Mycorrhizal specificity does not limit the distribution of an endangered orchid species

What factors determine the distribution of a species is a central question in ecology and conservation biology. In general, the distribution of plant species is assumed to be controlled by dispersal or environmentally controlled recruitment. For plant species which are critically dependent on mycorrhizal symbionts for germination and seedling establishment, specificity in mycorrhizal associations and availability of suitable mycorrhizal fungi can be expected to have a major impact on successful colonization and establishment and thus ultimately on a species distribution. We combined seed germination experiments with soil analyses and fungal assessments using 454 amplicon pyrosequencing to test the relative importance of dispersal limitation, mycorrhizal availability and local growth conditions on the distribution of the orchid species Liparis loeselii, which, despite being widely distributed, is rare and endangered in Europe. We compared local soil conditions, seed germination and mycorrhizal availability in the soil between locations in northern Belgium and France where L. loeselii occurs naturally and locations where conditions appear suitable, but where adults of the species are absent. Our results indicated that mycorrhizal communities associating with L. loeselii varied among sites and plant life cycle stages, but the observed variations did not affect seed germination, which occurred regardless of current L. loeselii presence and was significantly affected by soil moisture content. These results indicate that L. loeselii is a mycorrhizal generalist capable of opportunistically associating with a variety of fungal partners to induce seed germination. They also indicate that availability of fungal associates is not necessarily the determining factor driving the distribution of mycorrhizal plant species.     

Mycorrhizal association and symbiotic germination of the terrestrial orchid Bipinnula fimbriata (Poepp.) Johnst (Orchidaceae)

In this study mycorrhizal fungi were isolated from the roots of the endemic terrestrial orchid Bipinnula fimbriata. Seven isolates were previously identified as the form-genus Rhizoctonia, a polyphyletic group known to form mycorrhizal associations with Orchidaceae. Two other isolates were included in the study: #793 isolated from Chloraea crispa, and #1325 Rhizoctonia solani, isolated from potato. After morphological and molecular characterization of the nine isolates, they were divided into three groups, Ceratobasidium sp., Tulasnella calospora and Thanatephorus cucumeris, to determine the diversity between isolates. Consensus ITS sequences were used for a blast search on the GenBank database, which confirmed the results of the morphological observations. Once the isolates were identified, an in vitro germination test was done with four plates of oatmeal agar inoculated with each fungus, plus an asymbiotic control. The germination stages of the seeds were recorded 30 days after sowing. All isolates obtained from B. fimbriata, and the isolate #793 from Chloraea crispa, promoted seed germination. However, the isolate #1325 Rhizoctonia solani, which is known as both a pathogen and an orchid symbiont, did not promote germination. This shows that B. fimbriata is associated with more than one mycorrhizal fungus in its habitat and has a broader potential specificity in vitro. The results support the hypothesis that at least one fungal isolate promotes the germination of B. fimbriata, permitting the conservation of this species in ex situ conditions.     

Mycorrhizal acquisition of inorganic phosphorus by the green-leaved terrestrial orchid Goodyera repens

BACKGROUND AND AIMS: Mycorrhizal fungi play a vital role in providing a carbon subsidy to support the germination and establishment of orchids from tiny seeds, but their roles in adult orchids have not been adequately characterized. Recent evidence that carbon is supplied by Goodyera repens to its fungal partner in return for nitrogen has established the mutualistic nature of the symbiosis in this orchid. In this paper the role of the fungus in the capture and transfer of inorganic phosphorus (P) to the orchid is unequivocally demonstrated for the first time. METHODS: Mycorrhiza-mediated uptake of phosphorus in G. repens was investigated using spatially separated, two-dimensional agar-based microcosms. RESULTS: External mycelium growing from this green orchid is shown to be effective in assimilating and transporting the radiotracer (33)P orthophosphate into the plant. After 7 d of exposure, over 10 % of the P supplied was transported over a diffusion barrier by the fungus and to the plants, more than half of this to the shoots. CONCLUSIONS: Goodyera repens can obtain significant amounts of P from its mycorrhizal partner. These results provide further support for the view that mycorrhizal associations in some adult green orchids are mutualistic.     

