Genetic diversity of an endangered plant, Cypripedium macranthos var. rebunense(Orchidaceae)

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克隆的缺点英语作文

Cloning,a scientific process that has been the subject of much debate,presents a myriad of potential drawbacks that are often overlooked in the face of its potential benefits.Here are some of the key disadvantages associated with cloning:1.Ethical Concerns:Cloning raises significant ethical issues.The process essentially involves creating a genetic copy of an individual,which can be seen as a violation of the sanctity of life and the uniqueness of each human being.2.Genetic Diversity:One of the fundamental principles of natural selection is the importance of genetic diversity for the survival and adaptation of species.Cloning could lead to a reduction in genetic diversity,making populations more susceptible to diseases and less adaptable to environmental changes.3.Health Risks:Cloned animals have shown a higher incidence of health problems and abnormalities.This could be due to the cloning process itself,which may introduce errors or mutations in the genetic material.If applied to humans,these health risks could be significant and potentially lifethreatening.4.Psychological Impact:The psychological effects of being a clone are not well understood,but it is likely that clones would face unique challenges in terms of identity and selfperception.They may struggle with the knowledge that they are not unique and could experience a sense of depersonalization or loss of individuality.5.Social Implications:Society may struggle to integrate clones,leading to potential discrimination or social ostracism.The concept of family and lineage could be fundamentally altered,with implications for inheritance,social roles,and interpersonal relationships.6.Economic and Legal Issues:The commercialization of cloning could lead to a market for human clones,with serious implications for human rights and dignity.There are also concerns about the legal status of clones,including their rights and responsibilities.7.Potential for Abuse:Cloning technology could be misused for nefarious purposes,such as creating a workforce of clones or for military applications.This could lead to a devaluation of human life and an increase in exploitation.8.Environmental Impact:The mass production of clones could have unforeseen environmental consequences,particularly if clones are used for agricultural or industrial purposes.The ecological balance could be disrupted,leading to unforeseen consequences for other species and ecosystems.9.Moral and Religious Objections:Many religious and philosophical traditions oppose cloning on moral grounds,arguing that it is an affront to the natural order and to the divine.This opposition can create societal divisions and conflicts.10.Unpredictability:The longterm effects of cloning are not fully known,and there is a risk that unforeseen consequences could arise as clones age and interact with the world in ways that are different from naturally conceived individuals.In conclusion,while cloning may offer some potential benefits,such as the ability to reproduce endangered species or to provide organs for transplantation,the risks and drawbacks are significant and must be carefully considered.The potential for harm to individuals,societies,and the environment must be weighed against the potential benefits, and a cautious approach is warranted.。

动物园用于保护野生动物的必要性!英文作文

动物园用于保护野生动物的必要性!英文作文全文共3篇示例，供读者参考篇1The Necessity of Zoos for Protecting WildlifeIntroductionZoos play a crucial role in conserving and protecting wildlife. They provide a safe haven for endangered species and facilitate breeding programs to ensure their survival. In this essay, we will explore the importance of zoos in protecting wildlife and discuss the various ways in which they contribute to conservation efforts.Preservation of Endangered SpeciesOne of the primary functions of zoos is to preserve endangered species. Many animals face the threat of extinction due to habitat loss, poaching, and other human activities. Zoos provide a secure environment for these animals to live and breed, thereby increasing their chances of survival. By maintaining healthy populations of endangered species in captivity, zoos help to prevent their extinction and ensure that future generations will be able to admire these magnificent creatures.Breeding ProgramsZoos play a vital role in breeding programs for endangered species. Many animals bred in zoos are released into the wild to bolster wild populations and restore balance to ecosystems. These programs are essential for maintaining genetic diversity and preventing inbreeding, which can lead to health issues and reduced fertility in animals. By carefully managing breeding programs, zoos can help to ensure the long-term survival of endangered species.Education and AwarenessZoos also serve as centers for education and awareness about wildlife conservation. They provide opportunities for visitors to learn about different species, their habitats, and the threats they face in the wild. Through educational programs, interpretive displays, and interactive exhibits, zoos help to raise awareness about the importance of protecting wildlife and inspire action to safeguard the environment. By connecting people with nature, zoos play a crucial role in fostering a sense of responsibility towards the planet and its inhabitants.Research and ConservationZoos are actively involved in research and conservation efforts to protect wildlife. They collaborate with scientists, conservationists, and government agencies to study endangered species, develop conservation strategies, and implement sustainable practices. Research conducted in zoos helps to advance our understanding of animal behavior, biology, and ecology, which in turn informs conservation initiatives in the wild. By supporting research and conservation projects, zoos contribute valuable knowledge and resources to protect endangered species and their habitats.Global ImpactThe work of zoos has a global impact on wildlife conservation. Many species are threatened by environmental degradation, climate change, and other global challenges that require coordinated efforts to address. Zoos play a crucial role in international conservation networks, sharing expertise, resources, and best practices to protect endangered species on a global scale. By working together, zoos can make a significant difference in preserving biodiversity and safeguarding the future of wildlife.ConclusionIn conclusion, zoos are essential for protecting wildlife and conserving biodiversity. They provide a safe haven for endangered species, facilitate breeding programs, educate the public about conservation, conduct research and conservation initiatives, and collaborate with global partners to address pressing environmental challenges. By supporting zoos and their conservation efforts, we can help to ensure a future where wildlife thrives and ecosystems are healthy. Let us all work together to protect and preserve the magnificent creatures that share our planet.篇2The Importance of Zoos in Protecting Wild AnimalsZoos have long been controversial institutions, with some arguing that they confine animals in unnatural environments and others arguing that they play a crucial role in wildlife conservation. While there are valid arguments on both sides, it is clear that zoos can be valuable tools in protecting and preserving endangered species.One of the primary ways in which zoos contribute to wildlife conservation is through breeding programs. Many zoos participate in Species Survival Plans (SSPs), which arecooperative programs designed to manage the breeding of endangered species in captivity. By carefully selecting breeding pairs and monitoring the health and genetic diversity of captive populations, zoos can help to ensure the long-term survival of endangered species. In some cases, zoos have even successfully reintroduced captive-bred animals into the wild, bolstering populations that are struggling in their natural habitats.In addition to breeding programs, zoos also play a key role in education and public awareness. Zoos provide an opportunity for people to see and learn about animals that they may never have the chance to encounter in the wild. This first-hand experience can foster a sense of connection and empathy towards animals, inspiring visitors to care about conservation issues and take action to protect wildlife. Zoos often offer educational programs and resources for schools and community groups, further spreading awareness about the importance of protecting endangered species.Furthermore, zoos can serve as important research centers for studying wildlife biology and behavior. By closely observing animals in captivity, researchers can gain valuable insights into the needs and behaviors of endangered species. This information can inform conservation efforts in the wild, helpingto develop effective strategies for protecting and managingat-risk populations. Zoo research can also contribute to our understanding of animal welfare and husbandry practices, ensuring that captive animals receive the best possible care.Despite these benefits, it is important to acknowledge that not all zoos are created equal. Some facilities prioritize animal welfare and conservation, while others prioritize profit and entertainment. It is crucial for zoos to adhere to high standards of animal care and conservation ethics, ensuring that they are contributing positively to wildlife conservation efforts. Accreditation by organizations such as the Association of Zoos and Aquariums can help to ensure that zoos are meeting these standards and operating responsibly.In conclusion, zoos can be valuable institutions for protecting and conserving wild animals. Through breeding programs, education initiatives, and research efforts, zoos can play a vital role in ensuring the survival of endangered species. However, it is essential for zoos to prioritize animal welfare and conservation ethics in order to maximize their positive impact on wildlife conservation. by working together with government agencies, conservation organizations, and the public, zoos canhelp to protect and preserve the incredible diversity of life on our planet.篇3The Necessity of Zoos in Conserving WildlifeZoos play a crucial role in conserving and protecting wildlife, particularly endangered species. Despite some criticisms about animal welfare and captivity, zoos serve as important institutions that contribute significantly to the preservation of species at risk of extinction. In this essay, we will explore the importance and necessity of zoos in wildlife conservation.One of the main reasons why zoos are essential for protecting wildlife is that they provide a safe and controlled environment for animals that are endangered in the wild. Many species face threats such as habitat loss, poaching, and climate change, which put them at risk of extinction. Zoos play a vital role in breeding programs and conservation efforts to ensure the survival of these species. Through careful management and breeding, zoos help to increase populations of endangered animals and reintroduce them into the wild when conditions are suitable.Moreover, zoos also serve as educational institutions that raise awareness about the importance of wildlife conservation. By showcasing a variety of species from around the world, zoos help to educate the public about the diversity of life on Earth and the threats facing many species. Visitors to zoos can learn about the habitats, behaviors, and conservation status of different animals, which can inspire them to take action to protect wildlife and the environment.Furthermore, zoos provide research opportunities for scientists and conservationists to study and understand endangered species. By studying animals in captivity, researchers can learn more about their biology, behavior, and reproduction, which can inform conservation strategies in the wild. Zoos also play a role in supporting field conservation projects by providing funding, expertise, and resources to protect endangered species and their habitats.In addition, zoos are important for promoting the welfare and well-being of individual animals. While some critics argue that animals in captivity may suffer from stress and confinement, modern zoos place a strong emphasis on providing enriching environments and care for their animals. Zoos invest in creating habitats that replicate the natural environments of animals,provide mental and physical stimulation, and ensure proper nutrition and veterinary care.In conclusion, the necessity of zoos in conserving wildlife cannot be overstated. Zoos play a crucial role in protecting endangered species, raising awareness about conservation issues, supporting research and education, and promoting the welfare of individual animals. While challenges exist in maintaining high standards of animal welfare in captivity, zoos continue to evolve and adapt to ensure the well-being of the animals in their care. By supporting zoos and their conservation efforts, we can help to safeguard the future of wildlife and advance our understanding of the natural world.。

