Epipogium Aphyllum Research Articles

Evolution of Whirly1 in the angiosperms: sequence, splicing, and expression in a clade of early transitional mycoheterotrophic orchids.

The plastid-targeted transcription factor Whirly1 (WHY1) has been implicated in chloroplast biogenesis, plastid genome stability, and fungal defense response, which together represent characteristics of interest for the study of autotrophic losses across the angiosperms. While gene loss in the plastid and nuclear genomes has been well studied in mycoheterotrophic plants, the evolution of the molecular mechanisms impacting genome stability is completely unknown. Here, we characterize the evolution of WHY1 in four early transitional mycoheterotrophic orchid species in the genus Corallorhiza by synthesizing the results of phylogenetic, transcriptomic, and comparative genomic analyses with WHY1 genomic sequences sampled from 21 orders of angiosperms. We found an increased number of non-canonical WHY1 isoforms assembled from all but the greenest Corallorhiza species, including intron retention in some isoforms. Within Corallorhiza, phylotranscriptomic analyses revealed the presence of tissue-specific differential expression of WHY1 in only the most photosynthetically capable species and a coincident increase in the number of non-canonical WHY1 isoforms assembled from fully mycoheterotrophic species. Gene- and codon-level tests of WHY1 selective regimes did not infer significant signal of either relaxed selection or episodic diversifying selection in Corallorhiza but did so for relaxed selection in the late-stage full mycoheterotrophic orchids Epipogium aphyllum and Gastrodia elata. Additionally, nucleotide substitutions that most likely impact the function of WHY1, such as nonsense mutations, were only observed in late-stage mycoheterotrophs. We propose that our findings suggest that splicing and expression changes may precede the selective shifts we inferred for late-stage mycoheterotrophic species, which therefore does not support a primary role for WHY1 in the transition to mycoheterotrophy in the Orchidaceae. Taken together, this study provides the most comprehensive view of WHY1 evolution across the angiosperms to date.

Floristic diversity of the state natural sanctuary "Bushkovskiy forest" (the Kirov region, Russia)

The paper presents the results of the complex analysis of the flora structure of the state natural sanctuary "Bushkovskiy forest". The research had been made on the area of 9274.8 ha within the period of 2004–2023. Route recognition method was used together with geobotanical description of certain phytocoenoses on temporary sample plots made according to traditional methods. There vascular plant flora contains 270 taxon from 196 genera, 73 families, four classes and three divisio. On the territory of the sanctuary a rare species grows which is registered in the Red Data Book of the Russian Federation – Epipogium aphyllum Sw. The plants are distributed into 13 ecological-coenotic groups. As for the number of species, the floodplain meadow (21.9%), nemoral (17.0%), and boreal (12.6%) groups prevail. Among biological types hemicryptophythes prevail (49.6%); as for the biomorphological structure, dry ground grass polycarpics prevail (65.2%), and, among the latter, short rhizomatous (18.1%) and long rhizomatous (17.4%) grasses prevail. Plants of six hemerobia stages are represented in the flora, for a total, anthropophobic species (species of the a-o-m interval) account for 69.0%. Correlation of hemerobia stages, as well as a small share of adventitious species (3.0%), shows that the natural ecosystem of the sanctuary suffers from a certain anthropogenic load.

VASCULAR PLANTS OF THE UPPER YARAP RIVER (BADZHAL RANGE, KHABAROVSK TERRITOTY)

An annotated checklist of 250 vascular plants species in the local flora of the upper reaches of Yarap River (southern macroslope of the Badzhal Range) is given. For the first time, Aconitum subvillosum, Arctous erythrocarpa, Carex ussuriensis, Macrohystrix komarovii, Neottianthe cucullata, Poa arsenjevii, Salix rhamnifolia are recorded for the flora of the Badzhal Range; a total 20 species, three of which, Chrysosplenium krestovii, Silene badzhalensis and Calamagrostis burejensis, were previously described as new to science. In addition, rare and endemic Red Data Book species were found, there are: Aconitum baburinii, Aster woroschilovii, Calypso bulbosa, Cardamine tomentella, Epipogium aphyllum, Leontopodium blagoveshczenskyi, Neottianthe cucullata, Rhodiola rosea, Saussurea tomentosa, Phlomoides woroschilovii, Pentactina schlothauerae, Taraxacum badzhalense, Viola brachyceras, Weigela suavis. The vegetation of the study area is briefly characterized.

