Enhanced symbiotic seed germination of Cypripedium macranthos var. rebunense following inoculation after cold treatment

Cypripedium macranthos var. rebunense is a well-known wild orchid in Japan, and is considered to be a symbol for rare plant conservation. A fungus isolated from roots of C. macranthos var. rebunense induced symbiotic germination of the species in vitro. Cold treatment of the seeds at 4°C prior to fungal inoculation was required for the symbiotic germination. Changing the timing of inoculation of the fungus to the seeds greatly improved germination frequency. Maximum germination was attained after seeds were inoculated just after the cold treatment for 12 weeks, and approximately 20% of the seeds developed into protocorms more than 1 mm long. These results suggest that fungal inoculation takes place at the beginning of spring in nature, and the tough impervious seed coat may preserve the seed from the infection during autumn and winter seasons. The lengthy culture period of more than 16 weeks at 20°C on the same medium with the fungus caused gradual browning and rot of the protocorms. By elimination of the fungus with a fungicide and by transfer to a nutrient rich medium, approximately 20% of the protocorms developed into healthy plantlets. The methods obtained here appear to be applicable to symbiotic germination of many other threatened Cypripedium spp.     

Flowering Phenology, Pollination, and Fruit Set of Cypripedium macranthos var. rebunense, a Threatened Lady's Slipper (Orchidaceae)

Cypripedium macranthos var. rebunense, was preliminarily studied on Rebun Island, Hokkaido, Japan, for conservation of this threatened orchid. The functional longevity of individual flowers varied from 0 to 18 days, a determining factor being the wind conditions at the growing sites. The flower was self-compatible but needed a pollen vector for fruit production. It lacked nectar and adopted a deceptive pollination system. Queens of a bumblebee, Bombus pseudo-baicalensis, were found to be pollinators but their visits were infrequent. Fruit set was 8.3% at one study site but only 1.2% at another, probably due to interference from the many visitors who came to see the flowers. Based on the results, conservation and management of lady's slipper were discussed. The need for less human interference and for preservation of the interactions between lady's slipper and its pollinator or other plant species with floral rewards is stressed. Received 1 November 2000/ Accepted in revised form 15 March 2001     

Genetic differentiation in plants of the genus Cypripedium from Russia inferred from allozyme data

Ten gene loci of nine enzyme systems (PGI, 6-PGD, NADHD, SKDH, GDH, PGM, DIA, ADH, GOT-1, and GOT-2) were analyzed in Cypripedium calceolus, C. macranthon, C. shanxiense, and C. ventricosum plants from the south of the Russian Far East. Alleles of loci 6-PGD, NADHD, GDH, ADH, GOT-1, and PGIproved to be diagnostic for C. calceolus and C. macranthon. Plants of C. shanxiense from Primorye and Sakhalin Island were monomorphic at all of the loci examined, and their allelic structure can be regarded as diagnostic for the species. The allelic structure for fragments of the C. calceolus population from the western and eastern parts of the species range differed in two loci, PGl and SKDH: alleles absent in C. calceolus plants from the western part of the range occurred at a high frequency in the plants of this species from the eastern part of the range (28 and 55 plants or 41% and 68%, respectively). These alleles were found in C. shanxiense. The genetic structure of C. shanxiense was similar to that of C. calceolus from the eastern part of the range, i.e., the region when these species are sympartic. The additional alleles in C. calceolus from the eastern part of the range might have appeared as a result of hybridization with C. shanxiense. Our results indicate that C. calceolus plants occuring on the territory of Russia form two groups that represent two different units of genetic diversity preservation. We suggest that C. x ventricosum plants in southern Primorye were formed by hybridization between C. macranthon and C. calceolus x C. shanxiense hybrids. Thus, they differ from plants inhabiting the Urals and West Siberia, which originated by hybridization between C. macranthon and C. calceolus. The population of C. x ventricosum presumably also consists of two plant groups differing in genetic structure, which should be regarded as two different units of preservation of this taxon.     

