Chemical Insect Attractants Produced by Flowers of Impatiens spp. (Balsaminaceae) and List of Floral Visitors.

The study of the semiochemicals produced by the flowers of Impatiens spp. is an important topic that may explain the reason for the rapid expansion of some species in this genus. Impatiens L. belongs to the Balsaminaceae family, which includes several species considered to be invasive plants in Europe. This study aimed to characterize the phytochemistry of four naturally occurring plant species in Poland, including three invasive alien taxa (Impatiens parviflora, I. glandulifera, and I. capensis) and one native species (I. noli-tangere). Gas chromatographic techniques were used to assess phytochemical profiles of chemical attractant cues in their pollination biology. We detected differences in the scent profiles of the investigated species. All the examined Impatiens species produce various alcohols, i.e., heptacosanol, octacosanol, aldehydes (e.g., octadecanal, eicosanal, etc.), and fatty acids, as well as long-chain hydrocarbons such as dodecane, tricosane, petacosane, hexacosane, and farnesene. Impatiens parviflora, I. glandulifera, and I. capensis produce geraniol and linalool, which attract members of the Apidae family, including bumblebees and honeybees. Impatiens parviflora also produces linalool-derived monoterpenes (linalool oxide and 8-hydroxylinalool), which are a strong attractant for Diptera; this may clarify why the species is mainly visited and pollinated by syrphid flies. A list of insect visitors to the Impatiens species under study can be found in the article.

Floral Volatile Organic Compounds and a List of Pollinators of Fallopia baldschuanica (Polygonaceae).

Fallopia baldschuanica (Polygonaceae) is an Asian plant growing wild in parts of Europe and North and Central America as an introduced taxon, in many countries it is considered a potentially invasive species. This article presents the list of 18 volatile organic compounds (VOCs) emitted by the flowers of F. baldchuanica and identified by headspace gas chromatography/mass spectrometry (HS-GC/MS) analyzes, and a list of flower-visiting and pollinating insects that have been observed in the city center of Wroclaw (SW Poland). ß-ocimene, heptanal, nonanal, α-pinene, 3-thujene, and limonene, were detected as the floral scent's most important aroma compounds. F. baldschuanica also produces the aphid alarm pheromones, i.e., ß-farnesene and limonene, that repels aphids. Additionally, the pollinators of F. baldschuanica were indicated, based on two years of observations in five sites in the urban area. It was found, that the pollinators of this plant with the highest species stability are: Diptera from families Syrphidae (Chrysotoxum bicinctum, Eristalis pertinax, Eupeodes corollae, Episyrphus balteatus, Eristalis tenax, Syrphus ribesii, Eristalis intricaria), Muscidae (Musca domestica), Sarcophagidae (Sarcophaga spp.), Calliphoridae (Lucilia sericata, Lucilia caesar), Hymenoptera from families Vespidae (Vespula vulgaris), and Apidae (Apis sp., Bombus sp.). The key role of VOCs in adaptation to plant expansion is discussed.

Why does an obligate autogamous orchid produce insect attractants in nectar? - a case study on Epipactis albensis (Orchidaceae).

BACKGROUND: The flowers of some species of orchids produce nectar as a reward for pollination, the process of transferring pollen from flower to flower. Epipactis albensis is an obligatory autogamous species, does not require the presence of insects for pollination, nevertheless, it has not lost the ability to produce nectar, the chemical composition of which we examined by gas chromatography-mass spectrometry (GC-MS) method for identification of potential insect attractants. RESULTS: During five years of field research, we did not observe any true pollinating insects visiting the flowers of this species, only accidental insects as ants and aphids. As a result of our studies, we find that this self-pollinating orchid produces in nectar inter alia aliphatic saturated and unsaturated aldehydes such as nonanal (pelargonal) and 2-pentenal as well as aromatic ones (i.e., syringaldehyde, hyacinthin). The nectar is low in alkenes, which may explain the absence of pollinating insects. Moreover, vanillin and eugenol derivatives, well-known as important scent compounds were also identified, but the list of chemical compounds is much poorer compared with a closely related species, insect-pollinating E. helleborine. CONCLUSION: Autogamy is a reproductive mechanism employed by many flowering plants, including the orchid genus Epipactis, as an adaptation to growing in habitats where pollinating insects are rarely observed due to the lack of nectar-producing plants they feed on. The production of numerous chemical attractants by self-pollinated E. albensis confirms the evolutionary secondary process, i.e., transition from ancestral insect-pollinating species to obligatory autogamous.

The Epipactis helleborine Group (Orchidaceae): An Overview of Recent Taxonomic Changes, with an Updated List of Currently Accepted Taxa.