Contribution to the knowledge of the distribution of the rare orchid Epipogium aphyllum Swartz in Bosnia and Herzegovina

In 2020, a new site of the ghost orchid (Epipogium aphyllum Swartz) was found in the area of Mt. Jadovnik, near Drvar in the western part of Bosnia and Herzegovina. This mycoheterotrophic orchid belongs to one of the rarest and least known species of orchids in the world. The paper gives a brief description of the ecology of the species, a description of the habitat conditions in the site area, and an overview of the previous records for Bosnia and Herzegovina. It was found in beech and fir dominated primeval forest, in Visućka draga on Mt. Jadovnik. So far, it has been confirmed in only a few locations in Bosnia and Herzegovina.

Weak population spatial genetic structure and low infraspecific specificity for fungal partners in the rare mycoheterotrophic orchid Epipogium aphyllum

Some plants abandoned photosynthesis and developed full dependency on fungi for nutrition. Most of the so-called mycoheterotrophic plants exhibit high specificity towards their fungal partners. We tested whether natural rarity of mycoheterotrophic plants and usual small and fluctuating population size make their populations more prone to genetic differentiation caused by restricted gene flow and/or genetic drift. We also tested whether these genetic characteristics might in turn shape divergent fungal preferences. We studied the mycoheterotrophic orchid Epipogium aphyllum, addressing the joint issues of genetic structure of its populations over Europe and possible consequences for mycorrhizal specificity within the associated fungal taxa. Out of 27 sampled E. aphyllum populations, nine were included for genetic diversity assessment using nine nuclear microsatellites and plastid DNA. Population genetic structure was inferred based on the total number of populations. Individuals from 17 locations were included into analysis of genetic identity of mycorrhizal fungi of E. aphyllum based on barcoding by nuclear ribosomal DNA. Epipogium aphyllum populations revealed high genetic diversity (uHe = 0.562) and low genetic differentiation over vast distances (FST = 0.106 for nuclear microsatellites and FST = 0.156 for plastid DNA). Bayesian clustering analyses identified only two genetic clusters, with a high degree of admixture. Epipogium aphyllum genets arise from panmixia and display locally variable, but relatively high production of ramets, as shown by a low value of rarefied genotypic richness (Rr = 0.265). Epipogium aphyllum genotype control over partner selection was negligible as (1) we found ramets from a single genetic individual associated with up to 68% of the known Inocybe spp. associating with the plant species, (2) and partner identity did not show any geographic structure. The absence of mosaicism in the mycorrhizal specificity over Europe may be linked to preferential allogamous habit of E. aphyllum and significant gene flow, which tend to promote host generalism.