Comparative pollination biology in two sympatric varieties of Cypripedium macranthos (Orchidaceae) on Rebun Island,Hokkaido, Japan

Cypripedium macranthos sensu lato typically has purple‐pink flowers with no nectar and harvestable pollen. On Rebun Island, Hokkaido, Japan, purple‐pink‐flowered C. macranthos var. macranthos individuals rarely grow among numerous pale‐cream‐flowered C. macranthos var. rebunense plants. In both varieties, flower size is similar, their flowering periods completely overlap, and they share the same pollinator (Bombus pseudobaicalensis). However, in only one of 12 years from 2001 to 2012 did var. macranthos attain an annual fruit‐set ratio (an estimate of pollination success) higher than that of sympatric var. rebunense plants. These findings strongly suggest that in C. macranthos on Rebun Island, flower color results in the differential pollination success, because the pollinator prefers pale‐cream Cypripedium flowers and/or avoids purple‐pink ones, thereby producing pollinator‐mediated selection favoring pale‐cream flowers.     

Vegetation factors influencing the establishment and growth of the endangered Japanese orchid,Cypripedium macranthos var. rebunense

Cypripedium macranthos var. rebunense is a well-known endangered terrestrial orchid that is endemic to the cool-temperate Rebun Island off the coast of northwestern Hokkaido, Japan. The present study aimed to identify suitable methods for conservation of this orchid through management of the natural vegetation. We analyzed the vegetation types and plants growing sympatrically with this orchid using two-way indicator species analysis (TWINSPAN) and detrended correspondence analysis (DCA) ordination. Reproductive orchids can survive in various broad-leaved and short to medium-height herbaceous meadows with high plant species richness at a small scale in coastal regions. In order to maintain C. macranthos var. rebunense populations, succession to dense and tall large-leaved herbs, Sasa scrub, shrubs, and trees, should be suppressed. Favorable vegetation for the establishment of seedlings of this orchid includes narrow-leaved, medium-sized grasses, sedges, herbs, mosses, and prostrate mat-forming shrubs. The ground cover offered by these plants and their litter may favor stability of moisture, temperature, and suitable light conditions at the ground surface. In situ seed sowing at such ‘safe sites’ is a potential low-impact technique to enhance seedling recruitment of this orchid.     

Mitochondrial DNA phylogeography of the red fox (Vulpes vulpes) in northern Japan

Mitochondrial DNA variation in the cytochrome b (cyt b) gene and the control region was examined in the red fox Vulpes vulpes from Japan, with special focus on the population divergence between Hokkaido and northern Honshu. Resultant haplotypes from Hokkaido were subdivided into two distinct groups (I and II), with an average genetic distance of 0.027 for cyt b. Divergence time is roughly estimated to be 1-2 million years ago, given that the conventional divergence rate of the mammalian cyt b gene is 2% per million years. Notably, Group II was only found in Hokkaido, whereas Group I comprised haplotypes from Honshu, Kyushu (Japan), eastern Russia, and Europe, as indicated by a comparison of our own data to the literature. On the other hand, judging from constructed trees, Group I haplotypes from Hokkaido appeared to differ from those from other parts of Japan, i.e., Honshu and Kyushu. This implies that Blakiston's Line, which demarcates the boundary between Hokkaido and Honshu, has been an effective barrier and has allowed the structuring of genetic variation in maternal lineages. Thus, these results suggest that the Hokkaido population, which is sometimes referred to as the distinct subspecies V. v. schrencki, has its own genetic background with multiple migration events and differs from the parapatric subspecies V. v. japonica found in Honshu and Kyushu.     

