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Orchidaceae is one of the largest and most diverse families of flowering plants in the world but also one of the most threatened. Climate change is a global driver of plant distribution and may be the cause of their disappearance in some regions. Forest orchids are associated with specific biotic and abiotic environmental factors, that influence their local presence/absence. Changes in these conditions can lead to significant differences in species distribution. We studied three forest orchids belonging to different genera (Cephalanthera, Epipactis and Limodorum) for their potential current and future distribution in a protected area (PA) of the Northern Apennines. A Habitat Suitability Model was constructed for each species based on presence-only data and the Maximum Entropy algorithm (MaxEnt) was used for the modelling. Climatic, edaphic, topographic, anthropogenic and land cover variables were used as environmental predictors and processed in the model. The aim is to identify the environmental factors that most influence the current species distribution and the areas that are likely to contain habitats suitable for providing refuge for forest orchids and ensuring their survival under future scenarios. This will allow PA authorities to decide whether to invest more resources in conserving areas that are potential refuges for threatened species. [ABSTRACT FROM AUTHOR]

This work presents a summary of cytogenetic data, including new information, on several species within the tribe Neottieae, with an update of the karyotype for 23 species belonging to the genera Cephalanthera, Limodorum, Epipactis, and Neottia (including Listera). Each of these four genera also presents distinctive chromosomal features, such as bimodal karyotypes. Our research includes insights into the distribution of constitutive heterochromatin, measured using C-banding and, in some cases, specific fluorochromes for the detection of A-T- and G-C-rich DNA. In the Epipactis group, it is noteworthy that when using the Giemsa banding technique, certain species (e.g., E. placentina, E. meridionalis) with a chromosome number of 2n = 38 were observed to exhibit a conspicuous wide band of constitutive heterochromatin on the long arm of the third pair in a subcentromeric position, resembling what has been observed in E. helleborine. These differences also have the potential to contribute to the diversification of these species. Based on the karyological results obtained, a hypothesis regarding the origin of certain species within the E. helleborine group is proposed. Additionally, karyological analyses conducted on a specimen of E. microphylla revealed chromosome counts ranging from 36 to 40. Somatic metaphases exhibited evident structural alterations in certain chromosomes, showing rearrangements probably caused by translocation phenomena. Based on the data obtained from the species within the studied genera, it is conceivable that variations in chromosomes, both structurally and in the distribution of constitutive heterochromatin, exert a significant influence on the evolution of the karyotype. Moreover, in many entities belonging to the Neottieae tribe, these processes may also contribute to the diversification of the phenotype in some instances. [ABSTRACT FROM AUTHOR]

This work presents a summary of cytogenetic data, including new information, on several species within the tribe Neottieae, with an update of the karyotype for 23 species belonging to the genera Cephalanthera, Limodorum, Epipactis, and Neottia (including Listera). Each of these four genera also presents distinctive chromosomal features, such as bimodal karyotypes. Our research includes insights into the distribution of constitutive heterochromatin, measured using C-banding and, in some cases, specific fluorochromes for the detection of A-T- and G-C-rich DNA. In the Epipactis group, it is noteworthy that when using the Giemsa banding technique, certain species (e.g., E. placentina, E. meridionalis) with a chromosome number of 2n = 38 were observed to exhibit a conspicuous wide band of constitutive heterochromatin on the long arm of the third pair in a subcentromeric position, resembling what has been observed in E. helleborine. These differences also have the potential to contribute to the diversification of these species. Based on the karyological results obtained, a hypothesis regarding the origin of certain species within the E. helleborine group is proposed. Additionally, karyological analyses conducted on a specimen of E. microphylla revealed chromosome counts ranging from 36 to 40. Somatic metaphases exhibited evident structural alterations in certain chromosomes, showing rearrangements probably caused by translocation phenomena. Based on the data obtained from the species within the studied genera, it is conceivable that variations in chromosomes, both structurally and in the distribution of constitutive heterochromatin, exert a significant influence on the evolution of the karyotype. Moreover, in many entities belonging to the Neottieae tribe, these processes may also contribute to the diversification of the phenotype in some instances.