NOTEWORTHY RECORDS OF PLANTS, LICHENS AND FUNGI IN THE MURMANSK REGION. IV

Seventy nine important findings of 46 species of vascular plants, bryophytes, lichens and fungi acquired lately from the Kandalaksha District of the Murmansk Region are reported. The findings were considered important if they were red-listed in Russia and/or Murmansk Region, represented other particularly rare species known from not more than Ave locations in the Murmansk Region, or came from the northernmost locations in Europe or globally. Six fungal species ( Boletopsis grisea, Cystostereum murrayi, Irpex lacteus, Lenzites betulinus, Peniophora erikssonii, Steccherinum fimbriatum ) were found in the region for the second time. New locations in the Kandalaksha District were found for four rare fungal species ( Crustoderma corneum, Cystostereum murrayi, Phellodon melaleucus, Vuilleminia comedens ). New location of 35 species listed in the Red Data Book of the Murmansk Region ( Leptoporus mollis, Arctoparmelia subcentrifuga, Bryoria fremontii, Chaenotheca brachypoda, C. chlorella, C. gracillima, C. laevigata, C. subroscida, Chaenothecopsis nigra, Evernia divaricata, Lichenomphalia hudsoniana, Lobaria pulmonaria, Melanohalea exasperata, Pertusaria coronata, Phlyctis argena, Usnea glabrescens, Arnellia fennica, Metzgeria furcata, Scapania spitsbergensis, Crossocalyx hellerianus, Riccardia palmata, Buxbaumia aphylla, Asplenium viride, Botrychium lanceolatum, B. multifidum, Butomus umbellatus, Diplazium sibiricum, Epipogium aphyllum, Isoëtes echinospora, Nymphaea candida, Potamogeton filiformis, P. pectinatus, Ribes nigrum, Salixaurita, Viola selkirkii ) were found.

The Genomic Impact of Mycoheterotrophy in Orchids.

Mycoheterotrophic plants have lost the ability to photosynthesize and obtain essential mineral and organic nutrients from associated soil fungi. Despite involving radical changes in life history traits and ecological requirements, the transition from autotrophy to mycoheterotrophy has occurred independently in many major lineages of land plants, most frequently in Orchidaceae. Yet the molecular mechanisms underlying this shift are still poorly understood. A comparison of the transcriptomes of Epipogium aphyllum and Neottia nidus-avis, two completely mycoheterotrophic orchids, to other autotrophic and mycoheterotrophic orchids showed the unexpected retention of several genes associated with photosynthetic activities. In addition to these selected retentions, the analysis of their expression profiles showed that many orthologs had inverted underground/aboveground expression ratios compared to autotrophic species. Fatty acid and amino acid biosynthesis as well as primary cell wall metabolism were among the pathways most impacted by this expression reprogramming. Our study suggests that the shift in nutritional mode from autotrophy to mycoheterotrophy remodeled the architecture of the plant metabolism but was associated primarily with function losses rather than metabolic innovations.

Orchid Extinction over the Last 150 Years in the Czech Republic

Understanding temporal changes in the distribution and abundance of various species is one of the key goals of conservation biology. During recent decades, the abundance and distribution of many species of plants and animals have declined dramatically, mainly because of habitat loss and fragmentation. The purpose of this study is to analyze the rate of extinction of orchids at various sites in different 20-year time intervals over the last 150 years, determined according to changes in society. Using the dataset of the orchid records of the Nature Conservation Agency of the Czech Republic, we determined the disappearance rate of orchids from sites using a grid of 1 × 1 km. We found that the vast majority of orchids disappeared from many of their historical localities in all time intervals analyzed. The number of sites suitable for Czech orchids declined by 8–92%, depending on the species. The most threatened orchid species in the Czech Republic are Spiranthes spiralis, Anacamptis palustris, Epipogium aphyllum and Goodyera repens. This all seems to be closely related with changes in agricultural practices in the open as well as in forest habitats. Preserving suitable orchid habitats seems to be the key for keeping Czech orchid flora alive.

Еловые леса долины р. Копту (Республика Тыва) – реликт растительности позднего плейстоцена