Pollination of the Lady's slipper orchid (Cypripedium calceolus) in Scandinavia – taxonomic and conservational aspects

The Lady's slipper orchid Cypripedium calceolus L. is considered one of the most beautiful orchids of Europe. Consequently, the species has suffered from over-collecting and is now critically endangered in many countries. Although pollination success is suspected to influence the long-term survival of Cypripedium calceolus , relatively little is known about the identity of its pollinators in mainland Sweden – a region that comprises the largest European populations. In order to identify which species pollinate eight representative populations in mainland Sweden, we observed and sampled visitors to flowers using a standardized protocol. Specimens were identified and any pollen smear found on their body was examined for the presence of Cypripedium pollen. Nine species were recognized as effective pollen vectors ( Andrena cineraria, A. carantonica, A. haemorrohoa, A. helvola, A. nigroaenea, A. praecox, Colletes cunicularius, Lasioglossum fratellum and L. fulvicorne ), four of them for the first time in Scandinavia. This is the first time that a species of Colletes is reported to carry pollen of Cypripedium in this region. All but one specimens were females. Our results suggest a taxonomically heterogeneous pollinator fauna for Cypripedium calceolus and are discussed in light of the management of this species.     

An antifungal compound involved in symbiotic germination of Cypripedium macranthos var. rebunense (Orchidaceae)

Germination of orchid seeds fully depends on a symbiotic association with soil-borne fungi, usually Rhizoctonia spp. In contrast to the peaceful symbiotic associations between many other terrestrial plants and mycorrhizal fungi, this association is a life-and-death struggle. The fungi always try to invade the cytoplasm of orchid cells to obtain nutritional compounds. On the other hand, the orchid cells restrict the growth of the infecting hyphae and obtain nutrition by digesting them. It is likely that antifungal compounds are involved in the restriction of fungal growth. Two antifungal compounds, lusianthrin and chrysin, were isolated from the seedlings of Cypripedium macranthos var. rebunense that had developed shoots. The former had a slightly stronger antifungal activity than the latter, and the antifungal spectra of these compounds were relatively specific to the nonpathogenic Rhizoctonia spp. The level of lusianthrin, which was very low in aseptic protocorm-like bodies, dramatically increased following infection with the symbiotic fungus. In contrast, chrysin was not detected in infected protocorm-like bodies. These results suggest that orchid plants equip multiple antifungal compounds and use them at specific developmental stages; lusianthrin maintains the perilous symbiotic association for germination and chrysin helps to protect adult plants.     

Bee size and pollen transfer in Cypripedium calceolus (Orchidaceae)

In Denmark Cypripedium calceolus is found in only two small and isolated populations, and its fruit set is suspected to be pollinator-limited. Collection and observations of flower visitors were carried out between 1995 and 1997, and fruit set were estimated. Forty-four visitors were collected. The majority belonged to the genera Halictus, Lasioglossum and Andrena . The latter is known as pollinator of C. calceolus in other parts of Europe, but in this study Andrena spp. were both not regular visitors and too large to function as efficient pollinators. Medium-sized halictine species, especially females of Halictus tumulorum and Lasioglossum calceatum , were the most regular and frequent pollen vectors. Andrena jacobi and Lasioglossum rufitarse are recorded for the first time in Denmark.     

Genetic differentiation in plants of the genus <Emphasis Type="Italic">Cypripedium</Emphasis> from Russia inferred from allozyme data

Ten gene loci of nine enzyme systems (PGI, 6-PGD, NADHD, SKDH, GDH, PGM, DIA, ADH, GOT-1, and GOT-2) were analyzed in Cypripedium calceolus, C. macranthon, C. shanxiense, and C. × ventricosum plants from the south of the Russian Far East. Alleles of loci 6-PGD, NADHD, GDH, ADH, GOT-1, and PGI proved to be diagnostic for C. calceolus and C. macranthon. Plants of C. shanxiense from Primorye and Sakhalin Island were monomorphic at all of the loci examined, and their allelic structure can be regarded as diagnostic for the species. The allelic structure for fragments of the C. calceolus population from the western and eastern parts of the species range differed in two loci, PGI and SKDH: alleles absent in C. calceolus plants from the western part of the range occurred at a high frequency in the plants of this species from the eastern part of the range (28–41% and 55–68%, respectively). These alleles were found in C. shanxiense. The genetic structure of C. shanxiense was similar to that of C. calceolus from the eastern part of the range, i.e., the region when these species are sympartic. The additional alleles in C. calceolus from the eastern part of the range might have appeared as a result of hybridization with C. shanxiense. Our results indicate that C. calceolus plants occuring on the territory of Russia form two groups that represent two different units of genetic diversity preservation. We suggest that C. × ventricosum plants in southern Primorye were formed by hybridization between C. macranthon and C. calceolus × C. shanxiense hybrids. Thus, they differ from plants inhabiting the Urals and West Siberia, which originated by hybridization between C. macranthon and C. calceolus. The population of C. × ventricosum presumably also consists of two plant groups differing in genetic structure, which should be regarded as two different units of preservation of this taxon.     