The Epipactis helleborine (L.) Crantz group is one of the most taxonomically challenging species complexes within the genus Epipactis. Because of the exceptionally high levels of morphological variability and the ability to readily cross with other species, ninety different taxa at various taxonomic ranks have already been described within its nominative subspecies, but the taxonomic status of most of them is uncertain, widely disputed, and sometimes even irrelevant. The present review is based on results of the most recent research devoted to the E. helleborine group taxonomy. In addition, we analysed data about taxa belonging to this group presented in some research articles and monographs devoted directly to the genus Epipactis or to orchids in certain area(s). Based on the reviewed literature and data collected in four taxonomic databases available online, we propose an updated list of the 10 currently accepted taxa in the E. helleborine group (two species, six subspecies, and two varieties), which includes E. helleborine (L.) Crantz subsp. helleborine; E. helleborine subsp. bithynica (Robatsch) Kreutz; E. helleborine subsp. distans (Arv.-Touv.) R.Engel and P.Quentin; E. helleborine subsp. neerlandica (Verm.) Buttler; E. helleborine var. tangutica (Schltr.) S.C.Chen and G.H.Zhu; E. helleborine subsp. tremolsii (Pau) E.Klein; E. helleborine subsp. voethii (Robatsch) Jakubska-Busse, Zolubak, and Lobas, stat. nov.; E. condensata Boiss. ex D.P.Young; E. condensata var. kuenkeleana (Akhalk., H.Baumann, R.Lorenz, and Mosul.) Popovich; and E. cupaniana C.Brullo, D'Emerico, and Pulv.

Proteomics-based identification of orchid-associated bacteria colonizing the Epipactis albensis, E. helleborine and E. purpurata (Orchidaceae, Neottieae).

Using proteomics-based identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we conducted the first analysis of the composition of endophytic bacteria isolated from different parts of selected Epipactis species, i.e. the buds, the inflorescences and the central part of the shoots, as well as the rhizomes. We identified aerobic and anaerobic bacteria, including such taxa as Bacillus spp., Clostridium spp., Pseudomonas spp. and Stenotrophomonas spp., which may be considered as promoting plant growth. Because most of the indicated bacteria genera belong to spore-producing taxa (spores allow bacterial symbionts to survive adverse conditions), we suggest that these bacteria species contribute to the adaptation of orchids to the environment. We found clear differences in the microbiome between investigated closely related taxa, i.e., Epipactis albensis, E. helleborine, E. purpurata and E. purpurata f. chlorophylla. Some of the analysed orchid species, i.e. E. albensis and E. purpurata co-occur in habitats, and their bacterial microbiomes differ from each other.

Genetic History of the Remnant Population of the Rare Orchid Cypripedium calceolus Based on Plastid and Nuclear rDNA.

The lady's slipper orchid (Cypripedium calceolus), which inhabits shady deciduous and mixed forests and meadows, is now threatened with extinction in many European countries, and its natural populations have been dramatically declining in recent years. Knowledge of its evolutionary history, genetic variability, and processes in small populations are therefore crucial for the species' protection. Nowadays, in south-west Poland, it is only distributed in seven small remnant and isolated populations, which we examined. One nuclear (ITS rDNA) and two plastid (accD-psa1, trnL-F) markers were analyzed and compared globally in this study. Based on the nuclear marker, the most common ancestor of C. calceolus and Cypripedium shanxiense existed about 2 million years ago (95% HPD: 5.33-0.44) in Asia. The division of the C. calceolus population into the European and Asian lineages indicated by C/T polymorphism started about 0.5 million years ago (95% HPD: 1.8-0.01). The observed variation of plastid DNA, which arose during the Pleistocene glacial-interglacial cycles, is still diffuse in Poland. Its distribution is explained by the result of fragmentation or habitat loss due to human impact on the environment.

Genome Size Diversity in Rare, Endangered, and Protected Orchids in Poland.

Orchidaceae is one of the largest and the most widespread plant families with many species threatened with extinction. However, only about 1.5% of orchids' genome sizes have been known so far. The aim of this study was to estimate the genome size of 15 species and one infraspecific taxon of endangered and protected orchids growing wild in Poland to assess their variability and develop additional criterion useful in orchid species identification and characterization. Flow cytometric genome size estimation revealed that investigated orchid species possessed intermediate, large, and very large genomes. The smallest 2C DNA content possessed Liparis loeselii (14.15 pg), while the largest Cypripedium calceolus (82.10 pg). It was confirmed that the genome size is characteristic to the subfamily. Additionally, for four species Epipactis albensis, Ophrys insectifera, Orchis mascula, Orchis militaris and one infraspecific taxon, Epipactis purpurata f. chlorophylla the 2C DNA content has been estimated for the first time. Genome size estimation by flow cytometry proved to be a useful auxiliary method for quick orchid species identification and characterization.