Spruce forests of the Tuva Republic are not sufficiently described in literature. Spruce forests occupy small areas, but they are characterized by high species diversity. Enclaves of forest communities outside the forest belt are particularly interesting. The actual problem concerns species composition which forms plant communities existing under environmental and climate conditions that are not favorable for it. The aim of the research is to describe the communities of the valley dark coniferous forests in the model river valley in Central Tuva, to assess their diversity, to analyze species composition and to identify the main factors determining it. In the course of the fieldwork in 2020, we examined coniferous forests along the Koptu river, on the southern slope of the Academic Obruchev range (See Figures). In this part of the range, the foothills are occupied by steppe vegetation; forest-steppe landscapes prevail at altitudes of 800-1000 m; above there is the taiga belt, the upper border of the forest is about 1900 m above sea level. Two key areas were investigated; one of them is located in the middle course of the river within the taiga belt (51.90- 51.92°N; 95.45-95.48°E, 1400-1500 m above sea level), the other is in the lower course of the river in the forest-steppe belt (51.55-51.70°N; 95.34-95.45°E, 700-900 m above sea level). In total, 11 relevés were made on plots of 20×20 m by the standard method. We established that the forests are rich in species: 98 taxa were registered (See Table). It could be expected that coniferous forests in the river valley within the forest-steppe belt are a continuation of the above located communities, a continuation of communities from the taiga belt. However, our materials have demonstrated that it is not so. We revealed that, despite the physiognomic similarity, the dark coniferous communities growing in the river valley in its lower course differ significantly from those growing within the taiga belt (See Table). Larix sibirica is abundant everywhere; but in the taiga belt Pinus sibirica dominates in the valley communities, and Picea obovata prevailes in the river valley in the forest-steppe belt. Apart from it, the communities of these two key sites differ significantly in their species composition. Analysis of the ecology of differentiating species shows that the difference is due to a complex of factors. Four groups of species can be distinguished in the investigated forests: I. Taiga species which are only encountered in forests of the upper part and are absent below (Aegopodium alpestre, Carex iljinii, Cerastium pauciflorum, Vaccinium vitis-idaea, V. uliginosum). II. Meadow species, symmetrically, only occur in the forests of the lower part and are absent in the taiga belt (Galium boreale, Lathyrus pratensis, Rubus saxatilis and Vicia cracca, Achillea asiatica, Adonis sibirica, Artemisia laciniata, Iris ruthenica). The two groups mentioned above reflect the difference in altitude as well as the influence of the zonal environment. III. A series of species in the downstream valley forests reflect a waterlogging process, which takes place there due to the altitudinal gradient declining and surface flattening (Angelica tenuifolia, Caltha palustris, Carex cespitosa, C. dioica, Equisetum scirpoides, Delphinium crassifolium, Galium uliginosum, Lathyrus palustris, Parnassia palustris, Ribes procumbens; among mosses – Calliergonella cuspidata, Helodium blandowii, Timmia megapolitana, Tomentypnum nitens). IV. A number of species with mainly taiga or Arctic-Alpine distribution were recorded only in larch-spruce forests in the river valley in the forest-steppe belt and they are absent in the forests of the upper key area (Carex alba, Bistorta vivipara, Carex sedakowii, Orthilia obtusata, Pedicularis sceptrum-carolinum, Saussurea alpina, Schizachne callosa). For these species, it is impossible to explain their presence in the forest-steppe belt by modern ecological conditions. Their presence in the valley spruce forests has historical reasons and could be caused by the conditions of the Late Pleistocene cooling. So, we can draw a conclusion that the massif of valley spruce forests in the lower part of the Koptu river is a relict community formed in the Late Pleistocene periglacial environment on permafrost soils. In present conditions, it is in unstable equilibrium. The massif is the habitat of a number of rare species. In particular, the Orchidaceae taxa were found here, four of which are included in the Red Book of the Republic of Tuva (Cypripedium calceolus, Dactylorhiza sibirica, Epipogium aphyllum, Neottianthe cucullata). The valley forest of the lower part of the Koptu river can be nominated as a high conservation value forest and recommended to be included in the list of the Important Plant Areas of the Altai-Sayan region. The massif is under the strong anthropogenic pressure; in order to avoid extinction, it needs to be protected.