动物应该生活在大自然英语作文

动物应该生活在大自然英语作文英文回答：The wild, untamed landscapes of nature have always been the natural habitat of animals. From the vast savannas of Africa to the towering rainforests of the Amazon, these ecosystems have evolved to provide animals with the food, shelter, and resources they need to thrive. However, as human civilization has expanded, our encroachment uponthese natural habitats has forced many animals to adapt or face extinction.Confining animals to zoos or other artificial environments cannot replicate the complexities of their natural habitats. While captivity may provide some animals with protection from predators and a guaranteed food supply, it also deprives them of the freedom to roam, hunt, and interact with their own species. Studies have shown that captive animals often exhibit signs of stress, boredom, and abnormal behavior due to the lack of natural stimulationand social interaction.Beyond the ethical concerns of animal welfare, keeping animals in captivity can have detrimental effects on their physical and mental health. Restricted movement can lead to obesity, muscle atrophy, and joint problems. The absence of natural predators can disrupt animals' natural fear responses, making them more vulnerable to predators if they are ever released back into the wild. Furthermore, the lack of mental stimulation can result in cognitive decline and impaired problem-solving abilities.In addition to the welfare concerns, keeping animals in captivity also has significant financial and logistical challenges. Zoos, aquariums, and other captive facilities require substantial infrastructure, staffing, and ongoing maintenance to provide even a basic level of care for their animals. The costs of feeding, veterinary care, and enrichment activities can be astronomical, and there is often a need for specialized facilities to accommodate the unique needs of different species.The conservation value of captive breeding programs is often overstated. While it is true that some captive-bred animals have been successfully reintroduced into the wild, these programs are costly, time-consuming, and often have low success rates. The genetic diversity of captive populations is often limited, and animals raised in captivity may not have the necessary skills or instincts to survive in the wild. Reintroduction efforts are further complicated by the fact that many former habitats have been destroyed or fragmented by human activities.In contrast to captivity, allowing animals to live in the wild offers a multitude of benefits. Animals in their natural habitats are able to engage in their full range of natural behaviors, including hunting, foraging, mating, and raising young. They are also able to adapt to changing environmental conditions and make decisions that maximize their chances of survival. In natural ecosystems, animals play vital roles in maintaining biodiversity, controlling populations, and dispersing seeds.Protecting and restoring animal habitats is essentialfor the long-term survival of wildlife. By reducing our impact on the environment, we can help to ensure that animals have the space and resources they need to thrive. This includes reducing deforestation, pollution, and climate change, as well as working to reconnect fragmented habitats.In some cases, human intervention may be necessary to protect endangered species or manage populations that have become too large. This can include wildlife rehabilitation programs, population control measures, and habitat restoration projects. However, these interventions should always be conducted with the goal of restoring populations to a healthy and sustainable state in the wild.Animals are an integral part of our planet, and they deserve to live in their natural habitats. By protecting and restoring these habitats, and by respecting the inherent value of all living creatures, we can help to ensure a future where animals can thrive and flourish.中文回答：动物理应生活在野外。