Orchidaceae is one of the largest and most diverse families of flowering plants in the world but also one of the most threatened. Climate change is a global driver of plant distribution and may be the cause of their disappearance in some regions. Forest orchids are associated with specific biotic and abiotic environmental factors, that influence their local presence/absence. Changes in these conditions can lead to significant differences in species distribution. We studied three forest orchids belonging to different genera (Cephalanthera, Epipactis and Limodorum) for their potential current and future distribution in a protected area (PA) of the Northern Apennines. A Habitat Suitability Model was constructed for each species based on presence-only data and the Maximum Entropy algorithm (MaxEnt) was used for the modelling. Climatic, edaphic, topographic, anthropogenic and land cover variables were used as environmental predictors and processed in the model. The aim is to identify the environmental factors that most influence the current species distribution and the areas that are likely to contain habitats suitable for providing refuge for forest orchids and ensuring their survival under future scenarios. This will allow PA authorities to decide whether to invest more resources in conserving areas that are potential refuges for threatened species.

On the basis of numerous own observations, occasionally with information from third parties and with selected information from relevant literature, all orchid species of the island of Krk mentioned or recorded so far are described in detail. The basis for this is more than 2,000 entries of documented occurrences on the island of Krk, which were taken from our „data treasure“ with more than 12,000 entries on the orchid flora of the northeastern Adriatic (period of the years 1999 to 2022). The distribution maps, also based mainly on our own data, were supplemented by reports of findings in the Flora of Istria and, in some cases, with information from the Flora Croatica Database, thus providing an almost complete representation of the currently known distribution picture. [ABSTRACT FROM AUTHOR]

Cephalanthera, Limodorum and Neottia, which are known as primitive orchids, are rhizomatous orchids and commonly distributed in Turkey. This study aims to investigate orchid seed variation between some representatives of Neottieae, Cephalanthera rubra, Limodorum abortivum, Neottia nidus-avis, and Neottia ovata, naturally distributed in Turkey by using geometric morphometric analyses based on 2-dimensional landmarks. For this purpose, a total of 95 specimens were evaluated and photographed using scanning electron microscopy. Using software, 12 homologous landmarks were obtained to reflect the main aspects of the seed shape. Species were compared with various statistical methods by calculating the data obtained from shape and size variables with the Procrustes method. The difference in both size and shape were significant between the species. Shape differences were most prominent in the chalazal and micropylar regions of the seed as well as the whole seed width. Discriminant analysis and cross-validation scores were a highly powerful to distinguish the species with scores ranging from 60% to 88%. Regression analyses also revealed allometric effect of the size on seed shape with a similar trend across species. Based on current results, geometric morphometric analysis is encouraging in the research of structural variation within plant parts. The present study is also significant in terms of the widespread use of such studies in the field of botany, especially in the context of systematic or functional morphology. [ABSTRACT FROM AUTHOR]