How to Protect Natural Habitats of Rare Terrestrial Orchids Effectively: A Comparative Case Study of Cypripedium calceolus in Different Geographical Regions of Europe.

In this article we present and discuss the main factors that threaten natural populations of Cypripedium calceolus (lady's slipper orchid) in Europe, and we propose conservation strategies and directions for protective actions of its population on a regional scale. European C. calceolus populations have decreased significantly in the last two decades, in both number and size. A key result of the present study is an evaluation of the effectiveness of the Natura 2000 network across the European Union (EU) countries. Northern and/or mountainous countries present higher percentages of potentially suitable areas within the Natura 2000 network. Finland and the United Kingdom are the exceptions to this rule. It is predicted that, due to global warming, the coverage of niches suitable for C. calceolus will decrease in countries in which now-healthy colonies exist. However, as plant species can occur in micro-sites with suitable environmental conditions (e.g., microclimate, vegetation, soil factors) which cannot be predicted as suitable at coarser spatial resolutions, conservation efforts should be focused on management of local healthy populations. For the effective protection of C. calceolus in Natura 2000 sites, the participation of experts in botany, including orchid biology, is necessary at several stages.

How many type specimens can be stored in old lesser-known herbaria with turbulent histories? - A Juncus case study reveals their importance in taxonomy and biodiversity research.

Many herbarium sets in Europe are still being catalogued and it is likely that many old-type collections are yet to be discovered. This research has the potential to facilitate the study of the biodiversity of many regions, especially regions for which collections are extremely scarce. This has been confirmed by a case study using Juncus (Juncaceae) examining the turbulent history of botanical collections at the WRSL herbarium and the evaluation of its importance to the study of taxonomy and biodiversity since 1821. The analysis revealed that the WRSL collection is rich in types (ca. 3.6%) and we identified 76 (of 78) new, historically and nomenclaturally important specimens (types, original material and so-called "topotypes"). Some of these type specimens represent duplicates of these that were stored in Berlin and destroyed during World War II. Many of the type specimens are from the United States of America, South Africa, India, and Canada. The largest number of Juncus type specimens stored at WRSL originate from South Africa (42.3% of all type specimens), even though Juncus is rare in Africa. Our study highlights that uncatalogued old collections that are under-explored and under-exploited have the potential to facilitate the discovery of specimens important for the study of biodiversity, conservation, taxonomy and nomenclature.

A Revision of the Taxonomy and Identification of Epipactis greuteri (Orchidaceae, Neottieae).

Epipactis greuteri is an obligate autogamous orchid species. Due to large differences in the interpretation of the diagnosis of this species, it is often mistakenly identified by botanists, which results in erroneous information provided in the literature about its distribution in Europe. In the present paper we review its description, including flower details, gynostemium features, and papillae morphology and compare it to E. helleborine, with which it is often confused. Based on thorough study of herbarium material (including holotype and isotype) and field research in Greece, Romania, and Poland, we confirm that gynostemium of E. greuteri has strongly reduced clinandrium and does not produce viscidium. We also discuss taxonomic treatment of E. preinensis and E. flaminia, two recently described taxa related to E. greuteri. The results of genetic analyses, as well as the range of phenotypic variability of E. greuteri individuals from various regions of Europe were presented and discussed. The analysis based on the ITS (internal transcribed spacer) nuclear marker showed no differences among E. helleborine, E. purpurata, E. albensis, and E. greuteri, which probably indicates their close relationship. Based on the analysis of plastid regions, six haplotypes were detected in all investigated samples. An exhaustive description of morphological features of E. greuteri is provided.

Is the lady's-slipper orchid (Cypripedium calceolus) likely to shortly become extinct in Europe?-Insights based on ecological niche modelling.

Lady's-slipper orchid (Cypripedium calceolus) is considered an endangered species in most countries within its geographical range. The main reason for the decline in the number of populations of this species in Europe is habitat destruction. In this paper the ecological niche modelling approach was used to estimate the effect of future climate change on the area of niches suitable for C. calceolus. Predictions of the extent of the potential range of this species in 2070 were made using climate projections obtained from the Community Climate System Model for four representative concentration pathways: rcp2.6, rcp4.5, rcp6.0 and rcp8.5. According to these analyses all the scenarios of future climate change will result in the total area of niches suitable for C. calceolus decreasing. Considering areas characterized by a suitability of at least 0.4 the loss of habitat will vary between ca. 30% and 63%. The highest habitat loss of ca. 63% is predicted to occur in scenario rcp 8.5. Surprisingly, in the most damaging rcp 8.5 prediction the highest overlap between potential range of C. calceolus and its pollinators will be observed and in all other scenarios some pollinators will be available for this species in various geographical regions. Based on these results at least two approaches should be implemented to improve the chances of survival of C. calceolus. In view of the unavoidable loss of suitable habitats in numerous European regions, conservation activities should be intensified in areas where this species will still have suitable niches in the next 50 years. In addition, for C. calceolus ex-situ activities should be greatly increased so that it can be re-introduced in the remaining suitable areas.