新高一英语培优外刊阅读学案：濒危动物话题

高一英语培优外刊阅读班级：____________学号：____________姓名：____________外刊精选｜科学家克隆濒危动物，让33年前的雪貂重生去年12月，一只克隆雪貂在美国出生，它克隆的是一只已经去世32年，一直被冷冻保存的雪貂。

作为第一个被克隆的北美本土濒危物种，这一消息在美国引起不少关注。

人类几十年来一直在克隆技术领域不断探索，这只克隆雪貂的出生让人们看到了挽救濒危物种的新希望。

有很多物种都处于濒危状态，科学家们这次为什么会选择雪貂呢？Meet Elizabeth Ann, the First Cloned Black-Footed FerretBy Sabrina ImblerLast year, Ben Novak drove across the country to spend New Year's Eve with a black-footed ferret. Elizabeth Ann had just turned 21 days old — surely a milestone for any ferret but a particularly meaningful one for Elizabeth Ann, the first of any native, endangered animal species in North America to be cloned.Mr. Novak, the lead scientist of the biotechnology nonprofit Revive & Restore, spent less than 15 minutes with Elizabeth Ann, whose black mask, feet and tail were just beginning to show through her downy white fur. "It felt like time stopped," Mr. Novak said.Thankfully, time has not stopped for Elizabeth Ann, who now looks bigger, browner and considerably more like a ferret.Cloned siblings are on the way, and potential (cloned) mates are already being lined up. If successful, the project could bring needed genetic diversity to the endangered species. And it marks another promising advance in the wider effort to use cloning to retrieve an ever-growing number of species from the brink of extinction.The black-footed ferret, the first species to be reintroduced to former habitats with the help of artificial insemination, has long been a model species for new conservation technologies. So it is fitting that the ferrets have become the second species to be cloned for this type of genetic rescue. 【词汇过关】请写出下面文单词在文章中的中文意思。

大熊猫的状况,原因以及保护英语作文

大熊猫的状况,原因以及保护英语作文The Giant Panda, native to China, is one of the world's most famous and beloved animals. However, this iconic species is currently facing numerous threats that are putting its survival at risk. In this article, we will explore the current situation of the Giant Panda, the reasons behind its decline, and the efforts being made to protect and conserve this endangered species.The Current Situation of the Giant Panda:The Giant Panda, with its distinctive black and white markings, is a symbol of peace and conservation around the world. However, this charismatic species is currently classified as "vulnerable" on the IUCN Red List of Threatened Species, with an estimated population of around 1,800 individuals in the wild. The main population of Giant Pandas is found in the mountain ranges of central China, particularly in the provinces of Sichuan, Shaanxi, and Gansu.The decline in the population of Giant Pandas can be attributed to several factors, including habitat loss, fragmentation, and degradation. As China's human population continues to grow, more and more land is being converted for agriculture, infrastructure development, and logging, which hasresulted in the loss of bamboo forests – the primary food source of Giant Pandas. In addition, climate change is also impacting the distribution and availability of bamboo, further exacerbating the challenges faced by Giant Pandas.Furthermore, Giant Pandas also face threats from poaching and illegal trade. Despite being a protected species under Chinese law, poaching for their fur and body parts still occurs. The demand for Giant Panda products in the illegal wildlife trade market poses a significant threat to the survival of this species.Efforts to Protect and Conserve the Giant Panda:In response to the declining population of Giant Pandas, the Chinese government, conservation organizations, and local communities have implemented various conservation initiatives to protect and conserve this endangered species. One of the most significant efforts is the establishment of Giant Panda reserves and conservation areas, which provide protected habitats for Giant Pandas to thrive.In addition to habitat protection, conservation programs also focus on researching and monitoring the Giant Panda population, as well as conducting breeding and reintroduction programs. These programs aim to increase the genetic diversityof the captive population and reintroduce Giant Pandas into the wild to bolster wild populations.Furthermore, public awareness and education campaigns play a crucial role in raising awareness about the importance of conserving Giant Pandas and their habitats. Through educational programs and outreach activities, local communities are encouraged to participate in conservation efforts and support the protection of Giant Pandas.Looking Ahead:While significant progress has been made in the conservation of Giant Pandas, continued efforts are needed to ensure the long-term survival of this iconic species. By addressing the root causes of habitat loss, poaching, and climate change, and by promoting sustainable development practices, we can help secure a brighter future for the Giant Panda and ensure that this beloved species continues to thrive for generations to come.In conclusion, the protection and conservation of the Giant Panda are essential to preserving biodiversity and maintaining the delicate balance of ecosystems in China. By working together to address the threats facing this iconic species, we can make a positive impact on the future of Giant Pandas and contribute tothe conservation of wildlife worldwide. Let us all join hands to protect the Giant Panda and ensure a sustainable future for our planet.。