Местонахождение редких видов растений семейства Orchidaceae в Гайнском районе (Пермский край, Россия), перспективное для присвоения статуса особо охраняемой природной территории

The task of preserving biodiversity is closely related to the protection of rare and endangered species of organisms and the development of a system of specially protected natural areas. In 2020 and 2021, a reconnaissance survey of a site with a small open swamp near the village of Gayny (Perm Krai) was carried out in order to assess the prospects for giving it the status of a specially protected natural area. The site is characterized by a high diversity and abundance of orchids growing on it, which are one of the most vulnerable components of vegetation. The diversity of plant communities of the site was described and 11 orchids were recorded. All of them are included in the security lists of various levels. In addition, in the territory under consideration in 2015, a resident of the village of Gaina, Yu. A. Savelyev, recorded Epipogium aphyllum Sw., which was not encountered during a reconnaissance survey. The article presents some features of the distribution, biology and protection of all orchids noted on this territory. The main reason for the reduction in the number of orchids, as well as many other species of organisms, is the violation of habi-tats. Ecological and biological features of orchids enhance the effect of adverse factors. Therefore, today the conserva-tion of orchid habitats is considered as the most effective method of their protection. Based on this, it is concluded that it is advisable to assign the status of a specially protected natural area to the surveyed site in the vicinity of the village of Gainy, Perm Krai.

Plastome Evolution and Phylogeny of Orchidaceae, With 24 New Sequences.

In order to understand the evolution of the orchid plastome, we annotated and compared 124 complete plastomes of Orchidaceae representing all the major lineages in their structures, gene contents, gene rearrangements, and IR contractions/expansions. Forty-two of these plastomes were generated from the corresponding author's laboratory, and 24 plastomes—including nine genera (Amitostigma, Bulbophyllum, Dactylorhiza, Dipodium, Galearis, Gymnadenia, Hetaeria, Oreorchis, and Sedirea)—are new in this study. All orchid plastomes, except Aphyllorchis montana, Epipogium aphyllum, and Gastrodia elata, have a quadripartite structure consisting of a large single copy (LSC), two inverted repeats (IRs), and a small single copy (SSC) region. The IR region was completely lost in the A. montana and G. elata plastomes. The SSC is lost in the E. aphyllum plastome. The smallest plastome size was 19,047 bp, in E. roseum, and the largest plastome size was 178,131 bp, in Cypripedium formosanum. The small plastome sizes are primarily the result of gene losses associated with mycoheterotrophic habitats, while the large plastome sizes are due to the expansion of noncoding regions. The minimal number of common genes among orchid plastomes to maintain minimal plastome activity was 15, including the three subunits of rpl (14, 16, and 36), seven subunits of rps (2, 3, 4, 7, 8, 11, and 14), three subunits of rrn (5, 16, and 23), trnC-GCA, and clpP genes. Three stages of gene loss were observed among the orchid plastomes. The first was ndh gene loss, which is widespread in Apostasioideae, Vanilloideae, Cypripedioideae, and Epidendroideae, but rare in the Orchidoideae. The second stage was the loss of photosynthetic genes (atp, pet, psa, and psb) and rpo gene subunits, which are restricted to Aphyllorchis, Hetaeria, Hexalectris, and some species of Corallorhiza and Neottia. The third stage was gene loss related to prokaryotic gene expression (rpl, rps, trn, and others), which was observed in Epipogium, Gastrodia, Lecanorchis, and Rhizanthella. In addition, an intermediate stage between the second and third stage was observed in Cyrtosia (Vanilloideae). The majority of intron losses are associated with the loss of their corresponding genes. In some orchid taxa, however, introns have been lost in rpl16, rps16, and clpP(2) without their corresponding gene being lost. A total of 104 gene rearrangements were counted when comparing 116 orchid plastomes. Among them, many were concentrated near the IRa/b-SSC junction area. The plastome phylogeny of 124 orchid species confirmed the relationship of {Apostasioideae [Vanilloideae (Cypripedioideae (Orchidoideae, Epidendroideae))]} at the subfamily level and the phylogenetic relationships of 17 tribes were also established. Molecular clock analysis based on the whole plastome sequences suggested that Orchidaceae diverged from its sister family 99.2 mya, and the estimated divergence times of five subfamilies are as follows: Apostasioideae (79.91 mya), Vanilloideae (69.84 mya), Cypripedioideae (64.97 mya), Orchidoideae (59.16 mya), and Epidendroideae (59.16 mya). We also released the first nuclear ribosomal (nr) DNA unit (18S-ITS1-5.8S-ITS2-28S-NTS-ETS) sequences for the 42 species of Orchidaceae. Finally, the phylogenetic tree based on the nrDNA unit sequences is compared to the tree based on the 42 identical plastome sequences, and the differences between the two datasets are discussed in this paper.

