Endangered Epiphytic Orchid Research Articles

Diversity and specificity of orchid mycorrhizal fungi in a leafless epiphytic orchid, Dendrophylax lindenii and the potential role of fungi in shaping its fine-scale distribution

Orchids grow in diverse habitats worldwide with most (approximately 69%) growing on trees as epiphytes. Although orchid mycorrhizal fungi have been identified as potential drivers for terrestrial orchid distribution, the influence of these fungi on the fine-scale distribution of epiphytic orchids is poorly understood. In this study, we investigated the mycorrhizal fungal community and fine-scale distribution of Dendrophylax lindenii, a rare and endangered epiphytic orchid that is leafless when mature. We used amplicon sequencing to investigate the composition of orchid mycorrhizal fungi in the roots of 39 D. lindenii individuals in their natural habitat, the swamps of Florida. We compared the orchid mycorrhizal fungi of D. lindenii to those of co-occurring epiphytic orchids, as well as to the orchid mycorrhizal fungal communities of bark from potential host trees, with and without D. lindenii. Our results show that D. lindenii has a high specificity for a single Ceratobasidium species, which is widely distributed on phorophytes and detected in both wet and dry periods in the orchid’s habitat. This Ceratobasidium species was mostly absent or only recorded in low frequency in the roots of co-occurring epiphytic orchids. Phylogenetic analysis documented that this Ceratobasidium was conspecific with the strain that is used to germinate D. lindenii ex-situ. However, our findings suggest that laboratory germinated adult D. lindenii transplanted into the field had lower read abundances of this Ceratobasidium compared to naturally occurring plants. These findings suggest that this orchid mycorrhizal fungus may play a significant role in the fine-scale distribution of naturally occurring D. lindenii.

Dominant Dendrobium officinale mycorrhizal partners vary among habitats and strongly induce seed germination in vitro

Dendrobium officinale(Orchidaceae) is an endangered epiphytic orchid that has been well studied as a medicinal plant. Although previous studies have shown that various fungal isolates promoteD. officinaleseed germination and seedling developmentin vitro, mycorrhizal associations among its wild populations remain poorly understood. In this study, we identified mycorrhizal fungi associated withD.officinale(36 individuals from six sites) using Sanger sequencing and compared fungal communities among sites and habitats (lithophytic vs. epiphytic individuals). Among the obtained sequences, 76 belonged to orchid mycorrhizal fungi (OMF), among which Tulasnellaceae accounted for 45.8% and Serendipitaceae for 28.1%. The Serendipitaceae operational taxonomic unit (OTU) SE1 was the most dominant partner, accounting for 27.1% of all detected fungal sequences, followed by a Tulasnellaceae OTU, TU27, which accounted for 15.6%. The relative frequencies of Serendipitaceae and Tulasnellaceae differed greatly between lithophytic and epiphytic individuals. Serendipitaceae accounted for 47.3% of the OMF sequences among lithophytes, and Tulasnellaceae for 95.2% among epiphytes. Mycorrhizal community composition also varied among sites. We further conductedin vitrosymbiotic culture from seeds with six fungal isolates. Two Serendipitaceae and two Tulasnellaceae isolates, including SE1 and TU27, significantly promoted seed germination and seedling development. These results indicate thatD.officinaleis mainly associated with Tulasnellaceae and Serendipitaceae as its main fungal partners, which strongly induced seed germination and seedling developmentin vitro, suggesting their association withD.officinalethrough its life cycle.

Low Concentration of Anti-Auxin and Anti-Fungal Agent Accelerates the PLB Regeneration of Dendrobium okinawense under Green LED.

