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The distribution and abundance of Boloria selene (small pearl-bordered fritillary butterfly, SPBF) declined over recent decades in many parts of the UK. Availability of food plants, especially marsh violet (Viola palustris), for the caterpillars of the SPBF has been identified as one of the major causes of this decline. To achieve augmentation of existing colonies and develop new populations of SPBF large numbers of marsh violet propagules were required specifically to feed the larvae. The main objectives of the study were to produce thousands of good quality marsh violet propagules to restore selected habitats in the Heart of Durham, Northern England, using in vitro methods. Preliminary trials showed that in vitro multiplication of seedlings from wild collected seeds through conventional agar-based cultures was lengthy, expensive and turned out to be a non-viable route to achieve the objectives. This study explored the potential of bioreactor-based cloning and cost-effective one step rooting and weaning. Robust propagules, ready for transplantation following rapid propagation and one step rooting and weaning in vitro, were raised in a plug system for transplantation and establishment under field conditions. This was achieved by using simple and cost-effective methods to support the large-scale restoration exercise using 14,000 propagules. Application of high throughput micropropagation and low cost one step weaning systems for time-bound conservation and restoration projects are discussed in detail. This research highlights the important role of in vitro methods to support integrated biodiversity conservation of a native larval host plant and threatened butterfly.

Conservation Translocation (CT), which includes reintroduction, reinforcement and introduction, is gaining momentum, responding to serious population decline in many orchids. Orchid conservation underpinned by a greater emphasis on understanding biotic and abiotic factors of habitats is critical for successful recovery and restoration programmes. Anacamptis morio, commonly known as green-winged orchid (GWO), is a terrestrial orchid found throughout Europe, but populations in England rapidly declined in its native range in the last several decades. The current study explored the relationship between soil abiotic characteristics and the community composition of key fungal groups. Wild sites in Essex and Cumbria in England, where successful colonies are currently present, were compared to potential sites for CT in Cumbria. The Cumbrian sites managed by cattle grazing include the wild site and three potential CT sites, with two of them hosting no GWO plants. The Essex site, fOxley Meadow, where no cattle is used for grassland management, hosts the largest population of GWO in England. The aim of this study was to understand whether the community composition of fungi and soil characteristics of the potential CT sites in Cumbria are nearly compatible with that of Oxley Meadow. Oxley Meadow, with around 65,000 plants, stands out as a unique habitat compared to all Cumbria sites, as it showed low organic content. Nitrate and phosphate content were smaller for Oxley Meadow compared to other Cumbrian sites. The proportion of Basidiomycota fungi was greater in Oxley Meadow compared to all Cumbrian sites where Ascomycota dominated. The abundant fungal group found in Oxley Meadow was Agaricales. From Agariclaes, Hygrophoraceae or waxcaps fungi are considered an indicator group of fungi and were the most abundant group in Oxley Meadow. They have a negative correlation with targeted key fungal groups and abiotic parameters. More in-depth assessments using additional primers are essential to better understand the fungal diversity and how this diversity translates to the resilience of orchid habitats. This preliminary study points to future studies to assess whether sites are near-compatible to the wild site where large colonies are present using additional primers collected over different time scales. [ABSTRACT FROM AUTHOR]

The yellow early marsh orchid (Dactylorhiza incarnata ssp. ochroleuca) is a critically endangered terrestrial orchid in Britain. Previous attempts to translocate symbiotic seedlings to a site near the last remaining wild site demonstrated some success, with a 10% survival rate despite adverse weather conditions over a two-year period. However, to facilitate future reintroduction efforts or conservation translocations, a more comprehensive understanding of the fungal microbiome and abiotic soil characteristics at the final remaining wild site is required. Obtaining comprehensive information on both the fungal community and soil nutrient composition from wild sites has significant benefits and may prove critical for the success of future conservation translocations involving threatened orchids. This preliminary study, conducted at the last remaining wild site, revealed a significant correlation between the relative abundance of the orchid mycorrhizal fungal order Cantharellales and the concentrations of nitrate and phosphate in the soil. Another orchid mycorrhizal fungal group, Sebacinales, was found to be distributed extensively throughout the site. The composition of fungal communities across the entire site, orchid-hosting and non-orchid-hosting soils is discussed in relation to reinforcing the current population and preventing the extinction of this orchid. [ABSTRACT FROM AUTHOR]