Флористические находки редких видов растений в Республике Хакасии

The article contains an information on the locations for nine of rare plant species in the Republic of Khakassia. Utricularia ochroleuca, higher vascular species of hybridogenic origin, is shown for Siberia for the first time. One species from Marchantiophyta – Riccia fluitans – have been found in Khakassia for the first time. The information for new locations of rare species: orchids from Red List – Cypripedium macranthon, Epipogium aphyllum, Neottianthe cucullata and Orchis militaris, endemic from southern Siberia Saussurea parviflora subsp. purpurata, rare aquatic plants Nymphaea tetragona (previously considered extinct in Khakassia) and Zannichellia repens, is provided also.

Changes in mountain vascular plants of the ‘Jar rzeki Raduni’ nature reserve (Poland) within the last 50 years

Abstract ‘Jar rzeki Raduni’ is a landscape nature reserve created in 1972, which covers an area of 74,26 ha. The reserve protects the Radunia river gorge in the moraine upland of the Kashubian Lake District. In past, the reserve was an object of numerous botanical studies, and it was distinguished by a large share of mountain species. The main aim of this research was to define the current state of mountain species of the studied area compared to historical data. The study was carried out in 2017. Eight mountain species were noted: Aconitum variegatum, Alnus incana, Bupleurum longifolium, Chaerophyllum hirsutum, Dryopteris expansa, Huperzia selago, Pleurospermum austriacum and Ribes alpinum. The occurrence of five species was not confirmed: Coeloglossum viride, Epipogium aphyllum, Melampyrum sylvaticum, Polygonatum verticillatum and Valeriana sambucifolia.

RNA-seq highlights parallel and contrasting patterns in the evolution of the nuclear genome of fully mycoheterotrophic plants

BackgroundWhile photosynthesis is the most notable trait of plants, several lineages of plants (so-called full heterotrophs) have adapted to obtain organic compounds from other sources. The switch to heterotrophy leads to profound changes at the morphological, physiological and genomic levels.ResultsHere, we characterize the transcriptomes of three species representing two lineages of mycoheterotrophic plants: orchids (Epipogium aphyllum and Epipogium roseum) and Ericaceae (Hypopitys monotropa). Comparative analysis is used to highlight the parallelism between distantly related fully heterotrophic plants.In both lineages, we observed genome-wide elimination of nuclear genes that encode proteins related to photosynthesis, while systems associated with protein import to plastids as well as plastid transcription and translation remain active. Genes encoding components of plastid ribosomes that have been lost from the plastid genomes have not been transferred to the nuclear genomes; instead, some of the encoded proteins have been substituted by homologs. The nuclear genes of both Epipogium species accumulated nucleotide substitutions twice as rapidly as their photosynthetic relatives; in contrast, no increase in the substitution rate was observed in H. monotropa.ConclusionsFull heterotrophy leads to profound changes in nuclear gene content. The observed increase in the rate of nucleotide substitutions is lineage specific, rather than a universal phenomenon among non-photosynthetic plants.

Precipitation and timing of flowering in ghost orchids (Epipogium aphyllum Sw.)