Dendrobium okinawense is an endangered epiphytic orchid, and there has been no scientific report so far on its propagation. Protocorm is a mass of cells, and protocorm-like bodies (PLBs) are lookalike protocorms produced by vegetative explants in vitro. Regeneration of PLBs is a widely used technique for orchid micropropagation. We used different light-emitting diodes (LEDs) for the PLB regeneration of D. okinawense. The number of PLBs and fresh weight were increased by 81.1% and 80.8%, respectively, under green LED over the white fluorescent (WF) light. We added different concentrations of PCIB (p-Chlorophenoxyisobutyric acid, an anti-auxin) and HMI (3-Hydroxy-5-methyl isoxazole, an anti-fungal agent) in culture media. The number of PLBs was increased in media having 0.01 mg/L of PCIB (35.9%) compared to control (no PCIB), whereas 19.3% increased in media having 0.01 mL/L of HMI compared to control (no HMI). Green LED in combination with 0.01 mg/L of PCIB significantly increased the number of PLBs (69.0%) compared to the WF–without PCIB combination. In LEDs-PCIB and LED-HMI combinations, HMI did not show better PLBs regeneration compared with PCIB. The results suggested that a combination of low concentrations of PCIB and green LED have the potential to accelerate PLB regeneration of D. okinawense.

Mating system and female reproductive success of the endemic and endangered epiphyte Rhynchostele cervantesii (Orchidaceae) in a cloud forest in Michoacan, Mexico

Background and aims – The Orchidaceae family is vulnerable, because of the destruction of their habitat, as well as the extraction of individuals from natural populations. This is the case of the genus Rhynchostele Rchb.f.; among the actions considered important for appropriate conservation strategies for this genus is the generation of fundamental knowledge, such as on its reproductive biology. The objective of this work is to understand the mating system and reproductive success of Rhynchostele cervantesii, an endangered epiphytic orchid endemic to Mexico.Material and methods – Manual and open-pollination treatments were conducted during 2014 and 2015 in a cloud forest in Michoacan, Mexico. In each period, 30 to 40 randomly selected inflorescences were subjected to the following treatments: a) spontaneous-self-pollination, b) emasculation, c) self-pollination, d) cross-pollination, and e) open-pollination. The developed fruits were counted and harvested, the viability of the seeds was determined, through the observation and evaluation of embryos using microscopy.Key results – Significant differences were recorded between the treatments in both 2014 and 2015, with higher fruit production in cross-pollination than in self-pollination and natural-pollination. There were significant differences in seed viability, with higher values for seeds from open-pollination and cross-pollination and lower values for seeds from self-pollination.Conclusions – Rhynchostele cervantesii is a species that requires pollinators for sexual reproduction because there is no fruit production with spontaneous-self-pollination. Under pollen limitation, the fruit set of natural pollination was a lot lower than in cross-pollination although fruits were the same quality. R. cervantesii had a mixed mating system with a tendency to exogamy, presenting high values of female reproductive success compared to other tropical epiphytic orchid species reported in the literature.

Specialized pollination by honeybees in Cymbidium dayanum, a fall–winter flowering orchid

AbstractFloral biology and pollination mechanisms of Cymbidium dayanum, an endangered epiphytic orchid, were investigated in south Kyushu, Japan. The flowering period spanned approximately 4 months from fall to winter, with the inflorescences often blooming asynchronously on each plant and individual flowers commonly lasting for a month. The nectarless flowers are self‐compatible but cannot autonomously self‐pollinate; the orchid needs to attract pollinators by deceit for capsule production. The field observations showed that the flowers were pollinated exclusively by the Japanese honeybee Apis cerana japonica, despite its long flowering period. The worker bees pollinated flowers and/or received the pollinarium on the thorax, while escaping from the labellum chamber. The natural fruit‐set ratios at the population level, an estimate of pollination success, varied interannually but were generally low, as the result of infrequent flower visits by honeybee workers owing to the cold winter climate and the lack of a nectar reward. Because most flowers that were artificially pollinated in the winter successfully developed into capsules, the coldness was not considered a direct cause of the low fruit‐set ratios. Our results explicitly indicate that the bee pollination niche could be available to temperate plant species even during mid‐winter. We inferred advantages for the winter flowering of C. dayanum.