Polyploids have significantly influenced angiosperm evolution. Understanding the genetic consequences of polyploidy is advanced by studies on synthetic allopolyploids that mimic natural species. In Nicotiana, Burk (1973) and Kostoff (1938) generated synthetic tobacco (N. tabacum) using the parents [female] N. sylvestris x [male] N. tomentosiformis. We previously reported rapid genetic changes in the Burk material. Kostoff's material has 24 chromosomes of N. sylvestris origin (S-genome), 24 of N. tomentosiformis origin (T-genome), and a large intergenomic translocation, but not an additive distribution of ribosomal DNA (rDNA) families as expected from the parental contribution. Our new synthetic tobacco lines TR1 and TR2 are chromosomally balanced with no intergenomic translocations and are either sterile or have highly reduced fertility, supporting the nuclear cytoplasmic hypothesis that allopolyploid fertility is enhanced by intergenomic translocations. Two plants of TR1 (TR1- A, TR1-B) have the expected number, structure, and chromosomal distribution of rDNA families, in contrast to Burk's and Kostoff's synthetic tobaccos and to synthetic polyploids of Arabidopsis. Perhaps allopolyploids must pass through meiosis before genetic changes involving rDNA become apparent, or the genetic changes may occur stochastically in different synthetic allopolyploids. The lack of fertility in the first generation of our synthetic tobacco lines may have uses in biopharmacy. Key words: chromosomes; evolution; fertility; polyploidy; ribosomal DNA; synthetic tobacco.

The split between conservation science and real‐world application is an ongoing issue despite several calls for unification. Researchers are empowered to partially bridge the research‐implementation gap by making their findings more accessible. Cypripedium calceolus is the most recognizable orchid of the European flora, and is currently facing habitat change and fragmentation, in addition to threats from collectors and illegal traders. Although several studies have focused on the ecological and genetic features of the species, a comprehensive account of how such aspects can be translated into concrete conservation recommendations is still missing. In this study, we describe microsatellite genetic variation in 188 individuals from different Italian populations of C. calceolus. Our results indicate the need for immediate conservation action for the most isolated populations in the Central Apennines and north‐western Italy. Although our genetic findings are specific to the Italian populations, our aim is to review ecological and population genetic aspects in C. calceolus and their implications for conservation against the existing threats. Therefore, our detailed guidelines for translocation, habitat management and post‐translocation monitoring can be used to inform conservation strategies in threatened populations of C. calceolus across its range. [ABSTRACT FROM AUTHOR]

Cephalanthera rubra (L.) Rich., Red Helleborine, is a widespread orchid in Europe but known only from three very small populations in England. These populations are in decline with no natural seed setting for more than a decade. The species may become extinct in the UK soon unless viable strategies are in place for ex situ conservation, especially the use of symbiotic propagation. Because of the fragile nature of the populations in England mycorrhizal fungal diversity study is not feasible. Therefore, to understand the factors needed for healthy Red Helleborine populations, soil characteristics and diversity of culturable root-derived fungi of the populations from a small area in the Loire Valley in France were studied. Themain objectives of the study were: (1)Which culturable mycorrhizal fungi associated with C. rubra roots and (2) To what extent is variation in fungal communities related to variation in soil characteristics? Here, we report a significant difference in diversity of culturable mycorrhizal and non-mycorrhizal fungi depending on soil pH and phosphorus content. Mycorrhizal associations were favoured by plants in locations with low soil nutrient availability and comparatively higher pH. Our study shows that mycorrhizal fungi, both ecto and endo, can be cultured from roots of plants at different maturity stages. [ABSTRACT FROM AUTHOR]

Background: Tropical orchids need more study with respect to their mycorrhizal associations. For researchers in distant countries who aspire to study these orchids augmenting their conservation, the great distances involved, coupled with limited funds, pose formidable challenges. These challenges are sometimes exacerbated by political unrest, delays in securing permits, unexpected hardships, and the risk that the biological samples collected (e.g., roots harboring mycorrhizal fungi) will not survive long-distance transport. Results: We describe a protocol for the collection and transport of root samples from Madagascar orchids to labs in the United Kingdom (Kew) and the United States (Illinois) where Rhizoctonia-like fungi were subsequently isolated. Three separate trips were made spanning 4 years (2012-2015), with emphasis on the collection of roots from epiphytic, lithophytic, and terrestrial orchids inhabiting the Itremo Massif of the Central Highlands. Collectively, the trips to Madagascar resulted in the isolation of all major groups of Rhizoctonia-like fungi ( Ceratobasidium, Tulasnella, Sebacina) from all three orchid growth forms (terrestrials, epiphytes and lithophytes). Sampling of terrestrial and epiphytes during the rainy season (January) yielded best results. Conclusions: Our study demonstrates that peloton-forming fungi in root samples can retain viability up to 3 weeks after collection. [ABSTRACT FROM AUTHOR]

