fine-scale map of encyclopedia of DNA elements regions in the Korean population

The International HapMap Project aims to generate detailed human genome variation maps by densely genotyping single-nucleotide polymorphisms (SNPs) in CEPH, Chinese, Japanese, and Yoruba samples. This will undoubtedly become an important facility for genetic studies of diseases and complex traits in the four populations. To address how the genetic information contained in such variation maps is transferable to other populations, the Korean government, industries, and academics have launched the Korean HapMap project to genotype high-density Encyclopedia of DNA Elements (ENCODE) regions in 90 Korean individuals. Here we show that the LD pattern, block structure, haplotype diversity, and recombination rate are highly concordant between Korean and the two HapMap Asian samples, particularly Japanese. The availability of information from both Chinese and Japanese samples helps to predict more accurately the possible performance of HapMap markers in Korean disease-gene studies. Tagging SNPs selected from the two HapMap Asian maps, especially the Japanese map, were shown to be very effective for Korean samples. These results demonstrate that the HapMap variation maps are robust in related populations and will serve as an important resource for the studies of the Korean population in particular.     

Comparison of the morphology of the inner ear between newts and frogs in relation to their locomotory capability

The ultrastructural differences between the inner ears of Japanese red-bellied newts (Cynops pyrrhogaster) and black-spotted pond frogs (Rana nigromaculata) were investigated. Scanning electron microscopic observations showed apparent morphological differences in the shape of the ampulla cristae and the localization of the striola in the saccular macula. There were differences in the length of the kinocilia of the sensory hairs in each sensory region. In addition, the diameters of the bundles of stereocilia differed between the two species: the bundles of stereocilia in the semicircular cristae were thicker in frogs than in newts, while those of the utricular and lagenal maculae were thicker in newts than in frogs.