In vitro desiccation tolerance of the epiphytic Ghost Orchid, Dendrophylax lindenii (Lindl.) Benth x. Rolfe

The Ghost Orchid, Dendrophylax lindenii (Lindl.) Benth x. Rolfe, is a rare and endangered epiphytic orchid native to south Florida and Cuba. The orchid is considered difficult to propagate under greenhouse conditions, requiring high humidity and low air movement. In contrast, the orchid’s native habitat seasonally dries out with decreased precipitation and humidity. This suggests some level of desiccation tolerance. Ghost Orchid plants were assessed for potential desiccation tolerance and ability to recover from desiccation stress under in vitro conditions. In vitro-derived plants were placed into sterile baby food jars and transferred to chambers maintained at 10% relative humidity, which is extremely low compared to relative humidity levels (40–100%) recorded under natural field conditions. Plants were removed every week for 4 wk and recovered on P723 medium supplemented with banana powder for 4 wk. Data were collected at the initiation of the experiment, after the desiccation periods, and after 4 wk in vitro recovery. Ghost Orchid plants demonstrated extremely high desiccation tolerance. Even after 4 wk desiccation, plant survival was observed at 79.2% after recovery. Desiccated plants exhibited significant decreases in tissue water potential (− 18.44 MPa), fresh weight (65.5% loss), and water content (14.2%); however, high plant survival was still observed under these conditions similar to poikilohydric plants. Overall, the Ghost Orchid demonstrated high desiccation tolerance, which should be considered for future greenhouse culture and for its application in the direct field establishment of in vitro-derived plants without greenhouse acclimatization.

Host selection and distribution of Dendrobium okinawense, an endangered epiphytic orchid in Yambaru, Japan

AbstractWe investigated the epiphytic habit and distribution of Dendrobium okinawense, an endangered orchid in Yambaru (northern Okinawa), Japan. Fifty individuals of D. okinawense were detected on 46 host trees. From the canopy size in chronological aerial photographs, the habitat stands where D. okinawense was found were estimated to be mature. Host trees tended to have larger diameters. The most common host species was Castanopsis sieboldii, which dominates in Yambaru; several other tree species were described as hosts for the first time to our knowledge. However, the orchid was mainly associated with one of these, Distylium racemosum, irrespective of this tree's small basal area relative to other trees. We attribute this preference by D. okinawense to the external form and longevity of D. racemosum. The presence of D. okinawense and its host D. racemosum with large diameter is a good indicator of the existence of old‐growth forests containing endemic rare species in Yambaru.

Using In Situ Symbiotic Seed Germination to Restore Over-collected Medicinal Orchids in Southwest China.

Due to increasing demand for medicinal and horticultural uses, the Orchidaceae is in urgent need of innovative and novel propagation techniques that address both market demand and conservation. Traditionally, restoration techniques have been centered on ex situ asymbiotic or symbiotic seed germination techniques that are not cost-effective, have limited genetic potential and often result in low survival rates in the field. Here, we propose a novel in situ advanced restoration-friendly program for the endangered epiphytic orchid species Dendrobium devonianum, in which a series of in situ symbiotic seed germination trials base on conspecific fungal isolates were conducted at two sites in Yunnan Province, China. We found that percentage germination varied among treatments and locations; control treatments (no inoculum) did not germinate at both sites. We found that the optimal treatment, having the highest in situ seed germination rate (0.94-1.44%) with no significant variation among sites, supported a warm, moist and fixed site that allowed for light penetration. When accounting for seed density, percentage germination was highest (2.78-2.35%) at low densities and did not vary among locations for the treatment that supported optimal conditions. Similarly for the same treatment, seed germination ranged from 0.24 to 5.87% among seasons but also did vary among sites. This study reports on the cultivation and restoration of an endangered epiphytic orchid species by in situ symbiotic seed germination and is likely to have broad application to the horticulture and conservation of the Orchidaceae.

In vitro seed germination and seedling growth of an endangered epiphytic orchid, Dendrobium officinale, endemic to China using mycorrhizal fungi (Tulasnella sp.)