Last seen in 1958, the Ascension Island endemic fern, Anogramma ascensionis, was listed as extinct on the 2003 IUCN Red List. However, a 2009 survey rediscovered four plants on Green Mountain. Spores were collected and cultured in vitro at the Royal Botanic Gardens, Kew, where a living collection of thousands of gametophytes and hundreds of sporophytes has been developed. To gain further insights into the biology of this species and the potential implications of in vitro multiplication for conservation purposes, samples were characterized from the karyological point of view. Chromosome analysis of root tips has confirmed that the species is tetraploid, and flow cytometry assessments have revealed that haploid gametophytes produce diploid sporophytes, which confirms natural fertilization. In addition, an rbcL sequence from A. ascensionis has been generated and compared with those published for other Anogramma spp., suggesting a close relationship with A. chaerophylla from Brazil. Further surveys of Green Mountain have reported the presence of 40 A. ascensionis sporophytes in total. Vegetation community analyses have suggested that the present population may be confined to suboptimal habitats. We therefore propose that, prior to the dramatic transformation of the vegetation on the island as a result of the invasion of alien species (particularly Adiantum spp.), A. ascensionis may have flourished in more humid and shaded parts of the mountain. A multidisciplinary approach involving in vitro culture, invasive species clearance and controlled translocation is discussed as the future roadmap for the conservation of this critically endangered fern. Our experiences have also highlighted lessons more broadly applicable to the conservation of extremely rare species elsewhere in the world, especially on remote island systems. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 461-477. [ABSTRACT FROM AUTHOR]

Polyploids have significantly influenced angiosperm evolution. Understanding the genetic consequences of polyploidy is advanced by studies on synthetic allopolyploids that mimic natural species. In Nicotiana, Burk (1973) and Kostoff (1938) generated synthetic tobacco (N. tabacum) using the parents ♀N. sylvestris x ♂N. tomentosiformis. We previously reported rapid genetic changes in the Burk material. Kostoff's material has 24 chromosomes of N. sylvestris origin (S-genome), 24 of N. tomentosiformis origin (T-genome), and a large intergenomic translocation, but not an additive distribution of ribosomal DNA (rDNA) families as expected from the parental contribution. Our new synthetic tobacco lines TR1 and TR2 are chromosomally balanced with no intergenomic translocations and are either sterile or have highly reduced fertility, supporting the nuclear cytoplasmic hypothesis that allopolyploid fertility is enhanced by intergenomic translocations. Two plants of TR1 (TRI-A, TRI-B) have the expected number, structure, and chromosomal distribution of rDNA families, in contrast to Burk's and Kostoff's synthetic tobaccos and to synthetic polyploids of Arabidopsis. Perhaps allopolyploids must pass through meiosis before genetic changes involving rDNA become apparent, or the genetic changes may occur stochastically in different synthetic allopolyploids. The lack of fertility in the first generation of our synthetic tobacco lines may have uses in biopharmacy. [ABSTRACT FROM AUTHOR]

The yellow early marsh-orchid (Dactylorhiza incarnata subsp. ochroleuca) is critically endangered in the UK. Reintroduction of this threatened orchid to former haunts that have been restored is a long-term objective of this study. Identifying germination-specific mycorrhizal fungus lineages from closely related species is used as a method due to the extremely small number of plants left in the wild. A putative orchid mycorrhizal fungus of the family Tulasnellaceae, isolated from Dactylorhiza praetermissa, supported in vitro seed germination to produce reintroduction-ready seedlings. Reintroduced symbiotic seedlings survived over the winter months in the flooded reintroduction site (RS). The comparative soil analysis for key nutrients before reintroduction showed that phosphorus content in the RS is very low compared to the soil collected from the wild site (WS) where the last viable population exists. On the other hand, C:N ratio in the soil at the WS and RS were not significantly different. To our knowledge, this is the first-ever report on the reintroduction of symbiotic seedlings of a threatened orchid back to the wild in the UK. [ABSTRACT FROM AUTHOR]

About 90% of Cynorkis species are endemic to the biodiversity hotspot of Madagascar. This terrestrial habitat-specific genus received little study for fungal diversity to support conservation. We evaluated the diversity of culturable fungi of 11 species and soil characteristics from six sites spanning a >40 km radius in and along the region's inselbergs. Peloton-forming fungi were grown in vitro from root/protocorm slices and positively identified using DNA sequencing. The fungal diversity was then correlated with soil pH, NO3-N, P, and K. All species harbored either putative mycorrhizal associates in the Rhizoctonia complex or Hypocreales fungi. Tulasnella Operational Taxonomic Units (OTUs) were most prevalent in all soil types while Serendipita OTUs were found in species inhabiting granite/rock outcrops in moist soil (seepage areas). Most Cynorkis species were present in soil with low NO3-N and P levels with diversity of mycorrhizal fungi inversely correlated to NO3-N levels. Of the different orchid life stages sampled, only one species (Cynorkis fastigiata) yielded putative mycorrhizal fungi from juvenile stages. As diversity of mycorrhizal fungi of Cynorkis spp. was negatively correlated with NO3-N, and majority of the studied taxa were found in soils with low NO3-N and P contents, reintroduction studies must include analysis of N and P in detail. For the first time, we showed that the assemblage of culturable fungi in the roots of habitat-specific species of Cynorkis (Orchidaceae) are intimately tied to specific soil characteristics. [ABSTRACT FROM AUTHOR]