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Background: Cephalanthera nanchuanica is a terrestrial orchid species and has been red listed as a second-grade protected plant due to its limited distributions in China. Initially classified within a monotypic genus Tangtsinia, this species was later reassigned to Cephalanthera based on morphological and molecular data. However, previous phylogenetic analyses of Cephalanthera using several segment sequences exhibited a low discriminatory power in delineating its relationships., Results: In this study, we characterized and comparatively analyzed the complete chloroplast (cp) genome of C. nanchuanica with those of six previously reported Cephalanthera species. Our findings revealed that the cp genome of C. nanchuanica had the typical quadripartite structure, with a size of 161,365 bp and a GC content of 37.27%. A total of 113 unique genes were annotated, among which nearly half of protein-encoding genes (RSCU > 1) showed a preference in codon usage. No structural rearrangements were observed among the cp genomes of Cephalanthera species, except for C. humilis, which displayed structural alterations due to gene loss, relocation, and inverted repeat (IR) expansion/contraction. The cp genomes of Cephalanthera species were highly conserved, with only a small number of SSRs detected, most of which preferred A/T bases. Comparative analysis of cp genomes indicated that IR and coding regions were less divergent than single copy and non-coding regions and eight mutational hotspots were identified. Phylogenetic analysis suggested that the tribe Neottieae was a monophyletic group, divided into five clades. Palmorchis was the earliest-diverging lineage, followed by Cephalanthera. Diplandrorchis was deeply nested within Neottia, forming a clade. Aphyllorchis and Limodorum formed another clade, sister to Epipactis. Within the Cephalanthera clade, C. nanchuanica was sister to C. falcata with a strong support., Conclusions: This study demonstrated that the cp genome characters of C. nanchuanica are highly similar to those of other Cephalanthera species, except for the mycoheterotrophic species C. humilis. Although the cp genomes of Cephalanthera species (excluding C. humilis) exhibited conservation in genome structure and sequence, SSR repeats and mutational hotspots were identified, which could potentially serve as as molecular markers for distinguishing Cephalanthera species. The phylogenetic analysis based on the protein-coding genes provided high-resolution support for the infrageneric classification. Therefore, cp genome data will be instrumental in resolving the phylogeny of the genus Cephalanthera., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The complex biology and specialized relationships between orchids and both fungi and pollinators can complicate orchid conservation and management. Some terrestrial orchids are able to undergo prolonged periods of vegetative dormancy, likely depending on specific fungi for surviving belowground, which pose an additional challenge in monitoring and targeted conservation. This study tests the feasibility of using environmental DNA to detect orchid tissue from soil samples as a tool in surveillance of terrestrial orchid populations. We developed species-specific probe-based qPCR assays targeting ITS regions of the rare orchids Anacamptis pyramidalis and Cephalanthera rubra and tested these on 183 soil samples from four localities with current or previously recorded occurrence of the species. A subset of samples (22) were additionally analyzed by ITS2 metabarcoding for a small-scale comparison between methods, as well as for characterizing plant communities associated with the orchids. Although the developed assays were highly efficient and specific, orchid DNA was only detected in 21 of the tested soil samples, all within close vicinity to the nearest orchid, and most within 10 cm. No orchid DNA was detected in any sample from the localities with orchid occurrence a few years prior to the study. The metabarcoding analysis only detected orchid DNA in a single sample but proved informative in characterizing the plant communities at the four tested localities. The developed assays would be highly useful to apply in studies aiming at gaining knowledge on dormancy and number of belowground individuals in populations of high conservation concern. [ABSTRACT FROM AUTHOR]

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The SPME-GC-MS analysis of the aroma components of Cephalanthera damasonium, Cephalanthera rubra, and Cephalanthera longifolia were reported. The main components found in C. damasonium were pentadecane, hexadecane, heptadecane and α-farnesene. The same compounds were found in the scent of C. rubra. Although C. damasonium and C. rubra have different pollination mechanisms, their spectrum of volatile compounds surprisingly does not differ significantly. In C. longifolia pentadecane and heptadecane were found together with cis-β-farnesene. As C. longifolia is allogamous, the differences in scent composition may play a role in pollinator attraction. [ABSTRACT FROM AUTHOR]

The article informs that mixotrophic orchids such as Epipactis and Cephalanthera do not use photosynthesis for perennial underground organs. It mentions that orchids roots associate with ectomycorrhizal fungi that themselves associate with the surrounding trees, which are the ultimate carbon source of the whole consortium.

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Previous studies have shown that levels of genetic diversity in species of the genus Cephalanthera covary with the breeding system. In the southern part of the Korean Peninsula, the three self‐compatible terrestrial orchids Cephalanthera erecta, C. falcata and C. longibracteata flower synchronously in sympatric populations. The food‐deceptive C. falcata with bright yellow flowers is predominantly outcrossing, whereas autogamy is the dominant strategy in both C. erecta and C. longibracteata, whose white flowers do not open fully. We examined genetic diversity (by means of allozymes) of the three species in sympatric populations (600 × 600 m area) in the Yeonwhasan Provincial Park (YPP) and in non‐sympatric populations outside YPP, South Korea. Thirteen out of 20 putative loci were variable across the three species, but there was a complete lack of allozyme variation within each species and we found no evidence of hybridisation. Our results suggest that historical factors, i.e. the Quaternary climate oscillations, have played a major role in determining levels of genetic diversity in the three Cephalanthera species. The Korean populations of C. erecta (a warm‐temperate/temperate element) and C. falcata (a warm‐temperate element) may have been established by a single introduction from a genetically depauperate ancestral population, likely located outside the Korean Peninsula. On the other hand, since C. longibracteata is a boreal/temperate element, it may have survived the Last Glacial Maximum in microrefugia located in low elevation regions within the Peninsula where it has been subjected to population bottlenecks reducing its genetic diversity. [ABSTRACT FROM AUTHOR]