To date, tropical orchids appear to commonly associate with mycorrhizal fungi assignable to Tulasnella (Basidiomycota) for their seed germination and developmental needs in situ. In this study, two Tulasnella strains (JC-02 and JC-05) isolated from roots of an endangered species, Dendrobium nobile Lindl (Orchidaceae) collected from Yunnan province in China, were identified using the nuclear ribosomal internal transcribed spacer (ITS) and 5.8S rDNA sequences. Seed germination and plant growth were evaluated up to 11 weeks and 7 weeks after interaction with Tulasnella fungi, respectively. The results revealed that the two isolates could promote seeds germination up to stage 5 after sowing for 11 weeks, and the rates of germination were 98.47% and 99.05%, respectively, higher than that of control (81.05%). Without fungi, seed development was arrested at stage 2. After inoculating Tulasnella isolate to seedlings for 49 days, it was found that mycelium formed pelotons in the cortical cells of roots in the form of intact and degenerate pelotons. Significant differences were detected between the control group and the experimental group treated with Tulasnella isolates in dry weight and fresh weight of plants respectively. The study firstly demonstrated that two different strains of Tulasnella vary in their ability to facilitate germination/development of D. officinale in vitro.

Development of microsatellite markers for genetic diversity analysis of Dendrobium loddigesii Rolfe, an endangered orchid in China

Abstract Dendrobium loddigesii Rolfe is an endangered epiphytic orchid with high medicinal and horticulture value. To estimate genetic diversity and population structure of D. loddigesii , 12 new microsatellite markers of D. loddigesii were developed from an enriched library. A total of 98 alleles were detected with an average of 8.2 alleles per locus. The expected heterozygosity of each SSR locus varied from 0.454 to 0.857 with an average of 0.690. Polymorphism information content (PIC) of each SSR locus ranged from 0.358 to 0.838 with an average of 0.637. A high level of genetic diversity was detected. Analysis of molecular variance showed that the main variation component existed within populations (82.02%) rather than among populations (17.98%). Two main clusters were shown in UPGMA dendrogram using TFPGA, which was consistent with the result of principal coordinate analysis (PCA) using NTSYS. These microsatellite makers will contribute to research on individual identification, genetic diversity, population structure, genome mapping and conservation biology of D. loddigesii .

Asymbiotic Seed Germination, Induction of Calli and Protocorm-like Bodies, and In Vitro Seedling Development of the Rare and Endangered Nothodoritis zhejiangensis Chinese Orchid

Nothodoritis zhejiangensis Z. H. Tsi is a rare and endangered epiphytic orchid restricted to a narrow distribution in China. The species is threatened with extinction as a result of loss of suitable habitats. An efficient propagation system is part of this species’ conservation plan. Seed germination reached 64.7% on Knudson's C (KC) medium containing 1.0 mg·L−1 α-naphthaleneacetic acid (NAA), 10% coconut water, and 0.1% activated charcoal. After 50 days culture, most callus (71.3%) from seed-derived protocorms formed on KC medium with 1.0 mg·L−1 2,4-dichlorophenoxyacetic acid and 10% coconut water. Callus could be subcultured 12 times during ≈2 years with more than a 3.0-fold increase from the third to the twelfth subculture. Furthermore, 84% of callus from the tenth sub-culture on KC media supplemented with 1.0 mg·L−1 NAA, 5% coconut water, and 0.1% activated charcoal formed protocorm-like bodies (PLBs). Many (57%) protocorms on KC medium containing 1.0 mg·L−1 6-benzylaminopurine and 10% coconut water formed PLBs. Both callus and PLBs formed simultaneously from different protocorms on KC medium containing 0.01 to 1.0 mg·L−1 thidiazuron and 10% coconut water. PLBs were incubated 12 times during ≈2 years with an approximate 2.4-fold increase per sub-culture. Both callus and PLBs maintained their competence to regenerate plantlets. Hyponex N026 medium supplemented with 1.0 mg·L−1 NAA, 50 g·L−1 banana homogenate, and 0.1% activated charcoal was suitable for plantlet formation and growth of 95.5% of plantlets that developed from PLBs. The roots of plantlets 2 cm in height or taller were wrapped in Chilean sphagnum moss and fixed to a fir bark block; 69.3% of plantlets survived after 180 days in a greenhouse. This protocol is an efficient means for the large-scale propagation of this endangered orchid.