Genetic diversity of an endangered plant, <Emphasis Type="Italic">Cypripedium macranthos</Emphasis> var. <Emphasis Type="Italic">rebunense</Emphasis> (Orchidaceae): background genetic research for future conservation

Cypripedium macranthos var. rebunense is an endangered plant endemic to Rebun Island, Japan. A proper understanding of genetic diversity is needed when conducting conservation programs for rare and endangered species. We therefore examined the genetic diversity of C. macranthos var. rebunense using allozyme markers with a view to future conservation. Our study revealed that C. macranthos var. rebunense has relatively high genetic diversity (P was 0.62, n _a and n _e were 1.85 and 1.28 respectively, and H _o and H _e were 0.163 and 0.187, respectively) when compared with other plant taxa. The natural habitats of C. macranthos var. rebunense are geographically separated into northern and the southern populations. Disappearance of alleles and increase in homozygosity expected as a result of the bottleneck effect were observed, particularly in the southern populations composed of a small number of plants. As additional negative effects (inbreeding depression and further genetic drift) due to fragmentation are predicted in these populations, the southern populations may show deterioration of genetic diversity in the near future.     

Marine growth and survival of white-spotted charr,Salvelinus leucomaenis, in relation to smolt size

The relationships between marine growth and survival, and smolt sizes were examined for white-spotted charr (Salvelinus leucomaenis) populations in the Nairo River, Rebun Island, off northern Hokkaido Island, and the Haraki River, southern Hokkaido Island, Japan. Smolts in the Haraki River were much larger in size than those in the Nairo River, despite being similar size among age cohorts and between the sexes in each population. From scale analyses, smolt size-specific growth rates and survival were estimated by utilizing both observed and back-calculated size-frequencies. Inverse correlations between specific growth rates at sea and smolt size were found in both populations. However, the expected size increments at a given size of smolts in the Haraki River were significantly larger than of those in the Nairo River. For both populations, there were no significant effects of smolt ages (2 to 5 or 6 years) on growth rates at sea. Larger smolts had a consistent survival advantage, although the degree of size-dependent survival function seemed to differ between populations. The results suggested that growth and survival of individuals at sea are determined ultimately by size-, rather than age-, dependent factors depending upon local environmental conditions, supporting the hypothesis of a threshold size for smolting within populations.     

High specificity generally characterizes mycorrhizal association in rare lady's slipper orchids, genus Cypripedium

Lady's slipper orchids (Cypripedium spp.) are rare terrestrial plants that grow throughout the temperate Northern Hemisphere. Like all orchids, they require mycorrhizal fungi for germination and seedling nutrition. The nutritional relationships of adult Cypripedium mycorrhizae are unclear; however, Cypripedium distribution may be limited by mycorrhizal specificity, whether this specificity occurs only during the seedling stage or carries on into adulthood. We attempted to identify the primary mycorrhizal symbionts for 100 Cypripedium plants, and successfully did so with two Cypripedium calceolus, 10 Cypripedium californicum, six Cypripedium candidum, 16 Cypripedium fasciculatum, two Cypripedium guttatum, 12 Cypripedium montanum, and 11 Cypripedium parviflorum plants from a total of 44 populations in Europe and North America, yielding fungal nuclear large subunit and mitochondrial large subunit sequence and RFLP (restriction fragment length polymorphism) data for 59 plants. Because orchid mycorrhizal fungi are typically observed without fruiting structures, we assessed fungal identity through direct PCR (polymerase chain reaction) amplification of fungal genes from mycorrhizally colonized root tissue. Phylogenetic analysis revealed that the great majority of Cypripedium mycorrhizal fungi are members of narrow clades within the fungal family Tulasnellaceae. Rarely occurring root endophytes include members of the Sebacinaceae, Ceratobasidiaceae, and the ascomycetous genus, Phialophora. C. californicum was the only orchid species with apparently low specificity, as it associated with tulasnelloid, ceratobasidioid, and sebacinoid fungi in roughly equal proportion. Our results add support to the growing literature showing that high specificity is not limited to nonphotosynthetic plants, but also occurs in photosynthetic ones.     