介绍熊猫成和花的英语作文

介绍熊猫成和花的英语作文Pandas are one of the most beloved and iconic animals in the world. These gentle giants have captured the hearts of people across the globe, and their conservation has become a global priority. Among the many pandas, one particular panda has gained significant attention – the panda named Chengdu Hua.Chengdu Hua, also known as Hua Hua, is a giant panda born in 2016 at the Chengdu Research Base of Giant Panda Breeding in China. From the moment she came into the world, Hua Hua has been the subject of immense fascination and admiration.One of the most remarkable things about Hua Hua is her striking appearance. With her distinctive black and white fur, she is the epitome of the iconic panda look. Her large, round eyes and fluffy ears give her a captivating and endearing appearance that instantly draws in anyone who sees her.But Hua Hua's appeal goes far beyond her physical beauty. She is also known for her engaging and playful personality. Pandas are often described as gentle and docile, and Hua Hua is no exception. She is often observed engaging in playful behaviors, such as rollingaround in her enclosure, climbing trees, and even engaging in friendly interactions with her caretakers.One of the most heartwarming aspects of Hua Hua's story is her close bond with her mother, Cao Cao. Pandas are known to have a strong maternal instinct, and Cao Cao has been an exemplary mother to Hua Hua. From the moment Hua Hua was born, Cao Cao has been fiercely protective of her, ensuring that she receives the nourishment and care she needs to thrive.As Hua Hua has grown older, she has become an ambassador for her species, helping to raise awareness about the importance of panda conservation. Pandas are considered a vulnerable species, with their numbers in the wild steadily declining due to habitat loss and other threats. By showcasing Hua Hua's charm and personality, conservationists hope to inspire people around the world to support efforts to protect these magnificent creatures.One of the ways Hua Hua has contributed to panda conservation is through her role in the breeding program at the Chengdu Research Base. As a young, healthy panda, Hua Hua has the potential to contribute to the genetic diversity of the captive panda population, which is crucial for the long-term survival of the species.In addition to her role in the breeding program, Hua Hua has alsobecome a beloved figure in the public eye. She has been the subject of numerous documentaries and news articles, and her image has been used to promote panda conservation efforts around the world. Visitors to the Chengdu Research Base often flock to see Hua Hua, eager to catch a glimpse of this charismatic panda.But Hua Hua's story is not just about her own success and popularity. It is also a testament to the incredible work being done by panda conservationists around the world. The Chengdu Research Base, where Hua Hua was born and raised, is just one of many facilities dedicated to the preservation of the giant panda.Through their tireless efforts, conservationists have been able to slowly but steadily increase the panda population in the wild. In recent years, the International Union for Conservation of Nature (IUCN) has even reclassified the giant panda from "endangered" to "vulnerable," a testament to the progress that has been made.However, there is still much work to be done. Pandas continue to face threats to their habitat and well-being, and conservationists must remain vigilant in their efforts to protect these beloved creatures. By supporting organizations like the Chengdu Research Base and raising awareness about the importance of panda conservation, we can all play a role in ensuring that pandas like Hua Hua continue to thrive for generations to come.In conclusion, Chengdu Hua, or Hua Hua, is a remarkable panda who has captured the hearts and imaginations of people around the world. Her charming personality, striking appearance, and role in the conservation of her species have made her a true ambassador for the giant panda. As we continue to learn more about Hua Hua and other pandas, we can all be inspired to do our part in protecting these incredible animals and ensuring their survival for years to come.。

什么是生物多样性英语作文

什么是生物多样性英语作文Biodiversity: An Essential Component of Our PlanetBiodiversity, a term coined by Raymond in 1968, encapsulates the diversity and variability of life on Earth. It refers to the richness and variety of all living organisms, including animals, plants, and microorganisms, as well as the ecosystems they inhabit. Biodiversity is a cornerstone of our planet's existence, supporting numerous life processes and maintaining the delicate balance of nature.The Essence of Biodiversity•Biodiversity encompasses three key dimensions: ecosystem diversity, species diversity, and genetic diversity.•Ecosystem diversity refers to the variety of habitats and environments on Earth, from rainforests to deserts and oceans.•Species diversity describes the number and variety of species within an ecosystem or region.•Genetic diversity represents the range of genes and variations within a species.Why Biodiversity Is Important•Biodiversity is essential for the survival of all life on Earth. It provides us with food, water, medicine, and other vital resources.•Each species plays a unique role in its ecosystem, ensuring the smooth functioning of natural processes.•Biodiversity also contributes to the resilience of ecosystems, enabling them to adapt to changes and recover from disturbances.Threats to Biodiversity•Over the years, humans have severely impacted biodiversity. Thousands of species have become extinct, and many more are threatened with extinction.•Habitat destruction, pollution, climate change, and overexploitation are some of the major threats to biodiversity.•For example, in the United States alone, approximately 170 species are considered endangered.Protecting Biodiversity•It is crucial to take measures to protect biodiversity. This includes conserving habitats, reducing pollution, and addressing climate change.•Education and awareness-raising are also essential to promote sustainable practices and reduce the impact of human activities on biodiversity.In conclusion, biodiversity is a precious asset that we must cherish and protect. By understanding its importance and taking action to safeguard it, we can ensure the continued existence of a diverse and vibrant planet.。

Genetic Variation in Plant Species

Genetic Variation in Plant Species Genetic variation in plant species is a fascinating and crucial aspect of the natural world. It refers to the diversity of genes and alleles within a population or species, and it plays a fundamental role in the adaptation and evolution of plants. This variation can arise through a range of processes, including mutation, genetic recombination, and gene flow. Understanding genetic variation in plant species is essential for conservation efforts, agricultural practices, and the overall study of biodiversity.From an evolutionary perspective, genetic variation is the raw material upon which natural selection acts. It provides the diversity necessary for populations to adapt to changing environmental conditions, such as climate change or the introduction of new pathogens. In plant species, genetic variation can lead to differences in traits such as growth rate, resistance to disease, and tolerance to environmental stressors. This variation allows plants to thrive in a wide range of habitats and ensures the long-term survival of their populations.In the context of agriculture, genetic variation is of paramount importance. It forms the basis of crop improvement and breeding programs, allowing for the development of new varieties with desirable traits. By understanding and harnessing genetic variation, plant breeders can enhance crop yields, nutritional content, and resistance to pests and diseases. This, in turn, contributes to global food security and the sustainability of agricultural practices.Moreover, genetic variation in plant species is integral to the conservation of biodiversity. As human activities continue to impact natural habitats, many plant species face the threat of extinction. Preserving genetic variation within these species is crucial for their survival. It ensures that they have the potential to adapt to changing conditions and evolve over time. Conservation efforts often focus on protecting and maintaining the genetic diversity of endangered plant species, both in their natural habitats and in seed banks.On a more personal level, the beauty of genetic variation in plant species lies in the sheer diversity and complexity it encompasses. Each plant carries a unique genetic makeup, shaped by millions of years of evolution. This variation gives rise to the stunning array of colors, shapes, and sizes we see in the plant kingdom. It also serves as a reminder of the interconnectedness of all living organisms and the importance of preserving the natural world for future generations.In conclusion, genetic variation in plant species is a multifaceted and vital aspect of biology. It underpins the ability of plants to adapt and evolve, supports agricultural practices, and is essential for the conservation of biodiversity. Embracing and protecting genetic variation in plant species is not only a scientific endeavor but also a deeply meaningful pursuit that resonates with our own existence on this planet. As we continue to unravel the mysteries of genetic variation, we deepen our appreciation for the rich tapestry of life that surrounds us.。