In vitro propagation of the endangered orchid Laelia speciosa

Laelia speciosa is an endangered epiphytic orchid. The effects of various media components on germination of L. speciosa were evaluated. Pods were collected at 4, 7, and 9 months following hand-pollination, and seeds were germinated on Murashige and Skoog (MS) media with 30 g l−1 sucrose and five concentrations (0.0, 0.04, 0.22, 0.44, and 2.22 μM) of benzyladenine (BA) under light and dark conditions. Gibberellic acid (GA3; 0.0, 0.29, 1.44, 2.89, 14. 43 and 28.87 μM) with naphthaleneacetic acid (NAA; 0.0, 0.54, 1.34, 2.69, and 5.37 μM) were evaluated for in vitro subcultivation. MS medium with 30 g l−1 sucrose was effective for germination. The effects of BA and light on germination of L. speciosa seeds differed with pod maturity. All mature seeds germinated using 0.44 μM BA and light. The highest frequency of germinated seedlings (60%) was obtained using mature seeds grown on MS medium without BA and under light conditions. For subculture, MS with 30 g l−1 sucrose, 2.69 μM NAA, and 0.29 μM GA3 was effective. Plantlets of 5 cm in length were transplanted to the greenhouse, and a 77.5% of survival rate was obtained. A successful protocol for micropropagation by seed germination will contribute to the development of a sustainable management program for L. speciosa.

Reproductive biology of Cyrtopodium punctatum in situ: implications for conservation of an endangered Florida orchid

Abstract Cyrtopodium punctatum (Linnaeus) Lindley is an endangered epiphytic orchid restricted in the USA to southern Florida. This species has been extensively collected from the wild since the early 1900s, and today only a few plants remain in protected areas. As part of a conservation plan, a reproductive biology study was conducted to better understand the ecology of this species in Florida. Cyrtopodium punctatum relies on a deceit pollination system using aromatic compounds to attract pollinators. Nine aromatic compounds were identified as components of the fragrance of C. punctatum inflorescences, including two compounds that are known to be Euglossine bee attractants. However, this group of bees is not native to Florida. Of the four bee species observed to visit C. punctatum flowers in the present study, carpenter bees (Xylocopa spp.) are likely to be the main pollinators. Pollination experiments demonstrated that C. puntatum is self‐compatible, but requires a pollinator and thus does not exhibit spontaneous autogamy. In addition, the rates of fruit set were significantly higher for flowers that were outcrossed (xenogamy) than for those that were self‐crossed. Thus, the species has evolved a degree of incompatibility. Examples of natural pollination and fruit set were observed during the present study (2007–2008), but the rates of reproduction were modest as a consequence of the low plant numbers and possible changes in insect densities as a result of anthropogenic influences.

Asymbiotic seed germination and in vitro seedling development of Cyrtopodium punctatum: a propagation protocol for an endangered Florida native orchid

Cyrtopodium punctatum Lindley is an endangered epiphytic orchid restricted in the United States to southern Florida. Due to its ornamental value, the species was extensively collected from the wild during the past 100 years. Today, only a few plants remain in protected areas. As part of a conservation plan for the species, procedures for asymbiotic seed germination were developed. Five asymbiotic orchid seed germination media (PhytoTechnology Orchid Seed Sowing Medium, Knudson C, Malmgren Modified Terrestrial Orchid Medium, Vacin & Went Modified Orchid Medium, and ½-strengh Murashige & Skoog) were examined for their effectiveness in promoting seed germination and protocorm development under a 16/8 h L/D photoperiod and dark (0/24 h L/D). The influence of photoperiod on growth and development was also examined. Seeds were germinated under a 16/8 h, 12/12 h, 8/16 h L/D photoperiod, at 25 ± 3°C and allowed to develop in vitro for 10 weeks. After 10 weeks, developing seedlings were transferred to Sigma Phytatrays and returned to their assigned photoperiod treatments for continued seedling development for an additional 15 weeks. Highest germination occurred in 0/24 h L/D on PhytoTechnology Orchid Seed Sowing Medium and seedlings displayed more advanced development when cultured under 16/8 h L/D photoperiod after 15 weeks in Phytatrays. Thirty-five week old seedlings potted in coconut husk growing medium exhibited 90% survival following 5 weeks acclimatization to greenhouse conditions. This asymbiotic seed germination protocol for C. punctatum will facilitate future reintroduction projects involving this endangered species.