The inselbergs of the Central Highlands of Madagascar are one of many 'micro-hotspots' of biodiversity on the island, particularly for Orchidaceae. In this region are several genera that have a large number of endemic species that are in serious decline or edging towards extinction. Studies relating to diversity of orchids and their fungal partners (both mycorrhizal and non-mycorrhizal root associates) deserve more attention, as climate change and human induced decline in resilience of species in the wild is at an all-time high. Identification of mycorrhizal fungi (MF) via conventional seed baited-protocorms has limitations for large scale studies and its application for time-bound conservation projects. The paper describes the value of understanding fungal diversity in the roots of orchids at different stages of maturity. The first part of the study was a preliminary investigation mainly to identify culturable Rhizoctonia endophytes, and the second part looked at all life forms of available taxa together with associated soil characteristics. We isolated and identified 19 putative MF from 18 of the 50 taxa spread over an area of 250 sq. km, covering three life forms, growth phases of the orchid taxa, and habitat types. In the rest of the taxa, we were unable to detect any putative MF, but had varying numbers of non-mycorrhizal endophytes. We also found that diversity of putative MF was higher in plants from soils with the lowest P levels recorded. Putative mycorrhizal OTUs were predominantly from the Tulasnella lineage, followed by Ceratobasidium and Serendipita. Within a small subset of samples, a difference in colonised endophytes depending on the collection season was observed. In vitro germination studies using 10 OTUs of mycorrhizal fungi in 14 orchid species showed mostly generalist associations. When orchid seed and fungal sources were studied irrespective of habitat, life form, and distance from each other (orchid seed and fungal source), compatibility for symbiotic seed germination was observed in most cases. Issues with the identification of compatible MF and symbiotic system of seed germination are discussed. [ABSTRACT FROM AUTHOR]

The yellow early marsh-orchid ( Dactylorhiza incarnata subsp. ochroleuca ) is critically endangered in the UK. Reintroduction of this threatened orchid to former haunts that have been restored is a long-term objective of this study. Identifying germination-specific mycorrhizal fungus lineages from closely related species is used as a method due to the extremely small number of plants left in the wild. A putative orchid mycorrhizal fungus of the family Tulasnellaceae, isolated from Dactylorhiza praetermissa , supported in vitro seed germination to produce reintroduction-ready seedlings. Reintroduced symbiotic seedlings survived over the winter months in the flooded reintroduction site (RS). The comparative soil analysis for key nutrients before reintroduction showed that phosphorus content in the RS is very low compared to the soil collected from the wild site (WS) where the last viable population exists. On the other hand, C:N ratio in the soil at the WS and RS were not significantly different. To our knowledge, this is the first-ever report on the reintroduction of symbiotic seedlings of a threatened orchid back to the wild in the UK.

Background: Fungal endophytes are highly diverse ubiquitous asymptomatic microorganisms, some of which appear to be symbiotic. Depending on abiotic conditions and genotype of the plant, the diversity of endophytes may confer fitness benefits to plant communities., Methods: We studied a crop wild relative (CWR) of strawberry, along environmental gradients with a view to understand the cultivable root-derived endophytic fungi that can be evaluated for promoting growth and tolerating stress in selected plant groups. The main objectives were to understand whether: (a) suboptimal soil types are drivers for fungal distribution and diversity; (b) high pH and poor nutrient availability lead to fungal-plant associations that help deliver fitness benefits; and (c) novel fungi can be identified for their use in improving plant growth, and alleviate stress in diverse crops., Results: The study revealed that habitats with high pH and low nutrient availability have higher fungal diversity, with more rare fungi isolated from locations with chalky soil. Plants from location G were the healthiest even though soil from this location was the poorest in nutrients. Study of environmental gradients, especially extreme habitat types, may help understand the root zone fungal diversity of different functional classes. Two small in vitro pilot studies conducted with two isolates showed that endophytic fungi from suboptimal habitats can promote plant growth and fitness benefits in selected plant groups., Discussion: Targeting native plants and crop wild relatives for research offers opportunities to unearth diverse functional groups of root-derived endophytic fungi that are beneficial for crops., Competing Interests: The authors declare there are no competing interests.