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ORIGINAL RESEARCHGenetic diversity of an endangered plant,Cypripediummacranthos var.rebunense (Orchidaceae):background genetic research for future conservationTakeshi Izawa ÆTakayuki Kawahara ÆHideki TakahashiReceived:24April 2006/Accepted:5January 2007/Published online:15March 2007ÓSpringer Science+Business Media B.V.2007Abstract 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 conser-vation.Our study revealed that C.macranthos var.rebunense has relatively high genetic diversity (P was 0.62,n a and n e were 1.85and 1.28respectively,and H o and H e were 0.163and 0.187,respectively)when compared with other plant taxa.The natural habitats of C.macranthos var.rebunense are geographically sep-arated into northern and the southern populations.Disappearance of alleles and increase in homozygosity expected as a result of the bottleneck effect were ob-served,particularly in the southern populations com-posed 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.Keywords Genetic diversity ÁEndangered plant ÁCypripedium ÁAllozyme ÁConservationIntroductionThe biodiversity of the earth is rapidly decreasing due to human activity,and many wild species are in danger of extinction.In this critical situation,maintenance of biodiversity is a global issue.Biodiversity is catego-rized into three fundamental levels (ecological,species,and genetic)by the International Union for Conser-vation of Nature and Natural Resources (IUCN)(Mcneely et al.1990).The primary process of extinc-tion is through population fragmentation accompany-ing reduction in individual number.Fragmented populations may show gene ﬂow decline,inbreeding depression,and loss of genetic diversity (Frankham et al.2002).The loss of genetic diversity within popu-lations decreases adaptability to environmental chan-ges,and increases extinction risk.Even if the original cause of population decline is removed,the deleterious effect of population fragmentation remains.Extinction of an individual population removes any unique bio-logical characteristics that it may possess,inﬂuences overall species biodiversity,and may ultimately lead to species extinction.Maintenance of genetic diversity is therefore an important factor for the survival of endangered species,and assessment of genetic diver-sity is vital for their conservation management (Hamrick and Godt 1996).Several studies have reported that reduction in population size depresses genetic diversity (Young et al.1999;Rossum et al.2004),and may also lead todecreased ﬁtness (Luijten et al.2000;Ke´ry et al.2000).T.Izawa (&)Systematic Botany,Graduate school of agriculture,Hokkaido University,Sapporo 060-0810,Japan e-mail:ti@museum.hokudai.ac.jpT.KawaharaHokkaido Research Center,Forestry and Forest Research Institute,Sapporo 062-8516,JapanH.TakahashiSection of Fundamental and Curatorial Studies,Hokkaido University Museum,Hokkaido University,Sapporo 060-0810,JapanConserv Genet (2007)8:1369–1376DOI 10.1007/s10592-007-9287-1When decreasedﬁtness seriously affects species survival rate,self-recovery is likely impossible and human-aided conservation is needed.A typical exam-ple of such conservation is the case of the Mauna Kea Silversword(Argyroxiphium sandwicense ssp.sandwi-cense),which is endemic to the Mauna Kea Volcano on the Island of Hawaii.This silversword has experienced severe declines in both abundance and distribution, due to heavy predation by alien ungulates.The Hawaii State Government initiated conservation action for the silversword,which included an outplanting program in the1970s(USFWS1994).This outplanting accom-plished at least one of its goals—an outplanted popu-lation composed of over450plants was established. However,outplanting unintentionally resulted in a population bottleneck due to limited founder number (Robichaux et al.1997).This bottleneck could have been avoided by careful genetic study with a view to maintaining the maximum genetic diversity of the mother population before outplanting.This actual example serves as a good lesson for us;detailed study of genetic diversity should be carried out before beginning conservation activity.Cypripedium(known as‘‘Lady’s slipper’’),an her-baceous perennial plant of the genus Orchidaceae,has a widespread range in the Northern Hemisphere (Cribb1997).This genus is characterized by distinctive pouch-like labella,and is popular among horticultur-ists,making it a target for collectors.Both the popu-lation and individual numbers of Lady’s slipper have declined due to excessive worldwide collecting.Cyp-ripedium macranthos var.rebunense is endemic to Rebun Island,in northernmost Japan(Fig.1),and can be distinguished from C.macranthos var.macranthos by its cream-coloredﬂowers.Habitat range and pop-ulation size have rapidly declined,especially since the 1950’s,mainly due to illegal collecting for the purposes of horticultural use and sale(Environment Agency of Japan2000).Cypripedium macranthos var. rebunense is presently designated as an endangered plant in Appendix II of the Washington Convention (CITES:Convention on International Trade in Endangered Species of Wild Fauna and Flora).To date,the following conservation studies of C.macran-thos var.rebunense have been carried out.Miyabe and Kudo(1932)initially described this taxon as a variety of C.macranthos.However,Cribb(1997)and Ohwi (1965)claimed that the taxonomy of C.macranthos var.rebunense should be demoted from variety to form rank.Taniguchi et al.(2001)and Jo et al.(2005)sug-gested that C.macranthos var.rebunense is distin-guishable from C.macranthos by molecular data,but their samples are so limited that the range of genetic variation is not sufﬁciently clariﬁed and,thus,the taxonomical treatment remains controversial.Sugiura et al.(2001,2002),investigating the reproductive ecology of C.macranthos var.rebunense,pointed out the possibility that this taxon is aﬂoral mimic of Pedicularis schistostegia,and clariﬁed the importance of bumblebee as a pollinator.Shimura and Koda(2003, 2005)were successful in seed germination using sym-biotic fungi,and established an artiﬁcial culture method.At the time of the above research,genetic diversity in C.macranthos var.rebunense had not been investigated.We therefore undertook the present study of the genetic diversity of C.macranthos var. rebunense to contribute to the pool of background genetic information and to suggest a conservation program from the genetic viewpoint.Materials and methodsResearch sitesOur research sites for Cypripedium macranthos var.rebunense were established on Rebun Island, Hokkaido,in northernmost Japan(N:45.357284, E:141.038118).The natural habitat of C.macranthos var.rebunense has fragmented into northern and southern populations on Rebun Island,separated by approximately12km.The northern populations consist of two populations,N1and N2(Fig.1),that have a fairly large number ofﬂowering shoots of C.macranthos var. rebunense(~400in N1,>3,000in N2).Both populations were kept in sanctuaries and protected by fences.In