Search

The family Pyrolaceae (pyroloids) includes the group of evergreen herbs or subshrubs growing in coniferous, especially in pine forest of the northern hemisphere. Their typical features are mycoheterotrophy and mixotrophy. In the early stage of development the mycoheterotrophy is used to obtain the necessary resources because the seeds do not contain enough substances to germinate. Adult plants then switch to mixotrophy or autotrophy, except P. aphylla. Mixotrophy allows pyroloids to combine inorganic carbon gain obtained from photosyntesis and organic carbon from symbiotic fungi. Mixotrophy of this family can significantly affect other plant species growing in its vicinity through mycelial networks of mycorrhizal fungi and contribute to affecting the structure of the community. At the present, all pyroloids are among the endangered or declining species in our country and in the world. This bachelor project summarizes the knowledge about pyroloids in the field of phylogeny, mycorrhizal symbiosis, ecology, distribution and in vitro cultivation in the form of literature research. In the last chapter I deal with the causes of decline and the possibilities of pyroloid protection. Key words: Pyrolaceae, mixotrophy, mycoheterotrophy, ectomycorrhiza, Pyrola, Chimaphila umbellata, Moneses, Orthilia

International audience; Background and AimsPyroloids, forest sub-shrubs of the Ericaceae family, are an important model for their mixotrophic nutrition, which mixes carbon from photosynthesis and from their mycorrhizal fungi. They have medical uses but are difficult to cultivate ex situ; in particular, their dust seeds contain undifferentiated, few-celled embryos, whose germination is normally fully supported by fungal partners. Their germination and early ontogenesis thus remain elusive.MethodsAn optimized in vitro cultivation system of five representatives from the subfamily Pyroloideae was developed to study the strength of seed dormancy and the effect of different media and conditions (including light, gibberellins and soluble saccharides) on germination. The obtained plants were analysed for morphological, anatomical and histochemical development.Key ResultsThanks to this novel cultivation method, which breaks dormancy and achieved up to 100 % germination, leafy shoots were obtained in vitro for representatives of all pyroloid genera (Moneses, Orthilia, Pyrola and Chimaphila). In all cases, the first post-germination stage is an undifferentiated structure, from which a root meristem later emerges, well before formation of an adventive shoot.ConclusionsThis cultivation method can be used for further research or for ex situ conservation of pyroloid species. After strong seed dormancy is broken, the tiny globular embryo of pyroloids germinates into an intermediary zone, which is functionally convergent with the protocorm of other plants with dust seeds such as orchids. Like the orchid protocorm, this intermediary zone produces a single meristem: however, unlike orchids, which produce a shoot meristem, pyroloids first generate a root meristem.

Context: The leaves of Pyrola decorate H. Andr (Pyrolaceae), known as Luxiancao, have long been used for treating kidney deficiency, gastric haemorrhage and rheumatic arthritic diseases in traditional Chinese medicine. Objective: The phytochemicals and antioxidant capacities in vitro of P. decorate leaves were investigated. Materials and methods: Ethanol, petroleum ether, acetidin, n-butyl alcohol and aqueous extracts of Pyrola decorate leaves were prepared by solvent sequential process, and then isolated and purified to obtain phytochemicals. Cell viability was measured by MTT assay. PC12 cells were pretreated for 24 h with different extractions of P. decorate leaves at concentrations of 0.1, 0.5, 1, 5 and 10 mg/mL, then H2O2 of 0.4 mM was added in all samples for an additional 2 h. The antioxidant capacities of betulin, ursolic acid and monotropein were determined in PC12 cells against H2O2 induced cytotoxicity in vitro as well. Results: Nine compounds (1–9) were isolated and structurally determined by spectroscopic methods, especially 2D NMR analyses. Ethanol extract treated groups showed inhibitory activity with IC50 value of 10.83 mg/mL. Betulin, ursolic acid and monotropein were isolated from P. decorate, and demonstrated with IC50 values of 6.88, 6.15 and 6.13 μg/mL, respectively. Discussion and conclusions: In conclusion, Pyrola decorate is a potential antioxidative natural plant and worth testing for further pharmacological investigation in the treatment of oxidative stress related neurological disease.