Orchidaceous plants have symbiotic relationships with endophytic fungi, including mycorrhizal fungi, which play important roles in the seed germination and growth of the host plants. In this study, endophytic fungal communities isolated from the roots of Cephalanthera longibracteata collected from three different sites in Korea were analyzed, and it was determined whether fungal communities were preferentially correlated with the sites. The fungal isolates were identified by sequence analysis of the internal transcribed spacer regions of rDNA. In total, 30 species of endophytic fungi, including two species of mycorrhizal fungi belonging to the genus Tulasnella, were identified. Leptodontidium orchidicola showed the highest frequency and was isolated from all root samples. Species diversity and richness were not significantly different among sites. However, the community structure of the endophytic fungi significantly differed among sites, suggesting that the site characteristics affected the community composition of the endophytic fungi colonizing the roots of C. longibracteata. Our findings will aid in developing methods involving the use of symbiotic fungi for orchid conservation and restoration in native habitats. [ABSTRACT FROM AUTHOR]

Limodorumspecies are hard-to-cultivate, germinating with difficulty or not germinating at all on a number of media. The present work was focused on the effects of seed sterilization-scarification methods and of different nitrogen sources onin vitroseed germination and seedling development ofLimodorum abortivum, L. brulloiandL. trabutianum. Our results showed that sodium hypochlorite solution with a 1% concentration of available chlorine was ineffective for both sterilization and scarification ofLimodorumseeds, while a 5% concentration was found to be an effective sterilizing and scarifying solution forL. trabutianum. The positive effect of organic nitrogen in promoting asymbiotic germination in many terrestrial orchids was confirmed by this study forL. trabutianumandL. brulloi, which showed significantly higher germination percentages on Basal Medium modified than on Knudson C, which contains inorganic nitrogen. This is the first report ofin vitroasymbiotic germination of mature seeds ofL. brulloiandL. trabutianum, especially successful forL. trabutianumthat showed high germination percentages (>63%) and an active seedling development. These results may be useful for conservation purposes, sinceL. brulloiis a narrow Italian endemics andL. trabutianumis threatened with extinction both in Italy and in France. [ABSTRACT FROM AUTHOR]

The group of Epipactis helleborine s.l. includes several subspecies. A new subspecies Epipactis helleborine subsp. moratoria was determined in Slovenia in 2015. It thrives in mixed wood in a region of Gorica at Raztez. Morphologic and phenomenological comparison confirmed clear differences between E. helleborine subsp. moratoria and E. helleborine subsp. helleborine. The characteristic differences seen in E. helleborine subsp. moratoria are the stem which is more or less bent at the level of the leaf base, the plants are smaller and more slender than E. helleborine, there are also differences in the flowers and the leaves. The ovary in E. moratoria is often in a horizontal position, especially at the time of fruiting, wheras in E. helleborine ovary usually hangs down. All of the wild orchids in Slovenia are protected species and among them it is Epipactis helleborine subsp. moratoria which, up till now is only known at one site. [ABSTRACT FROM AUTHOR]

The diversity of mycorrhizal fungi among four Japanese C ephalanthera ( Orchidaceae) species was examined at a total of seven sites, based on sequence variation in nuclear ribosomal DNA. This is the first report to detect mycorrhizal fungi in C. subaphylla. Two patterns of mycorrhizal associations were confirmed from our results. C. falcata has lower fungal specificity, associating with Russulaceae, Sebacinales and Thelephoraceae. By contrast, the three C ephalanthera species C. erecta, C. subaphylla, and C. longifolia were associated mainly with Thelephoraceae fungi. There is no large difference found in mycobionts between green and albino individuals of C. falcata and no distinct preference of Japanese C ephalanthera species for a specific fungal group of Thelephoraceae. [ABSTRACT FROM AUTHOR]