A new threespine stickleback,Gasterosteus nipponicus sp. nov. (Teleostei: Gasterosteidae), from the Japan Sea region

Gasterosteus nipponicus sp. nov. is described from the holotype, 35 paratypes, and 60 additional specimens. The species differs from congeners in the following combination of characters: lateral plates complete, abruptly reducing in size above the anus, depth of lateral plate above the anus < 60 % that of the deepest plate; caudal keels thin, membranous. The new species is distributed in coastal Japan facing the Sea of Japan from Kyushu to Hokkaido Islands, along the Pacific coast of northern Japan from the Chiba Prefecture to Hokkaido, along the Sea of Okhotsk of Hokkaido, west to the southern and eastern coasts of Korean Peninsula, the Maritime Territory and north to Sakhalin Island, Russia.     

Ancient Mitochondrial DNA Reveals the Origin of Sus scrofa from Rebun Island, Japan

The Kabukai A site (5 to 8C A.D.) of the Okhotsk cultural area is on Rebun Island, a small island near the coast, north–northwest of Hokkaido, Japan. Specimens of Sus scrofa, called the Sakhalin pig, were discovered in five cultural layers at the Kabukai A site. Ancient DNA was extracted from the remains of 42 Sakhalin pig bones. Thirty-nine nucleotide sequences of the 574-bp mitochondrial DNA control region, estimated to have originated from at least 21 individuals, were amplified and analyzed phylogenetically. Nine distinct haplotypes (A1, A2, A3, B1, B2, C1, C2, D1, and D2) from this site were classified into four haplotype groups (A, B, C, and D) by parsimonious network analysis. Phylogenetic analysis of 9 ancient and 55 modern haplotypes indicated that the population of Sakhalin pigs at the Kabukai A site belonged to two distinct clusters; haplotype groups A and B formed a cluster comprised only of themselves, and haplotype groups C and D belonged to the cluster of one of the two genetic groups of Japanese wild boars uniquely distributed in the western part of Japan, including one northeast Mongolian wild boar. Analysis of the haplotype distribution among three archaeological sites and their historical transitions among the five layers reflecting the cultural periods at the Kabukai A site suggests that the Sakhalin pig populations were introduced from Sakhalin island and the Amur River basin in the northeastern Eurasian continent together with some cultural influences. Received: 18 April 2000 / Accepted: 24 November 2000     

Long-term occurrence of hybrids between Japan Sea and Pacific Ocean forms of threespine stickleback, <Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis>, in Hokkaido Island,Japan

The distribution patterns of two genetically divergent forms (Japan Sea and Pacific Ocean forms) of Gasterosteus aculeatus and their hybrids in the Hokkaido Island, Japan were investigated. Hybrid frequencies and the mating pattern of natural F1-hybrids and backcrosses, were examined by using allozyme analysis. The distribution patterns matched those previously reported. All three sympatric localities in eastern Hokkaido Island included hybrids between the two forms in each year examined. A low frequency of hybrids of sticklebacks has been maintained in areas of sympatry of the two forms in Hokkaido Island for several years. The proportion of F1-hybrids was significantly lower than expected by random mating in six samples from four sympatric populations, suggesting form-assortative mating. Mating crosses of natural F1-hybrids existed in both patterns. Natural F1-hybrids backcrossed more frequently with Pacific Ocean form than Japan Sea form. However, the proportions of backcross individuals in two of three samples were not significantly different from the expected proportions by random mating between F1-hybrids and their parent forms. In other samples, despite the Pacific Ocean form predominant in a population, F1-hybrid predominantly backcrossed with the Japan Sea form. These results support the hypothesis of gene flow from Pacific Ocean to Japan Sea forms.     