addition,nature guards were stationed during the ﬂowering period of C.macranthos var.rebunense to guard against illegal collection.The southern popula-tions consist of three populations,S1,S2,and S3 (Fig.1),that have a relatively small number ofﬂowering shoots compared to the northern populations(~50in S1, 15in S2,and10in S3).The conservation activity tar-geting the southern populations is considerably lower than that targeting the northern populations.Detailed information of the locations of the southern populations is omitted to avoid the threat of illegal collection. SamplingLeaf tissue samples were collected in June2005from ﬁve populations of C.macranthos var.rebunense, carefully avoiding duplicated sampling.Samples were collected at random from the northern populations;94 from N1and79from N2.In contrast,samples were collected from all clusters in the southern populations;40from S1,12from S2and 6from S3.Samples were transported and stored in cold storage.ElectrophoresisA total of 231samples were studied for allozyme var-iation,using 10enzyme systems.The following enzymes were analyzed;alcohol dehydrogenase (ADH),glutamate oxaloacetate transaminase (GOT),leucine aminopeptidase (LAP),menadion reductase (MNR),6-phoshogluconate dehydrogenase (6PG),phosphoglucoisomerase (PGI),phosphoglucomutase (PGM),shikimate 5-dehydrogenase (SDH),triose-phosphate isomerase (TPI),and UDP-glucose pyro-phosphorylase (UGP).Electrophoresis was conducted following the general methods of Gottlieb (1981)and Soltis et al.(1983),using the Tris–HCl grinding buffer-PVP solution described by Gottlieb (1981).Three buffer systems were used to analyze enzymes.System 1was the gel and electrode buffer system 8of Soltis et al.(1983),and was used to analyze PGI and TPI.System 2was the method of Cardy et al.(1981),and was used to analyze 6PG and UGP.System 3was the method used for acrylamide electrophoresis described by Tsumura et al.(1989),and was used to analyze ADH,GOT,LAP,MNR,PGM,and SDH.Data analysisFor estimation of the genetic structure of C.macran-thos var.rebunense ,the following genetic statistics were calculated:proportion of polymorphic loci (P ),mean number of alleles per locus (n a ),effective number of alleles per locus (n e )(Kimura and Crow 1964),average expected (H e )and observed (H o )het-erozygosity (Nei 1973),and Nei’s genetic distance among populations (Nei 1972)using the program TFPGA (Miller 1997),inbreeding coefﬁcient (F is )(Weir and Cockerham 1984),and Nei’s genetic differ-entiation (G st )(Nei 1973)using the program FSTAT (Goudet 1995).Statistical differences in H e among the populations were tested using the Kruskal–Wallis test.Deviations from Hardy–Weinberg equilibrium (devia-tion of F is value from 0)were examined using permu-tation.For estimation of the correlation between genetic and geographic distance,the Mantel test was employed.UPGMA clusters based on genetic and geographic distance were also compared.Results Taxon levelThe following 13putative loci were investigated:Adh,Got-1,Got-2,Got-3,Lap,Mnr,6pg,Pgi,Pgm,Sdh,Tpi-1,Tpi-2,and Ugp.The zymogram of GOT is shown in Fig.2as an example.Of the 13loci,8were polymorphic and 5were monomorphic.The number of samples and the allele frequency at the polymorphic loci in every population and overall populations of C.macranthos var.rebunense are shown in Table 1.These genetic statistics are shown in Table 2for each C.macranthos var.rebunense population:P was 0.62;n a and n e were 1.85and 1.28,respectively;H e and HoFig.1Map showing locations of the Rebun Island and the researched sites.The location of the Rebun Island is indicated with an arrow.The northern populations of Cypripedium macranthos var.rebunense are indicated by two ﬁlled circles.The southern populations exist within the shaded circle;the detailed location of these southern populations is not identiﬁed to prevent from illegalcollectionFig.2Photograph of zymogram of Glutamate-oxaloacetate transaminase (GOT).Locus 1and 3are monomorphic.Locus 2is polymorphic with 4alleles,a,b,c and dwere0.187and0.163,respectively;F is was0.066,indi-cating no signiﬁcant deviation from Hardy–Weinberg equilibrium(P>0.01);and G st was0.085.Population levelSeveral genetic statistics were also calculated for each population:P,n a,n e,H e,H o,and F is(Table3).Vari-ations in H e and H o were detected using the Kruskal–Wallis test,and deviation from Hardy–Weinberg equilibrium of F is was determined using the permuta-tion test.Neither statistical test detected any signiﬁcant differences or deviations.The lowest values of P(0.54), n a(1.54),and H o(0.092)were observed in the S2 population.H o in S2was particularly low compared with the overall value(0.163),and this was reﬂected in the value of F is(0.568).This F is value shows no sig-niﬁcant deviation from Hardy–Weinberg equilibrium in any population(P>0.05),but was the highest value in populations of C.macranthos var.rebunense. Genetic distance showed signiﬁcant correlation to geographic distance(r=0.683,P=0.042).However, low correlation was detected between the southern populations when compared with northern populations (Fig.3).DiscussionInterspeciﬁc comparisonsCypripedium macranthos var.rebunense has a rela-tively high genetic diversity when compared to other plant taxa.The comparisons below were conducted in order to elucidate the current situation of genetic diversity of C.macranthos var.rebunense.In compar-ison with our results,lower genetic diversities have been reported in other rare Lady’s slippers.Case et al.Table1Allele frequencies at eight polymorphic loci and number of samples in the examined populations for C.macranthos var. rebunenseLoci and alleles North South OverallN1N2S1S2S3Got-2a0.058––––0.021 b0.0710.019–––0.033c0.5380.7470.962 1.0000.7500.724d0.3330.2340.038–0.2500.222 Lap a0.0800.0250.0260.083–0.048 b0.8210.8800.9490.9170.8330.871c0.0990.0950.026–0.1670.081 Mnr a0.8800.9490.4870.5000.6670.808 b0.1200.0510.5130.5000.3330.192 6pg a0.9940.9230.8380.8180.8000.929 b0.0060.0770.1620.1820.2000.071 Pgi a0.4890.6410.5510.4170.1670.540 b0.5110.3590.4490.5830.8330.460 Pgm a0.9350.8700.7180.8750.5000.861 b0.0650.1300.2820.1250.5000.139 Sdh a0.5750.6650.8210.6250.9170.664 b0.4260.3350.1790.3750.0830.336 Tpi-2a0.0320.0490.1670.3640.1000.081 b0.9680.9510.8330.6360.9000.919 Number in sample947940126231 Table2The comparison of C.macranthos var.rebunense and other plant taxa(Hamrick and Godt1989)P n a n e H e G st C.macranthos var.rebunense0.62 1.846 1.2800.1870.085 Endemic species0.40 1.800 1.1500.0960.248 Monocots0.49 1.790 1.2700.1810.231 Long-lived herbaceous0.40 1.420 1.2800.2050.213 Mixed-animal pollination0.40 1.680 1.1600.1200.216 Seed dispersal by wind0.55 2.100 1.1900.1440.143 P=proportion of polymorphic loci;n a=mean number of alleles per locus;n e=effective number of alleles per locus;H e=expected heterozygosity;G st=genetic differentiation(1998)reported low-level genetic diversity(P=0.25, n a=1.33,H e=0.050)in C.kentuckiense.Aagaard et al.