The evolution of mycoheterotrophy has been accompanied by extreme reductions in plant leaf size and photosynthetic capacity. Partially mycoheterotrophic plants, which obtain carbon from both photosynthesis and their mycorrhizal fungi, include species with leaves of normal size and others that are tiny-leaved. Thus, plant species may lose their leaves in a gradual process of size reduction rather than through a single step mutation. Little is known about how the degree of mycoheterotrophy changes during reductions in leaf size. We compared the degree of mycoheterotrophy among five Japanese Cephalanthera species, four with leaves of normal size ( Cephalanthera falcata, Cephalanthera erecta, Cephalanthera longibracteata and Cephalanthera longifolia), one with tiny leaves ( Cephalanthera subaphylla), and one albino form of C. falcata (as reference specimens for fully mycoheterotrophic plants). The levels of mycoheterotrophy were determined by stable isotope natural abundance analysis. All Cephalanthera species were relatively enriched in C and N in comparison with surrounding autotrophic plants. Cephalanthera subaphylla was strongly enriched in C and N to levels similar to the albinos. Species with leaves of normal size were significantly less enriched in C than C. subaphylla and the albinos. Thus, C. subaphylla was strongly mycoheterotrophic, obtaining most of its carbon from mycorrhizal fungi even though it has tiny leaves; species with leaves of normal size were partially mycoheterotrophic. Hence, during the evolutionary pathway to full mycoheterotrophy, some plant species appear to have gained strong mycoheterotrophic abilities before completely losing foliage leaves. [ABSTRACT FROM AUTHOR]

Evolutionary losses of photosynthesis in terrestrial plants all originate in photosynthetic ancestors. The adaptive context under which this transition happens has remained elusive because of the rarity of plants in which both photosynthetic and nonphotosynthetic forms exist as a polymorphism. Here, we report on demographic patterns in photosynthetic ('green') and nonphotosynthetic ('albino') individuals within populations of two such species, Cephalanthera damasonium and C. longifolia, which also acquire nutrition from their mycorrhizal hosts (partial mycoheterotrophy). We hypothesized that demographic shifts in albinos relative to greens would include compensatory patterns with respect to fitness, such that maladaptive changes to survival or reproduction would be adaptively countered by changes to other parameters, such as growth probabilities. We tracked individuals in two populations of C. damasonium for 3 yr, and in one population of C. longifolia for 14 yr. We then analyzed vital rates for both phenotypes using general linear mixed models ( GLMMs) and multi-state capture mark-recapture models ( CMR), and used these models to develop size-classified, function-based population projection matrices. We estimated fitness as the deterministic population growth rate (λ) for each phenotype, and explored the impact of shifts in demographic patterns to albinism via life table response experiments ( LTREs). Mortality differed between greens and albinos, but not similarly across species. Albinos generally sprouted less than greens, and flowered more when small but less at other times. Albinos typically had a higher probability of fruiting, although their lower flower numbers yielded lower numbers of fruits overall. Fitness did not differ significantly among phenotypes. Thus, we did not find significant evidence that albinism is adaptive or maladaptive; however, if in fact it is the latter, then we did find evidence of incomplete compensation for declines in survival and reproduction from growth transitions, particularly to small flowering size classes in C. damasonium, and to large vegetative size classes in C. longifolia. These patterns indicate some support for the idea that albinism may lead to the speciation of mycoheterotrophic plants. [ABSTRACT FROM AUTHOR]

A new orchid species, Cephalanthera ericiflora Szlach. and Mytnik, sp. nov. is described and illustrated. Its taxonomic position is briefly discussed

A revision of the genus Cephalanthera in the Iberian Peninsula and NW of Africa has been done. Three species have been accepted. These species, C. rubra, C. longifolia and C. damasonium, have been listed with their synonyms in the study zone, a detailed description of each, and distribution maps. The indumentum pollen and seeds have been studied by SEM. C. damasonium has been typified.

