Gametophyte and Embryonic Ontogeny: Understanding the Reproductive Calendar of Cypripedium Japonicum Thunb
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Ghimire et al. BMC Plant Biology (2020) 20:426 https://doi.org/10.1186/s12870-020-02589-9 RESEARCH ARTICLE Open Access Gametophyte and embryonic ontogeny: understanding the reproductive calendar of Cypripedium japonicum Thunb. (Cypripedoideae, Orchidaceae), a lady’s slipper orchid endemic to East Asia Balkrishna Ghimire1 , Sungwon Son2, Jae Hyeun Kim2 and Mi Jin Jeong2* Abstract Background: The genus Cypripedium L. is one of the five genera of the subfamily Cypripedioideae, members of which are commonly known as lady’s slipper orchids. Cypripedium japonicum is a perennial herb native to East Asia, specifically China, Japan, and Korea. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List. Results: We investigated gametophyte development, including complete embryogenesis, in C. japonicum. The complete reproductive cycle is presented based on our observations. Anther development begins under the soil, and meiosis of pollen mother cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid–late June. The pattern of embryo sac formation is bisporic, and there are six nuclei: three forming the egg apparatus, two polar nuclei, and an antipodal cell in the mature embryo sac. Triple fertilization results in the endosperm nucleus, which degenerates when the proembryo reaches the eight-to-sixteen-cell stage. Conclusion: Our overall comparisons of the features of gametophyte and embryo development in C. japonicum suggest that previous reports on the embryology of Cypripedium are not sufficient for characterization of the entire genus. Based on the available information, a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared. Keywords: Cypripedium japonicum, Gametophyte development, Embryology, Reproductive calendar, Endangered species Background and the diverse morphology of the suspensor is one of The family Orchidaceae is one of the largest families of the most distinctive features of the whole family [1, 2]. flowering plants, along with Asteraceae [1]. The orchid The characteristic suspensor development, patterns of taxa exhibit an unusual pattern of embryo development, polyembryony, and vast diversity of megagametophyte development in orchids provide continual fascination for plant biologists investigating the embryology of this fam- * Correspondence: [email protected] – 2Division of Plant Resources, Korea National Arboretum, Yongmun 12519, ily [2 5]. The extraordinarily diverse pattern of suspen- South Korea sor morphology in orchids motivated Swamy [4]to Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ghimire et al. BMC Plant Biology (2020) 20:426 Page 2 of 14 conceive a classification scheme for embryo develop- and humus-rich soil in forests or on shady slopes along ment in Orchidaceae. In addition, anthers usually de- ravines at an altitude of 1000–2000 m. Due to its limited velop much earlier than ovules in orchid species; at distribution and small populations in its natural habitat, pollination, when anthers release mature pollen grains this species has been categorized as critically endangered containing fully developed vegetative and generative at the national level in Korea (although it is endangered cells, the ovule has yet to produce megaspore mother globally) [24]. Correspondingly, in China and Japan, the cells (MMCs [6, 7]). It has been suggested that ovule de- species is at a high risk of extinction due to habitat loss velopment is activated by pollination, after which the and anthropogenic activities such as overcollection for plant takes several weeks to produce a female gamete horticultural and medicinal purposes [25, 26]. [8]. Zhang and O’Neill [9] believed that the commonly Empirical knowledge of plant reproductive biology, observed prolonged periods of ovule development in the particularly for rare orchids, could help us determine family makes Orchidaceae an intriguing subject for em- whether inadequacy in the recruitment cycle controls bryological study. successful reproduction and constrains population The genus Cypripedium L. is one of the five genera of growth [27]. A few recent efforts have been made to the subfamily Cypripedioideae, commonly known as study the pollination biology and reproductive char- lady’s slipper [1, 10]. Habitat destruction and overcollec- acteristics of C. japonicum [28, 29]. In the early tion have demographic implications, leading to the de- twentieth century, Pace [30] wrote a comprehensive cline and regional extinction of populations of account of fertilization in the genus Cypripedium, al- Cypripedium spp. [11–13]. On the other hand, low con- though C. japonicum was not examined. Later, Sood version rates of flowers to fruit remain a major issue in and Rao [31] studied the embryology of Cypripedium the reproductive ecology of existing populations of Cyp- cordigerum and confirmed that the embryological ripedium spp. [14]. All the species of this group have a characteristics and pericarp structure in this species pouch-like lip, two fertile stamens, a shield-like stamin- were typical orchidaceous types. Although a majority ode, and a synsepal with fused lateral sepals, and this of orchids follow a monosporic pattern of embryo specific flower morphology does not offer an easy re- sac development [32, 33], a bisporic pattern has been ward to pollinators. A unique feature of Cypripedioideae described for Cypripedium and Paphiopedilum [34, orchids is the one-way trap flower, which offers a fixed 35]. While there was no structural or developmental and unidirectional route to pollinators (see [15]). Despite examination of antipodal cells in Pace’s[30]report, this fixed pollination route, slipper orchids exhibit a re- Poddubnaya-Arnoldi [36] described the well- markable degree of diversity in food mimesis traits such developed antipodal cells that undergo secondary as scent, color and flower size for their pollinators [15– multiplication in some Cypripedium species. In 19]. addition, most orchids fail to form a functional The genus Cypripedium comprises approximately 45– endosperm, although some degree of endosperm de- 50 species that are mostly found in temperate zones of velopment with a multinucleate stage in some spe- the Northern Hemisphere, mainly in temperate regions cies has been reported [30, 31, 36]. This failure to of Asia and North America, extending to the Himalayan form a triploid endosperm is one of the unique fea- regions and Central America [1, 10, 20]. The center of tures in orchid seed development. In recent years, diversity of the genus lies in eastern Asia, where 38 spe- several studies have been carried out focusing on the cies have been reported; in particular, China alone has embryology of different orchid species [2, 37–40]. an astonishing diversity of this genus, harboring 36 spe- However, little is known about the embryology and cies of which 25 species are endemic [20]. Many species reproductive biology of C. japonicum.Knowledgeof of Cypripedium in North America and Europe have be- gametophyte, embryo, and seed development is es- come rare due to overexploitation and illegal trade in sential for understanding successful reproduction in past centuries [12, 21]. The status of Cypripedium in this endangered orchid and for formulating a restor- northeastern Asia is no different. Twenty-four Asian ation strategy. Cypripedium species are currently included in the En- We investigated the detailed embryology and seed for- dangered or Critically Endangered category of the IUCN mation processes in this popular lady’s slipper orchid. Red List [22]. In addition, the Convention on Inter- The primary objectives of this study were (1) to investi- national Trade in Endangered Species of Wild Fauna gate male and female gametophyte development in artifi- and Flora (CITES) currently lists all Cypripedium species cially pollinated C. japonicum; (2) to understand the in CITES Appendix II [23]. fertilization, embryo, and seed development processes in Cypripedium japonicum Thunb. is an endemic species C. japonicum; and (3) to develop a complete reproduct- of the endangered East Asian lady slipper orchid distrib- ive calendar of the species showing key reproductive uted across China, Japan, and Korea. It grows in damp events leading to seed development. Ghimire et al. BMC Plant Biology (2020) 20:426