Mitochondrial DNA variation among worldwide populations of gypsy moths, Lymantria dispar

Gypsy moth populations from Japan, mainland Asia, Europe, Tunisia, and North America were analyzed for variation in mitochondrial DNA (mtDNA) sequences from three gene regions. These samples resolve into four groups, representing gypsy moths from (1) Okinawa, Japan, (2) Hokkaido, Japan, (3) Honshu and Kyushu, Japan and mainland Asia, and (4) Europe, Tunisia, and North America. Some patterns of geographic variation observed for mtDNA (for example, the distinctiveness of gypsy moths from Hokkaido, Japan) coincide with those observed by Goldschmidt from analyses of morphology, life history, and intersexuality. Other patterns (relative sequence homogeneity across Asia, Honshu, and Kyushu and reduced levels of variation in mainland Japan) do not.     

A complex B chromosome system in the Korean field mouse, Apodemus peninsulae

Information on B chromosomes of six subspecies of A. peninsulae Thomas, 1906, from 79 local populations of Russia (Siberia, Altai, Buryatia and the Far East), Mongolia, China, Korea and Japan (Hokkaido) is reviewed. The frequency of animals with B chromosomes is higher in this taxon than in other mammals and ranges from 0.4 up to 1.0, excluding two insular populations (Sakhalin Island and Stenin Island, Primorye) where Bs were not found. The B chromosome polymorphism shows four levels of variation in number (intraindividual mosaicism, intrapopulational and interpopulational), as well as variability in size, morphology and differential staining. Geographic variation was found among populations for these cytogenetic characteristics and, in some cases, it coincided with subspecies distribution. Comparative chromosome banding of micro and macro Bs illuminates possible pathways for their origin.     

中国大杜鹃的鸣声

一直以来认为广布欧洲大陆和亚洲大陆的大杜鹃 ,其鸣声在地区之间无差异 ,而分布在亚洲的大杜鹃的鸣声至今未有系统的研究报道。本文以分布在中国北部的大杜鹃指名亚种和新疆亚种的广告鸣叫为主要研究对象 ,将其与在四川和云南省分布的大杜鹃华西亚种以及分布在朝鲜、日本、俄罗斯和英格兰的大杜鹃指名亚种的广告鸣叫进行了初步比较。在中国分布的大杜鹃有五种鸣叫类型。中国所有采集地大杜鹃的广告鸣叫都由两个音节组成 ,并且与英国分布种群的广告鸣叫很相似。各地大杜鹃的领域鸣唱呈现出个体质的高度保守性和数量性状的明显差异     

Genetic evidence supporting the existence of two distinct species in the genusGasterosteus around Japan

Synopsis The genetic and morphological features ofGasterosteus aculeatus were investigated for 29 populations around Japan. Allozyme analyses recognized two groups (Pacific Ocean group and Japan Sea group) that had distinct characteristic features, and showed high genetic differentiation between them (D = 0.482). The Pacific Ocean group had a wide range, from North America to Japan, along the Pacific coast. The distribution of the Japan Sea group was limited around the Sea of Japan and the Sea of Okhotsk. The distribution of these groups were found to be sympatric on the Pacific coast of Hokkaido Island, Japan. From this area, genetic analyses demonstrated that the sympatric populations of the two groups formed independent breeding stocks, and it is considered that the two groups were reproductively isolated from each other. Additionally, each group had distinctive morphological features of lateral plates and caudal keels in the sympatric area. These results suggested that these two groups of the threespine stickleback comprise different species and that the Japan Sea group is taxonomically distinguishable fromG. aculeatus.