Cephalanthera is an orchid genus that harbors mixotrophic species obtaining carbon compounds from nearby trees via ectomycorrhizal fungi. Cephalanthera species show variable levels of fungal dependency and specificity. We identified mycobionts from C. longibracteata . A total of 142 root samples were collected from 68 C. longibracteata individuals at five sites in Japan, and their associated mycobionts were identified based on the DNA sequences of nuclear ribosomal internal transcribed spacer regions using a molecular taxonomical method. Several groups of ectomycorrhizal fungi, including Russulaceae, Sebacinaceae, and Thelephoraceae were identified from the roots of C. longibracteata , suggesting that C. longibracteata has lower fungal specificity than other Cephalanthera species examined previously. [ABSTRACT FROM AUTHOR]

Cephalanthera rubra ( L.) Rich., the red helleborine, is described and illustrated with an historical plate. Details of its distribution, relationships, pollination, patterns of hybridization and its history and conservation in England are provided. Notes relating to the difficulty of propagating and cultivating this species are also included. [ABSTRACT FROM AUTHOR]

Mixotrophy, obtaining carbon by mycoheterotrophy and photosynthesis, has been suggested in Cephalanthera species (Orchidaceae) by analyses on stable isotopes of carbon. In this study, we examined the growth of Cephalanthera falcata in pot cultured tripartite symbioses with Thelephoraceae fungi and Quercus serrata. Mycorrhizal fungi were isolated from roots of C. falcata in natural habitats. Two fungal isolates identified as Thelephoraceae were cultured and inoculated to fine roots of non-mycorrhizal seedlings of Q. serrata (Fagaceae). After the ectomycorrhizal formation, non-mycorrhizal seedlings of C. falcata were co-planted. The pots with tripartite symbioses were cultured in greenhouse for 30 months, and growth of C. falcata seedlings was examined. Fresh weight of C. falcata seedlings was significantly increased by the tripartite symbioses even in those with no shoot, thus providing further evidence for the mycoheterotrophic nature of this orchid. The achievement of seedling culture in tripartite symbioses would be valuable for conserving many forest orchids and for conducting experiments to understand their physiology and ecology. [ABSTRACT FROM AUTHOR]

Copyright of Natura Croatica is the property of Natura Croatica and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Achlorophyllous variants of some forest orchids are known to reach almost the same size as their green forms. These vegetative albino forms cover their entire carbon (C) demand through fungi that simultaneously form ectomycorrhizae with trees, while green variants partially draw on C from photosynthesis and C from fungal hosts. Here, we investigate whether the amount of C derived from either source is proportional to leaf chlorophyll concentration. The discovery of two Cephalanthera damasonium populations with variegated leaves enabled a continuous bridging of leaf chlorophyll concentrations between green and albino forms. Leaves of 27 green, variegated and albino individuals of C. damasonium were compared for chlorophyll concentrations, C sources (as characterized by 13C abundances) and total C and nitrogen (N) concentrations. We found a linear relationship between leaf chlorophyll concentrations and the proportional reliance on fungi as a C source. Furthermore, we show that the shift in C gain through mycoheterotrophic means significantly changes leaf total C and N concentrations. Our results document that partial mycoheterotrophy in C. damasonium is not a static nutritional mode but a flexible mechanism related inter alia to leaf chlorophyll concentrations. The change in proportional reliance on fungi as a C source affects leaf chemical composition. [ABSTRACT FROM AUTHOR]

Several green orchids of the Neottieae tribe acquire organic carbon both from their mycorrhizal fungi and from photosynthesis. This strategy may represent an intermediate evolutionary step towards mycoheterotrophy of some non-photosynthetic (albino) orchids. Mixed populations of green and albino individuals possibly represent a transient evolutionary stage offering opportunities to understand the evolution of mycoheterotrophy. In order to understand the emergence of albinos, we investigated patterns of spatial and genetic relationships among green and albino individuals in three mixed populations of Cephalanthera damasonium and one of C. longifolia using spatial repartition and Amplified fragment length polymorphism (AFLP) markers. Two of these populations were monitored over two consecutive flowering seasons. In spatial repartition analyses, albino individuals did not aggregate more than green individuals. Genetic analyses revealed that, in all sampled populations, albino individuals did not represent a unique lineage, and that albinos were often closer related to green individuals than to other albinos from the same population. Genetic and spatial comparison of genets from the 2-year monitoring revealed that: (i) albinos had lower survival than green individuals; (ii) accordingly, albinos detected in the first year did not correspond to the those sampled in the second year; and (iii) with one possible exception, all examined albinos did not belong to any green genet from the same and/or from the previous year, and vice versa. Our results support a scenario of repeated insurgence of the albino phenotypes within the populations, but unsuccessful transition between the two contrasting phenotypes. Future studies should try to unravel the genetic and ecological basis of the two phenotypes. [ABSTRACT FROM AUTHOR]