In-vitro seed germination ofCalanthe sieboldii, an endangered orchid species

Immature seeds from unripe capsules ofCalanthe sieboldii, were sown on one of three sterilized media: MS; modified MSH [i.e., the inorganic salts of MS plus the organic elements of Schenk and Hildebrandt]; Hyponex. Germination and protocorm development occurred on the MS and MSH media within eight weeks, but percent germination was low. The addition of putrescine (1 mg L^-1) or adenine sulfate (25 mg L^-1) to the MSH medium enhanced germination. Resultant protocorms were subcultured on the Hyponex medium, where they developed into plantlets after 12 weeks of additional culture. Plantlets were then successfully transferred to community pots.     

Conservation genetics of an endangered orchid in eastern Canada

Platanthera leucophaea, the Eastern Prairie Fringed-orchid, is a globally imperilled species native to the USA and Canada, with its Canadian distribution limited to the Province of Ontario. In Canada, where P. leucophaea is listed as endangered, approximately 40 % of populations have been extirpated in recent years, and many remaining populations have experienced substantial declines. In this study, we investigated whether reduced population sizes have led to low genetic diversity and inbreeding. We also investigated the extent to which hybridization with Platanthera psycodes may be threatening the genetic integrity of P. leucophaea populations. We found that overall, genetic diversity is low, and inbreeding is high. This is despite evidence of regular gene flow between proximate populations, although more distant populations show high levels of genetic differentiation. At sites where P. leucophaea is sympatric with P. psycodes, interspecific hybridization occurs in a bidirectional manner, i.e. with both parental species acting as either pollen donor or pollen recipient. Inbreeding and low genetic diversity in all populations, and hybridization in some populations, may pose future threats to P. leucophaea, and should be considered in the future by biodiversity managers.     

Gavilea gladysiae (Chloraeeae: Orchidaceae), a new orchid from southern Argentina and Chile

A new species, Gavilea gladysiae, is described and illustrated. This rare species inhabits the humid forests of Santa Cruz (southern Argentina) and the Magellan region (southern Chile). This species has been mistakenly identified as Gavilea kingii in herbarium collections and previous floristic works.     

Phylogenetic and conservation status of the endangered terrestrial orchid Nervilia nipponica (Orchidaceae) in Korea

The putative Japanese endemic Nervilia nipponica was discovered recently on Jeju Island in Korea. However, Japanese populations of N. nipponica have since been shown to comprise two polyphyletic taxa. To confirm the taxonomic status of the Korean Nervilia populations, we compared ITS1, matK and trnL intron sequence data with that from the two cryptic Japanese taxa. Our results confirmed all Korean populations as belonging to the true N. nipponica s.s, which must shed its endemic status. Korean accessions showed no distinct genotypic patterning, with samples from all populations and representing two distinct colour forms forming a single monophyletic clade. The extremely low level of genetic diversity detected within N. nipponica on Jeju Island underscores the urgent need for measures to secure the species from local extirpation. We conclude that N. nipponica is a priority for conservation and recommend that systematic controls and sustained efforts are required to ensure its protection.     

The phylogenetic relationship and non‐specific symbiotic habit of mycorrhiza fungi from a terrestrial orchid (Cymbidium)

To investigate the specificity of the symbiotic relationship between Cymbidium plants and their mycorrhiza fungi, thirty mycorrhiza fungi were isolated from roots of six terrestrial Cymbidium species. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) with universal fungal primers ITS1/ITS4. All fungal strains isolated from natural roots of orchids were inoculated into the rhizomes of in vitro Cymbidium goeringii. Phylogenetic analysis indicated fungal isolates of different cluster could be obtained from a special terrestrial Cymbidium species. Observation of light microscope and scanning electron microscope showed that fungi entered the cortical tissue by destroying cell wall of epidermal cells, where they formed hyphal knots in the cortical cells and were digested gradually. A large number of small protuberances were visible on cross sections of the rhizome. There was no strict inter‐species specificity between the isolated mycorrhiza fungi and terrestrial Cymbidium.     