Isolation and characterization of microsatellite loci in Phalaenopsis gigantea

This paper describes the characterization of the population structure of an ex situ population of Phalaenopsis gigantea, an endangered epiphytic orchid endemic to Sabah, Malaysia, using 30 polymorphic microsatellite loci. The objective of this investigation was the estimation of the genetic distance between accessions within an ex situ population on the basis of which a conservation strategy aimed at selective breeding and restoration was developed.

Isolation, characterization and cross‐amplification of polymorphic microsatellite loci in Laelia speciosa (Orchidaceae)

Laelia speciosa is an endangered epiphytic orchid endemic to central Mexico. Here, we report on the development of 14 perfect and imperfect microsatellite repeat loci for this species. Numbers of alleles ranged from two to 16 and levels of observed heterozygosities among the 14 loci ranged from 0.28 to 1.00 across two widely divergent populations. All loci were also tested for cross-amplification in four other Laelia species and other selected genera of the subtribe Laeliinae.

Conservation-driven Propagation of an Epiphytic Orchid (Epidendrum nocturnum) with a Mycorrhizal Fungus

Seeds of an endangered epiphytic orchid from Florida (Epidendrum nocturnum Jacquin) germinated in vitro with a mycorrhizal fungus [Epulorhiza repens (Bernard) Moore] using a technique normally applied to terrestrial orchids (symbiotic seed germination). Seeds from two sources (Fakahatchee Strand, Fla. Panther NWR) were sown on either modified oats medium (MOM) or standard oat medium (SOM) and inoculated with the fungus. Significant differences in germination were detected between the two seed sources. MOM had a significant effect on mean leaf length during incubation in vitro (F (1278) = 23.81, P > 0.000), but media had no significant effect on leaf number. After 48 days in vitro, all leaf-bearing seedlings were exposed to light and then transferred to greenhouse conditions ex vitro on sterile Sphagnum moss with or without half-strength Miracle-Gro (Scotts, Port Washington, N.Y.) commercial fertilizer. After 163 days ex vitro, seedlings on Sphagnum without Miracle-Gro displayed highest survivorship (>90%), whereas Miracle-Gro-exposed seedlings from standard oat agar experienced low (44%) survivorship. Healthy seedlings with a mycotrophic capability were obtained 1 year after sowing. A total of 43 seedlings were subsequently reintroduced into the Florida Panther NWR in Nov. 2005, 16 months after sowing. The symbiotic technique may, therefore, have practical merit for conservation of E. nocturnum and other epiphytic orchids threatened with extinction.

Conservation genetics of an endemic and endangered epiphyticLaelia speciosa(Orchidaceae)

We used isozymes (16 loci in 11 enzymatic systems) from Laelia speciosa, an endemic and endangered epiphytic orchid of Mexico, to assess the genetic diversity and population genetic structure in nine populations distributed along its geographic range, as well as to detect those populations that are genetically unique and therefore deserve high-priority protection. On average, the genetic diversity was high (percentage of polymorphic loci, P(p) = 76%, mean number of alleles per locus, A = 3.34, the average observed heterozygosity H(O) = 0.302, the average expected heterozygosity H(E) = 0.382). Moderate levels of inbreeding ( f = 0.216, 95% confidence interval = 0.029-0.381) were found. Low levels of genetic differentiation were observed among populations ((p) = 0.040); however, there was a significant correlation between geographic and genetic distances among the populations (Mantel test: r(2) = 0.43, P < 0.05). Populations located within the same mountain range were genetically more similar. Private alleles were found, so proper management requires protection and maintenance of genetic diversity throughout its range. In case of reintroduction, we suggest using individuals propagated from seeds from as many capsules as possible, from close populations. An ex situ conservation strategy also is proposed.