Plastid microsatellite loci developed for Cephalanthera longifolia were used to examine the level of genetic variation within and between populations of the three widespread Cephalanthera species ( C. damasonium, C. longifolia and C. rubra). The most detailed sampling was in C. longifolia (42 localities from Ireland to China; 147 individuals). Eight haplotypes were detected. One was detected in the vast majority of individuals and occurred from Ireland to Iran. Three others were only found in Europe (Ireland to Italy, England to Italy and Austria to Croatia). Two were only found in the Middle East and two only in Asia. In C. damasonium, 21 individuals from 10 populations (England to Turkey) were sampled. Only one haplotype was detected. In C. rubra, 34 individuals from eight populations (England to Turkey) were sampled. Although it was not possible to amplify all loci for all samples of this species, nine haplotypes were detected. Short alleles for the trnS-trnG region found in two populations of C. rubra were characterized by sequencing and were caused by deletions of 26 and 30 base pairs. At this level of sampling, it appears that C. rubra shows the greatest genetic variability. Cephalanthera longifolia, C. rubra and C. damasonium have previously been characterized as outbreeding, outbreeding with facultative vegetative reproduction and inbreeding, respectively. Patterns of genetic variation here are discussed in the light of these reproductive system differences. The primers used in these three species of Cephalanthera were also demonstrated to amplify these loci in another five species ( C. austiniae, C. calcarata, C. epipactoides, C. falcata and C. yunnanensis). Although it is sometimes treated as a synonym of C. damasonium, the single sample of C. yunnanensis from China had a markedly different haplotype from that found in C. damasonium. All three loci were successfully amplified in two achlorophyllous, myco-heterotrophic species, C. austinae and C. calcarata. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 181–193. [ABSTRACT FROM AUTHOR]

Five mycoheterotrophic species of Cephalanthera have been proposed from tropical Asia. Until recently, all of them were only known from the type specimens, for which reason it has been difficult to judge if some of them were actually conspecific. The recent discovery of two large populations of C. exigua in Thailand made it possible to describe the range of variation of this species in much more detail, and we then found it to be well distinguished from the others. A phylogenetic analysis based on nuclear rDNA ITS sequences corroborated the systematic position of C. exigua at the generic level. Examination of the previously neglected type of C. pusilla from Myanmar lead to the conclusion that this taxon of disputed generic affinity likewise belongs to Cephalanthera, and that it is a clearly distinct species. Finally, we accept that also C. ericiflora from Laos as well as C. gracilis and C. calcarata from Yunnan are probably distinct species. Cephalanthera exigua grows in hill evergreen forest, but we found little floristic similarity between its two localities in Thailand (ISS=22.5%). Ectomycorrhizal trees of the families Fagaceae and Ulmaceae are probably the ultimate carbon source for C. exigua at both sites, as the mycorrhizal partners of this orchid (basidiomycetes of the family Thelephoraceae) obtain their carbon nutrition through ectomycorrhizal association with forest trees. Pollination experiments demonstrated that the nectarless flowers of C. exigua are not spontaneously autogamous. Based on the pollination syndrome and previously published observations on other species of the genus, we suggest that C. exigua has an insect-operated breeding system acting by deceit, and that the pollinators are probably solitary bees. [ABSTRACT FROM AUTHOR]