濒危植物独花兰的形态变异及其适应意义

采用ANOVA和UPGMA等方法对濒危植物独花兰(Changnieniaamoena)分布于庐山、新宁和神农架3个地点的12个自然居群的形态变异进行了研究,探讨其形态多样性水平和地理变异式样及其可能的适应机制。结果表明,庐山居群13个性状的平均观测值均高于新宁和神农架居群。在物种水平上各个性状存在较丰富的变异,变异系数(CV值)为0.02–0.30。尽管同一性状在不同居群中的变异程度较大,但总体而言,花部器官的变异性比营养器官低。单因素方差分析显示3个地区间多个形态性状存在极显著差异(P<0.01);UPGMA聚类分析也表明这3个地区分别形成明显不同的分支。值得注意的是,神农架地区龙门河和关门山两个地点间存在明显的形态分化,而这种形态分化是以传粉者为媒介自然选择的结果。相关性分析显示,营养器官之间、花部器官之间,以及营养器官与花部器官之间均存在较高的相关性。上述结果对进一步了解独花兰的适应机制以及制定科学的保护策略提供了有益的资料。     

Molecular evidence on the species status and phylogenetic relationships of Spiranthes parksii, an endangered orchid from Texas

New genetic information on the taxonomic status of an endangered species may have strong implications for its continued protection and that of other listed taxa with questionable species status. While analyzing a more comprehensive project on the molecular phylogeny of North American Spiranthes (Orchidaceae), lack of genetic differentiation between endangered S. parksii, endemic only to Texas, and a common local congener, S. cernua, was discovered. To confirm these results in a comparative context, we focused parsimony analyses on a subset of data from local Spiranthes and the same congeners found elsewhere, expanded by additional local samples of the two focal species. Four DNA sequence segments (two chloroplast, one mitochondrial, one nuclear ribosomal) totaling 3191 base pairs were used separately and together to verify that S. parksii is nested within the same clade as S. cernua, and thus likely to be the same species. Our results concur with another recent investigation using AFLP and microsatellite data that also suggests S. parksii is not unique genetically. Therefore, based on empirical data and the phylogenetic species concept, endangered S. parksii is merely an aberrant local phenotype of and a synonym for S. cernua sensu lato. Our results should be considered during the current review of this orchid’s listing status, despite some recent policy changes on consideration of genetic data after listing.     

Genetic variation and conservation of Changnienia amoena, an endangered orchid endemic to China

Nuclear DNA content (2C) is used as a new criterion to investigate nearly all species of the genus Nerine Herb. The species have the same chromosome number (2n = 2x = 22), with the exception of three triploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with propidium iodide, is demonstrated to range from 18.0–35.3 pg. This implies that the largest genome contains roughly 2 × 10¹⁰ more base pairs than the smallest. The species, arranged according to increasing genome size, fell apart in three groups if growth cycle and leaf width were also considered. A narrow-leafed, evergreen group with a DNA content between 18.0 and 24.6 pg contains thirteen species, a broad-leaved winter growing group with four species has a DNA content from 25.3–26.2 pg and a broad-leafed summer growing group has a DNA content of 26.8–35.3 pg and contains six species. If the presence of filament appendages and hairiness of the pedicels were also considered, the thirteen evergreen species could be further divided into a group without filament appendages or hairy pedicels with a DNA content of 18.0–18.7 pg. A second group without filament appendages but with hairy pedicels had a DNA content of 19.7–22.3 pg. And a third group with both filament appendages and hairy pedicels had a DNA content of 22.0–24.6 pg. The exception is N. marincowitzii that, despite a low DNA content and narrow leaves is summer growing. The broad-leafed group is further characterised by the absence of filament appendages and the absence of strongly hairy pedicels. The exception here is N. pusilla that, despite a high DNA content, has narrow leaves and minutely hairy pedicels. Nuclear DNA content as measured by flow cytometry is shown to be relevant to throw new light on the relationships between Nerine species.     