(1999)also reported low-level genetic diversity (P=0.17–0.25,n a=1.17–1.25,H e=0.040)in C.fas-ciculatum.In the case of C.arietinum,an extremely endangered Lady’s slipper,Bornbusch et al.(1994)and Case(1994)documented a total lack of genetic diver-sity(P=0.00).However,similar or higher genetic diversities were reported in C.calceolus by Brzosko et al.(2002,P=0.46,n a=1.73,H e=0.184)and Case (1994,P=0.75,n a=2.50,H e=0.244).Although all the Lady’s slippers cited above are,to varying degrees, rare species,they show great genetic diversity.Even in comparison with other taxa with the same ecological or life history characters,genetic diversity of C.macranthos var.rebunense can be regarded as rel-atively high level.Hamrick and Godt(1989)reviewed and categorized a number of allozyme diversities in plants by studying their life history characters(see Table2).With the exception of the G st value,all other genetic statistical values of C.macranthos var.rebun-ense were higher than the mean values of other taxa categorized by the same ecological and/or life history characters.Differences between the mean values of endemic species and the values of C.macranthos var. rebunense as regards to genetic diversity are striking in comparison(Table2).The values of P,n a,n e,and H e in this plant are higher than the mean value in endemic species—the H e value in particular is almost double.The G st value of C.macranthos var.rebunense is one third of the mean value of endemic species. Endemic species generally tend to show low-level genetic diversity and high genetic differences among populations due to the bottleneck effect and genetic drift associated with population fragmentation.One of the possible reasons for the low G st value of C.mac-ranthos var.rebunense is the fairly restricted distribu-tion area(Rebun island is approximately81km2).It is not,however,known why genetic diversity of C.mac-ranthos var.rebunense is of a high level despite the fact that it is an endemic and endangered plant.Low-level genetic diversity of endemic and/or endangered plants has been reported(Hamrick et al.1979;Karron1987; Soltis and Soltis1991).However,in agreement with our observations,relatively high-level genetic diversity in spite of endemism and/or endangered status has also been reported(Lewis and Crawford1995;Ranker 1994;Sharma et al.2003;Zhang et al.2005).More-over,Karron(1991)found great variability in a rare plant,as per our results.Intraspeciﬁc comparisonWhen genetic statistics P,n a,n e,H e,and H o were compared among the populations,no signiﬁcant dif-ferences were detected(Table3).The differences we found occurred mainly between the northern and southern populations.Firstly,allele disappearance wasTable3The comparative table of each population for C.macranthos var.rebunenseP n a n e H e H o F is Sample number N10.62 1.846 1.3090.1770.1730.02794N20.62 1.769 1.2310.1590.1480.07979S10.62 1.692 1.2870.1860.195–0.03440S20.54 1.538 1.3270.1990.0920.56812S30.62 1.615 1.2890.1940.2000.0666Overall0.62 1.846 1.2800.1870.1630.066231P=proportion of polymorphic loci;n a=mean number of alleles per locus;n e=effective number of alleles per locus;H e=expectedheterozygosity;H o=observed heterozygosity;F is=inbreedingcoefﬁcientFig.3Theﬁgure showing UPGMA phenograms,upper one indicates of the genetic distance and lower one indicates the geographic distance.The digits in bifurcation point exhibit each internode both,genetic and geographic(m)distancedetected(Table1).The numbers of alleles lost were1, 2,4,and3in populations N2,S1,S2,and S3,respec-tively.Allele disappearance was striking in Locus Got-2,where absolute loss of polymorphism was observed in population S2.Secondly,increase in genetic distance was found among populations.Genetic distance devi-ated from geographical distance(Fig.3)and was striking among southern populations as well as between northern and southern populations;however, it was not observed between N1and N2populations. Finally,when comparing homozygosity across all populations,increased homozygosity was observed in population S2.Since the size of the S2and S3popu-lations was very small,genetic drift or inbreeding may have played an important role in the increase of homozygosity.F is values showed no signiﬁcant devia-tion from Hardy–Weinberg equilibrium,but the F is value of population S2(0.568)was remarkably higher than in other populations.Possible causes of these differences include(1)the bottleneck or founder effect in the distant past,and(2) change of genetic composition due to illegal collection (genetic disturbance).According to the islanders,the populations were previously much larger,and as such the latter factor may have affected the change in population size that brought about genetic change.If this were indeed the case,genetic drift and inbreeding depression associated with population decrease and fragmentation have not yet conspicuously damaged the chances of population survival;however survival also depends on the life history character of Cypripedium.Nicole´et al.(2005)stated that C.calce-olus populations have remarkably slow and stable dynamics due to extremely long individual life spans (mean longevity from110to350years).C.acaule also has a long life span of about26years(Ehrle´n and Lehtila¨2002).Even though accurate estimation of the longevity of Cypripedium is difﬁcult due to the fre-quent cycle of dormancy and budding,we can easily infer that C.macranthos var.rebunense has similar longevity to other Cypripedium,namely several dec-ades or hundreds of years.The decrease in individual numbers seen since the1950’s has not occurred in a sufﬁciently long time period to produce bottleneck effects in this taxon.Although the genetic disturbance reported in the present study is possibly a temporary phenomenon,we speculate that the genetic diversity of C.macranthos var.rebunense will decline with time.The natural distribution area of C.macranthos var. rebunense has become divided into northern and southern populations due to decreasing individual numbers as a result of human activity,and this population fragmentation between the northern and southern populations may cause reduction of gene ﬂow.Geneﬂow between the northern and southern populations occurs by one of only two methods;(1) pollen movement by insect pollinators,and(2)seed dispersal by the wind.Considering the former method, we expect that pollen transfer by insects is not likely due to the geographical distance between the northern and southern populations(approximately12km). Pollination of C.macranthos var.rebunense takes place via a queen bee of Bombus pseudobaicalensis (Sugiura et al.2001).As there is no data concerning the foraging range of the queen,we have no exact information on the pollination range of C.macranthos var.rebunense.However,the foraging range of a bumblebee worker is approximately several hundred meters only(Knight et al.2005);thus it is very improbable that the queen bee of B.pseudobaicalensis can forage around12km,well beyond the range of a worker.Considering the latter geneﬂow method through seed dispersal by the wind,while this may be possible,it would account for only a low rate in total geneﬂow between the northern and southern popula-tions.Although orchids are generally known to show wind seed dispersal over a wide range,seed germina-tion of orchids is highly dependent on the help of symbiotic mycorrhizal fungus under natural conditions. This fungus may be native to the habitat of C.mac-ranthos var.rebunense.However,mutual seed trans-portation between the northern and southern populations may be fairly restricted due to the distance involved and the relatively small proportion of habitat area to potential seed dispersal area.It is highly probable that geographical distance is a barrier to gene ﬂow between the northern and southern populations. We believe it is necessary to focus our attention on the low level of geneﬂow caused by fragmentation across the northern and southern populations of C.macran-thos var.rebunense.However,as Forrest et al.