We investigated the spatial distribution and taxonomic identity of mycorrhizal fungi colonizing the root systems of two threatened Cephalanthera species, C. falcata and C. erecta, in naturally regenerated forests. Peloton formation was observed in both plant species, confirming the existence of orchid mycorrhizas. For C. falcata, mycorrhization was significantly different among individuals, ranging from 14 to 63%, and no significant difference among C. erecta individuals was detected (57–68%). Mycorrhization among three growth directions of roots and between orchid species was not significantly different. The spatial distribution of mycorrhizas in both orchids showed significant differences, being most frequent at an apical position. Based on DNA sequencing and phylogenetic analyses, we inferred that the families Thelephoraceae and Sebacinaceae were mycobionts for C. falcata and Thelephoraceae for C. erecta. Our findings indicated that mycorrhizal colonization occurs at a distal position from the base of these orchid root systems and that mycorrhizal fungi are restricted to few ectomycorrhizal fungal families. [ABSTRACT FROM AUTHOR]

• While measurements of tissue stable isotope signatures and isotope mixing models have suggested that the green orchid Corallorhiza trifida is photosynthetically active and hence only partially mycoheterotrophic, these assumptions have not been validated by direct analysis of carbon assimilation. • The photosynthetic capabilities of three orchid species assumed on the basis of the indirect methods or chlorophyll content to have differing trophic strategies: Neottia nidus-avis (fully mycoheterotrophic), Cephalanthera damasonium (partially autotrophic), C. trifida (partially autotrophic), as well as saplings of an autotrophic tree, Fagus sylvatica, were investigated by combining the determination of chlorophyll content and fluorescence, with direct measurement of the potential for CO2 assimilation using 13C isotope tracers in the field. • Chlorophyll content and fluorescence values were indicative of ineffective photochemical processes in Neottia and reduced efficiency of photochemical processes in Corallorhiza. These differences are reflected in the mean assimilation rates of 13CO2 of 594 ± 129, 331 ± 72, 12.4 ± 2.4 and 7.3 ± 0.9 µg g−1 h−1 for Fagus, Cephalanthera, Corallorhiza and Neottia, respectively. • Our study, while confirming the fully mycoheterotrophic status of Neottia and the partially autotrophic condition in Cephalanthera, also demonstrates under field conditions that Corallorhiza is physiologically closer to the fully mycoheterotrophic condition than has previously been recognized. [ABSTRACT FROM AUTHOR]

• Background and Aims Many Orchidaceous species are threatened globally by development and over-collection from their natural habitats for horticultural purposes. Artificial propagation from seeds is difficult in most terrestrial orchids native to temperate regions. Seed production is another limiting factor in the artificial propagation for these species because of the lessened probability of pollination and the destruction of fruit by insect larvae. Members of the genus Cephalanthera are distributed across Europe, Asia and North America. C. falcata is a temperate species of East Asia and an endangered species in Japan. As successful propagation from seeds of this species has never been reported, a reproducible method is described here for seed production in situ and propagation using immature seeds in asymbiotic culture in vitro. [ABSTRACT FROM PUBLISHER]

We investigated an Estonian population of the orchid Cephalanthera longifolia (L.) Fritsch. (Neottieae tribe), which harbours green and achlorophyllous individuals (= albinos), to understand albino survival and compare mycorrhizal associates, development, and nutrition of the two phenotypes. Albinos never changed phenotype over 14 years and had development similar to green individuals; their chlorophyll content was reduced by 99.4%, making them heterotrophic. Molecular typing by polymerase chain reaction amplification of fungal intergenic transcribed spacer and microscopic analyses showed that Thelephoraceae (Basidiomycetes, usually forming ectomycorrhizae with trees) were mycorrhizal on both phenotypes. Molecular typing also demonstrated that additional fungi were present on roots, including many endophytes (such as Helotiales) and various ectomycorrhizal taxa, whose role and pattern of colonization remained unclear. Mycorrhizal colonization was increased in albinos by about twofold, but no obvious difference in fungal partners compared with green individuals was demonstrated. Analysis of stable isotope composition (N and C) showed that albinos were dependent on their fungi for carbon (mycoheterotrophy), while green individuals recovered 33% of their carbon from fungi (mixotrophy). Surrounding trees, which formed ectomycorrhizae with at least one Thelephoraceae found in orchids, were likely the ultimate carbon source. These data are discussed in the framework of evolution of mycoheterotrophy in orchids, especially in Neottieae. [ABSTRACT FROM AUTHOR]