In vitro culture of Chloraea gavilu Lindl., an endemic terrestrial orchid from Chile

Orchids are the second most diverse plant family, recognized for their importance as ornamental species; this has driven research development in propagation. One of the most common culture methodologies is in vitro asymbiotic germination, in which the nutritional conditions that provide orchids with a fungal partner are emulated. Although Chile possesses more than 60 terrestrial orchid species, in vitro cultivation protocols have only been developed for Chloraea crispa. In Southern Chile, Chloraea gavilu stands out due to its floral characteristics. We evaluate different explants and cultivation conditions for C. gavilu. The best germination and development results were achieved in the MM medium +0.1% yeast extract +1% sucrose +0.454 µmol l^?1 TDZ, using immature seeds of 24–30 days after pollination, which we cultivated into seedlings in order to be acclimatized and mycorrhized. In addition, induction of protocorm-like bodies was achieved from germinated seeds, using the same culture media as in the germination and development of immature C. gavilu seeds. This resulted in the successful asymbiotic germination of immature seeds, along with the micropropagation of a terrestrial, temperate orchid. We hope to use our protocol in the commercial production of Chilean orchid species as well as to propagate threatened species.     

Conservation genetics of the endangered terrestrial orchid Liparis japonica in Northeast China based on AFLP markers

Levels of genetic diversity and population genetic structure of the rare, endangered terrestrial orchid Liparis japonica were examined for eight natural populations (n = 185) in Northeast China using six AFLP primer pairs, where this species has experienced severe habitat loss and fragmentation. Based on 406 DNA bands, a high level of genetic diversity was found at the species level with the PPB of 85.47 %, while the genetic diversity at the population level was low (PPB = 47.48 %). A significantly high degree of population differentiation was found with 42.69 % variation existed among populations as measured by AMOVA, indicating potential restricted gene flow. The genetic distances between populations were independent of the corresponding geographic distances, and the genetic relationship of individuals had no significant correlation with their spatial distribution. The restricted gene flow might be impacted by reduced population size, habitat destruction and fragmentation. The results in this study suggested that habitat protection and keeping a stable environment are critical for the conservation of L. japonica species.     

Isolation and characterization of 24 microsatellite loci in Paphiopedilum rothschildianum, an endangered slipper orchid

This paper describes the characterization of 24 polymorphic microsatellite loci in Paphiopedilum rothschildianum, an endangered slipper orchid endemic to Sabah, Malaysia. A total of 36 clones containing microsatellite repeat motifs yielded 41 dinucleotide, 20 tetranucleotide and 30 cryptic simple repeat sequences. Thirty microsatellite loci were randomly selected to characterize the population, of which 24 were found to be polymorphic. The expected heterozygosity ranged from 0.0813 to 0.7279 (mean 0.4533) and the observed heterozygosity ranged from 0 to 1 (mean 0.3800). The results indicated that there was limited migration between the three subpopulations and a significant degree of genetic differentiation (F_ST = 0.5098) between populations implying that gene flow has been disrupted as a result of habitat fragmentation. The molecular markers have a potential application in population management, CITES enforcement and the forensic detection of specimens in international trade.     

Axenic rhizome culture and genetic fidelity assessment of Eulophia dabia (D. Don) Hochr: an endangered terrestrial orchid species

In Vitro Cellular & Developmental Biology - Plant - Eulophia dabia (D. Don) Hochr is an endemic, critically endangered, terrestrial orchid species having profound importance in traditional...     