(2004) reported a relatively wide range of geneﬂow in orchid plants,seed dispersal level in orchids may be much higher than our predictions.Seed dispersal should therefore be monitored using other molecular markers, such as microsatellite markers,in C.macranthos var. rebunense.The conservation programA serious conservation problem has developed in the southern populations.Fragmentation appears to be causing negative effects,namely inbreeding depression and allele disappearance,leading to decline of genetic diversity(Frankham et al.2002).The southern popu-lations are particularly susceptible to these negativeeffects,because these effects are more apparent in smaller populations.A population with an effective size of50is considered to be the minimum to retain sufﬁcient allelic richness,while an effective size of500 individuals is required to counteract the effect of ge-netic drift and permit evolutionary change(Frankel et al.1995).Accordingly,almost all the populations investigated in the present study might not maintain their own genetic diversity if they remain separate from the N2population.Even if geneﬂow were high within the northern and southern populations,a critical situation might be reached in the southern populations because the sizes of the southern populations are apparently lower than the effective size needed to maintain genetic diversity.Consequentially,there is the possibility of population extinction,which is itself connected to species level extinction.We therefore recommend that some kind of conservation activity be employed to preserve the southern populations.Although the preservation of endangered species understandably relies on the natural recovery potential of the species and area,artiﬁcial reintroduction may have to be considered in extreme cases(Pavlik1994). Our study clariﬁed the alarming situation for the southern populations.To conserve them,mature seed or pollen transfer from the northern populations via human intervention is needed and this creation of an ‘‘artiﬁcial geneﬂow’’may,in fact,be the most effective method.As mentioned in the Introduction,the out-planting population of Mauna Kea Silversword has experienced a major population bottleneck.To over-come this bottleneck,manual pollen transfer was car-ried out(Robichaux et al.1997)and this artiﬁcial gene ﬂow produced good results.In the case of C.calceolus in Britain,artiﬁcial geneﬂow between a conserved wild population and a cultivated population has been planned(Ramsay1998).Major points to note in planning rehabilitation by such artiﬁcial geneﬂow methods are(1)loss of regional unique genetic variation,and(2)outbreeding depression due to introgression from a different origin population(Adams et al.2002;Hufford and Mazer 2003).These impediments may not matter in our case because C.macranthos var.rebunense showed a rather low value of G st across all populations studied.How-ever,careful attention should be paid when planning artiﬁcial geneﬂow.Our study revealed genetic diver-sity at the allozyme level,but it is not enough merely to consider the following practical questions without considering genetic background:Which plants should be chosen as parents?How can we conduct follow-up research to examine the recovery of genetic diversity? To answer these questions,we need to use a genetic marker with a higher resolution than allozymes. We have just started developing microsatellite markers that we hope will prove to be a powerful tool for monitoring the achievements of the conservation pro-gram for C.macranthos var.rebunense. Acknowledgements We thank M.Somata,S.Miyamoto, Y.Saito and other islanders for their kind help during the ﬁeldwork on Rebun Island.We also thank Soya Forestry Man-agement Ofﬁce and Rebun Town.Theﬁrst author thanks Y.Sato,Y.Kato,G.Sarwar and ckner for their help during ﬁeldwork and grammatical revision of the manuscript.This study was supported by the Ministry of Environment of Japan(Kogai-boshi-to shiken kenkyu)and the Ministry of Education,Culture, Sports,Science and Technology of Japan(Grant-in-Aid for Scientiﬁc Research,16310157).ReferencesAagaard JE,Harrod RJ,Shea KL(1999)Genetic variation among populations of rare clustered lady-slipper orchid (Cypripedium fasciculatum)from Washington A Nat Areas J19:234–238Adams JM,Piovesan G,Strauss S,Brown S(2002)The case for genetic engineering of native and landscape trees against introduced pests and diseases.Conserv Biol 16:874–879Bornbusch AH,Swender LA,Hoogerwerf DL(1994)Genetic variation in Massachusetts populations of Cypripedium arietinum R.Brown in Ait.and C.acaule Ait.(Orchida-ceae).Rhodora96(888):354–369Brzosko E,Ratkiewicz M,Wroblewska A(2002)Allozyme differentiation and genetic structure of the Lady’s slipper (Cypripedium calceolus)island populations in north–east Poland.Bot J Linn Soc138:433–440Cardy BJ,Stuber CW,Goodman MM(1981)Techniques for starch gel electrophoresis of enzyme from maize(Zea mays),revised.Instl.Stat.Mimeogr.Ser.1317.North Carolina State UniversityCase MA(1994)Extensive variation in the levels of genetic diversity and degree of relatedness amongﬁve species of Cypripedium(Orchidaceae).Am J Bot81(2):175–184 Case MA,Mlodozeniec HT,Wallace LE,Weldy TW(1998) Conservation genetics and taxonomic status of the rare Kentucky lady’s slipper:Cypripedium kentuckiense(Orchid-aceae).Am J Bot85(12):1779–1786Cribb P(1997)The genus Cypripedium.Timber Press,Portland Ehrle´n J,Lehtila¨K(2002)How perennial are perennial plants?OIKOS98:308–322Environmental Agency of Japan(2000)Threatened wildlife of Japan,Red data book2nd ed.,vol8,Vascular plants.Japan Wildlife Research Center,TokyoForrest AD,Hollingsworth ML,Hollingsworth PM,Cydes C, Bateman RM(2004)Population genetic structure in Euro-pean populations of Spiranthes romanzofﬁana set in the context of other genetic studies on orchids.Heredity 218–227Frankel OH,Brown AHD,Burdon JJ(1995)The conservation of plant biodiversity.Cambridge University Press,Cam-bridgeFrankham R,Ballou JD,Briscoe DA(2002)Introduction to conservation genetics.Cambridge University Press,Cam-bridge。