Morphology and genome size of Epipactis helleborine (L.) Crantz (Orchidaceae) growing in anthropogenic and natural habitats.

BACKGROUND: The process of apophytism or spreading native species to human-made habitats is one of the main elements in the creation of plant cover in anthropogenic areas. Lately, an increase of anthropogenic localities with valuable flora has been observed. Apophytes are also members of the family Orchidaceae, especially from the genus Epipactis. The aim of the study was to (i) determine and compare the phenotypic variation of E. helleborine (L.) Crantz plants in anthropogenic and natural habitats, (ii) compare the genome size of plants growing in natural and anthropogenic habitats. The results reported in this study may indicate that a habitat influences morphological characteristics of plant species. METHODS: Field studies were conducted on four native stands and four stands in anthropogenic areas of E. helleborine in Poland in years 2011-2013. Biometrical analyses were performed on shoots and flowers. The flowers were characterised by 25 biometric features and measured using a Nikon SMZ 800 binocular, microscopic Moticam-1SP cameras and the MIPlus07 programme (Conbest Co.). The nuclear DNA content was determined in fresh and young leaves of E. helleborine, collected from four natural and four anthropogenic populations. RESULTS: We observed that in anthropogenic populations: (i) shoots were higher than shoots from natural populations, (ii) flowers differed significantly in terms of ten biometric features between habitats, (iii) the genome size of some population differed significantly between plants growing in natural and anthropogenic habitats. DISCUSSION: According to some researchers, the presence of phenotypic variability and the occurrence of ecotypes are adaptation strategies of plants to environmental changes. In our opinion, in the case of the studied anthropogenic habitats (roadside) in which the E. helleborine populations grew, we can talk about ecofen due to the often repeated set of characteristic features, i.e., high shoots, long inflorescence and long, broad leaves. We agree, however, that it is difficult to isolate a taxonomic unit for ecofen due to the lack of experimental research.

Leaf arrangements are invalid in the taxonomy of orchid species.

The selection and validation of proper distinguishing characters are of crucial importance in taxonomic revisions. The modern classifications of orchids utilize the molecular tools, but still the selection and identification of the material used in these studies is for the most part related to general species morphology. One of the vegetative characters quoted in orchid manuals is leaf arrangement. However, phyllotactic diversity and ontogenetic changeability have not been analysed in detail in reference to particular taxonomic groups. Therefore, we evaluated the usefulness of leaf arrangements in the taxonomy of the genus Epipactis Zinn, 1757. Typical leaf arrangements in shoots of this genus are described as distichous or spiral. However, in the course of field research and screening of herbarium materials, we indisputably disproved the presence of distichous phyllotaxis in the species Epipactis purpurata Sm. and confirmed the spiral Fibonacci pattern as the dominant leaf arrangement. In addition, detailed analyses revealed the presence of atypical decussate phyllotaxis in this species, as well as demonstrated the ontogenetic formation of pseudowhorls. These findings confirm ontogenetic variability and plasticity in E. purpurata. Our results are discussed in the context of their significance in delimitations of complex taxa within the genus Epipactis.

Juncus quartinianus (Juncaceae, sect. Ozophyllum): A Neglected Species from the Horn of Africa and Its Re-Description Based on Morphological SEM Studies.

Juncus quartinianus (Juncaceae sect. Ozophyllum) was described by Richard in 1851 from Ethiopia. Some authors have treated this species as a synonym of J. fontanesii and others as a synonym of J. oxycarpus. Based on morphological analyses of flowers, fruit and seeds, we propose to restore J. quartinianus as a distinct species from both these taxa. Its detailed re-description and an identification key to the morphologically similar species of Juncus sect. Ozophyllum are provided.

Shapes of leaves with parallel venation. Modelling of the Epipactis sp. (Orchidaceae) leaves with the help of a system of coupled elastic beams.

Static properties of leaves with parallel venation, with particular emphasis on the genus EpipactisZinn, 1757 (Orchidaceae, Neottieae) have been modelled with coupled quasi-parallel elastic "beams." The non-linear theory of strongly bended beams have been employed. The resulting boundary-value problem has been solved numerically with the help of the finite-difference method. Possible dislocations resulting in additional Dirac-delta like forces have been take into account. Morphological similarity of the model and real leaves has been obtained. In particular, the concentrated forces have been shown to cause undulation in the leaves.