Fungi associated with terrestrial orchid mycorrhizas,seeds and protocorms

The identity and ecological role of fungi in the mycorrhizal roots of 25 species of mature terrestrial orchids and in 17 species of field incubated orchid seedlings were examined. Isolates of symbiotic fungi from mature orchid mycorrhizas were basidiomycetes primarily in the generaCeratorhiza, Epulorhiza andMoniliopsis; a few unidentified taxa with clamped hyphae were also recovered. More than one taxon of peloton-forming fungus was often observed in the cleared and stained mycorrhizas. AlthoughCeratorhiza andEpulorhiza strains were isolated from the developing protocorms, pelotons of clamped hyphae were often presents in the cleared protocorms of several orchid species. These basidiomycetes are difficult to isolate and may be symbionts of ectotrophic plants. The higher proportion of endophytes bearing clamp connections in developing seeds than in the mycorrhizas is attributed to differences in the nutritional requirements of the fully mycotrophic protocorms and partially autotrophic plants. Most isolates ofCeratorhiza differed enzymatically fromEpulorhiza in producing polyphenol oxidases. Dual cultures with thirteen orchid isolates and five non-orchid hosts showed that some taxa can form harmless associations with non-orchid hosts. It is suggested that most terrestrial orchid mycorrhizas are relatively non-specific and that the mycobionts can be saprophytes, parasites or mycorrhizal associates of other plants.     

Mycorrhizal diversity,seed germination and long‐term changes in population size across nine populations of the terrestrial orchid Neottia ovata

In plant species that rely on mycorrhizal symbioses for germination and seedling establishment, seedling recruitment and temporal changes in abundance can be expected to depend on fungal community composition and local environmental conditions. However, disentangling the precise factors that determine recruitment success in species that critically rely on mycorrhizal fungi represents a major challenge. In this study, we used seed germination experiments, 454 amplicon pyrosequencing and assessment of soil conditions to investigate the factors driving changes in local abundance in 28 populations of the orchid Neottia ovata. Comparison of population sizes measured in 2003 and 2013 showed that nearly 60% of the studied populations had declined in size (average growth rate across all populations: ?0.01). Investigation of the mycorrhizal fungi in both the roots and soil revealed a total of 68 species of putatively mycorrhizal fungi, 21 of which occurred exclusively in roots, 25 that occurred solely in soil and 22 that were observed in both the soil and roots. Seed germination was limited and significantly and positively related to soil moisture content and soil pH, but not to fungal community composition. Large populations or populations with high population growth rates showed significantly higher germination than small populations or populations declining in size, but no significant relationships were found between population size or growth and mycorrhizal diversity. Overall, these results indicate that temporal changes in abundance were related to the ability of seeds to germinate, but at the same time they provided limited evidence that variation in fungal communities played an important role in determining population dynamics.     

濒危植物盘龙参种子的非共生萌发及种苗的快繁研究

以盘龙参种子为材料,筛选离体条件下适宜种子非共生萌发、种苗快繁的培养基。结果表明:盘龙参种子在无植物生长物质的培养基中能够萌发,但不能发育成苗;在含有1.0mg·L-1 KT、0.1mg·L-1 IAA和0.1mg·L-1 GA3的培养基中萌发,并能进一步发育形成种苗;在较高浓度生长素(1.2mg·L-1 NAA)的培养基中不能萌发。种苗在较高浓度细胞分裂素和较低浓度生长素配比的培养基中能够增殖,最高增殖系数可达到2.8,转入壮苗生长培养基中培养80d后可以移栽温室。最适增殖培养基为1/2MS+12.0mg·L-1 6-BA+0.1mg·L-1 NAA+10.0mg·L-1腺嘌呤;最适壮苗生根培养基为1/2MS+1.0mg·L-1 KT+0.1mg·L-1 IAA+10.0mg·L-1腺嘌呤。