The rare, fully mycoheterotrophic Ghost Orchid, Epipogium aphyllum is only visible during its short flowering and fruiting season, which lasts for a few weeks between May and October. Due to the apparent unpredictability of its flowering, decades may pass between subsequent observations at the same locality. The factors affecting timing of flowering in this enigmatic species remain largely unexplored. In Hungary, it is an extremely rare species: between 1924 and 2014 only 25 dated observations from 15 locations are known. Hungary is located on the edge of the species’ distribution area where higher precipitation may occur only in higher regions of mountains. Hence, the spatial and temporal pattern of precipitation might limit the emergence of generative shoots. In this paper we compared rainfall patterns in relation with the Ghost Orchids’ observations to multiannual precipitation averages. The year of flowering and the month preceding flowering (but not the year before flowering and the month of flowering) were characterised by significantly more rainfall than the multi-annual average precipitation. These results suggest that the appearance of the species in Hungary is precipitation-dependent.

IUCN guidelines using for assessment of plants from the Red Book of Russian Federation at regional level: a case study for the Republic of Mordovia (Russia)

AbstractThere are 15 plant species included in the Russian Red Book, which occur in the Republic of Mordovia:Najas tenuissima,Koeleria sclerophylla,Stipa dasyphylla,S. pennata,S. pulcherrima,S. zalesskii,Fritillaria ruthenica,Iris aphylla,Cypripedium calceolus,Cephalanthera rubra,Epipogium aphyllum,Neottianthe cucullata,Orchis militaris,Neotinea ustulata,Thymus cimicinus. Of these, onlyC. calceolusis included in the Global Red List as Critically Endangered. Threat status for studied species in Mordovia was assessed, and their distribution dynamics in this region throughout 12 years was described. Recent floristic studies, inspections of herbariums and literature were used for searches all provided data. Grid mapping and IUCN criteria were used. Nine taxa were determined as Critically Endangered, three as Endangered, one as Vulnerable, one as Near Threatened and one as Data Deficient. The areas of occupancy and extent of occurrence in Mordovia were assessed for each taxon. All studied species should be included in the next edition of the Mordovian Red Book.F. ruthenica,E. aphyllum,N. ustulata,T. cimicinusrequire additional studies to confirm earlier observations.

Evidence for mixed sexual and asexual reproduction in the rare European mycoheterotrophic orchid Epipogium aphyllum, Orchidaceae (ghost orchid).

Despite their significant capacity to propagate vegetatively, most orchids reproduce via seeds. Sexual reproduction via seed is commonly reported, in contrast to apomixis, whereby seeds are clones of the mother. Although insect pollination and autonomous self-pollination exist in mycoheterotrophic plants, the reproductive embryology of these plants remains under-studied. This paper provides evidence for the co-occurrence of both sexual and apomictic reproduction in a population of mycoheterotrophic plants - Epipogium aphyllum We investigated seed formation via open pollination, induced autogamy, autogamy sensu stricto and autonomous apomixis. The study was performed on a population of E. aphyllum located in northern Poland. The research included studies of the micromorphology, histochemistry and embryology of four types of reproductive systems. Scanning, fluorescence and light microscopy accompanied by graphical and statistical analyses were employed. We observed gametophyte development, from the one-nucleate stage to maturity, in unpollinated flower buds. The lack of zygotes in flower buds indicated that fertilization did not occur at this stage. Manual self-pollination led to a zygote, followed by embryo formation. Fertilization and embryo development derived from embryogenesis via open pollination is delayed compared with hand pollination. Isolation from external pollination resulted only in structures resembling zygotes that may originate either sexually or independent of fertilization. Parthenogenetic structures that resembled zygotes were observed in flowers that were emasculated and isolated from pollination. Zygotes formed at significantly higher frequencies via open pollination and induced autogamy in comparison to the parthenogenetic structures formed in other treatments. We showed the absence of pre-zygotic barriers for autogamy in E. aphyllum Self-pollination and self-fertilization are possible; however, natural self-pollination is unlikely or rare due to the position of the pollinia. Incidental parthenogenesis in E. aphyllum is very likely, given the biology of ovule development of this mycoheterotrophic orchid. This species therefore has the potential to produce seeds via both sexual and asexual means, although the contribution of apomixis to this process appears largely negligible.

New records of noteworthy vascular plants from Leningrad Region

The article includes information about new localities of protected species of vascular plants, which are included into Red Data Book of Leningrad Region. The majority of the findings were done within protected areas (PAs) of Leninrad Region, viz. wildlife sanctuaries «Ivinskiy Razliv» (Epipogium aphyllum, Lonicera pallasii, Petasites frigidus, Ranunculus subborealis, Rubus humulifolius), «Kuznechnoye» (Cardamine parviflora, Carex bohemica, Nymphaea tetragona, Tripleurospermum subpolare, Woodsia ilvensis), «Upper Reaches of River Sondala» (Epipogium aphyllum, Lathyrus laevigatus, Lonicera pallasii, Ranunculus subborealis, Rubus humulifolius), «Golomenskiy Mokh» (Epipogium aphyllum). Also we report few localities of plants (Allium scoenoprasum, Epipogium aphyllum, Isatis tinctoria, Petasites frigidus, Rumus humulifolius) which are situated outside PAs. The detailed data about the only one locality of arcto-alpine species Poa alpina L. in Leningrad Region are also given. In Leningrad Region, its localities are situated beyond the limits of its main distribution. We have recorded a small population of P. alpina on the shore of Ladoga Lake in the eastern part of Sosnovyi Island. Earlier this species was known in Leningrad Region in the only one locality on the shore of Onega Lake near Shcheleyki village, but in 2008 this locality has been destroyed. Refs 9.

Protection of Norwegian orchids – a review of achievements and challenges

Norway has a rich and diverse orchid flora consisting of 36 species. Orchids are found throughout the country, but most of the species are confined to calcareous or base-rich substrates. Important orchid-rich types of vegetation include rich pine and spruce forests, rich deciduous forests, open calcareous meadows, rocky outcrops and screes, hay meadows and calcareous mires and fens. Many species are rare, and 17 species and 3 subspecies are red listed. 13 species are generally protected. Both the orchids and their habitats are susceptible to various disturbances such as e.g. building activities, road construction, quarrying, drainage, forestry and changes in agricultural practices (less intense grazing, termination of mowing), which has resulted in the continuation of the previously inhibited succession. Most of the types of habitat mentioned are important conservation sites and thus many orchid occurrences (e.g. of Cypripedium calceolus , Epipogium aphyllum , Epipactis palustris and Ophrys insectifera ) are protected by a network of nature reserves designated for these habitats. However, there is an urgent need to secure species with small populations in some of the mire reserves, and succession is also a problem in many of the reserves. Protection of the few existing localities for some species is also needed. A more detailed discussion of the status of the red listed species is presented.

Exploring the limits for reduction of plastid genomes: a case study of the mycoheterotrophic orchids Epipogium aphyllum and Epipogium roseum.

The question on the patterns and limits of reduction of plastid genomes in nonphotosynthetic plants and the reasons of their conservation is one of the intriguing topics in plant genome evolution. Here, we report sequencing and analysis of plastid genome in nonphotosynthetic orchids Epipogium aphyllum and Epipogium roseum, which, with sizes of 31 and 19 kbp, respectively, represent the smallest plastid genomes characterized by now. Besides drastic reduction, which is expected, we found several unusual features of these “minimal” plastomes: Multiple rearrangements, highly biased nucleotide composition, and unprecedentedly high substitution rate. Only 27 and 29 genes remained intact in the plastomes of E. aphyllum and E. roseum—those encoding ribosomal components, transfer RNAs, and three additional housekeeping genes (infA, clpP, and accD). We found no signs of relaxed selection acting on these genes. We hypothesize that the main reason for retention of plastid genomes in Epipogium is the necessity to translate messenger RNAs (mRNAs) of accD and/or clpP proteins which are essential for cell metabolism. However, these genes are absent in plastomes of several plant species; their absence is compensated by the presence of a functional copy arisen by gene transfer from plastid to the nuclear genome. This suggests that there is no single set of plastid-encoded essential genes, but rather different sets for different species and that the retention of a gene in the plastome depends on the interaction between the nucleus and plastids.