Pyroleae species are perennial understory shrubs, many of which are partial mycoheterotrophs. Most fungi colonizing Pyroleae roots are ectomycorrhizal (ECM) and share common mycobionts with their Pyroleae hosts. However, such mycobiont sharing has neither been examined in depth before nor has the interspecific variation in sharing among Pyroleae species. Here, we examined root-associated fungal communities in three co-existing Pyroleae species, including Pyrola alpina, Pyrola incarnata, and Orthilia secunda, with reference to co-existing ECM fungi on the surrounding trees in the same soil blocks in subalpine coniferous forests. We identified 42, 75, and 18 fungal molecular operational taxonomic units in P. alpina, P. incarnata, and O. secunda roots, respectively. Mycobiont sharing with surrounding trees, which was defined as the occurrence of the same mycobiont between Pyroleae and surrounding trees in each soil block, was most frequent among P. incarnata (31 of 44 plants). In P. alpina, sharing was confirmed in 12 of 37 plants, and the fungal community was similar to that of P. incarnata. Mycobiont sharing was least common in O. secunda, found in only 5 of 32 plants. Root-associated fungi of O. secunda were dominated by Wilcoxina species, which were absent from the surrounding ECM roots in the same soil blocks. These results indicate that mycobiont sharing with surrounding trees does not equally occur among Pyroleae plants, some of which may develop independent mycorrhizal associations with ECM fungi, as suggested in O. secunda at our research sites. Electronic supplementary material The online version of this article (doi:10.1007/s00572-017-0788-6) contains supplementary material, which is available to authorized users.

The family Pyrolaceae is represented in Bulgaria by six species, belonging to three genera. The viewpoints for the taxonomical position of the species in this family are not clearly unilateral. The aim of this study is to find molecular markers, allowing the objectively differentiation of the genera in this family, without use of morphological characters. Three species of the family were examined, representing the tree genera distributed in Bulgaria: Moneses uniflora (L.) A.Gray, Orthilia secunda (L.) House and Pyrola minor L. DNA was isolated from leafs and amplified via PCR reactions using 4 primers. The results were processed using cluster analysis and non-metric multidimensional scalling. The consequent cladogram, obtained by the mean euclidean distances, displayed grouping of the samples by genera, independently of their locality. The independent multitudes confirm the accuracy of the method for determination of the species in Pyrolaceae.

Starch gel electrophoresis was used to estimate genetic diversity and population structure of Pyrola fauriena H. Andr. in Korea. The percentage of polymorphic loci within enzymes was . The values of genetic diversity at the species level and at the population were higher than average values for herbaceous with similar life history traits (Hes : 0.149; Hep

Five new phenolic glycosides, 2-beta-D-glucopyranosyloxy-5-hydroxyphenylacetic acid methyl ester (4), 4-hydroxy-2-[3-hydroxy-3-methylbutyl]-5-methylphenyl beta-D-glucopyranoside (5), 4-hydroxy-2-[(E)-4-hydroxy-3-methyl-2-butenyl]-5-methylphenyl beta-D-glucopyranoside (7), 4-hydroxy-2-[(2E,6Z)-8-beta-D-glucopyranosyloxy-3,7-dimethylocta-2,6-dien-1-yl]-5-methylphenyl beta-D-glucopyranoside (8), and 2,7-dimethyl-1,4-dihydronaphthalene-5,8-diol 5-O-beta-D-xylopyranosyl(1--6)-beta-D-glucopyranoside (10), were isolated from the whole plants of Pyrola japonica (Pyrolaceae), together with androsin, (-)-syringaresinol glucoside, homoarbutin, pirolatin, hyperin, monotropein and chimaphilin.

Floral scent was collected by headspace methods from intact flowers, petals, and stamens of four species ofPyrolaceae. The scent samples were analyzed by coupled gas chromatography-mass spectrometry (GC-MS). The floral scent inPyrola spp. is differentiated into a characteristic petal scent—phenyl propanoids and a characteristic stamen scent—methoxy benzenes. InMoneses the scent is characterized by isoprenoids and benzenoids, with a larger proportion of benzenoids in the stamens compared to the petals. Specific anther scents may promote foraging efficiency in buzz-pollinated species and enhance flower fidelity. Variation in floral scent composition is consistent with the taxonomic relationships among the genera and species examined.

Several forest understorey achlorophyllous plants, termed mycoheterotrophs (MHs), obtain C from their mycorrhizal fungi. The latter in turn form ectomycorrhizas with trees, the ultimate C source of the entire system. A similar nutritional strategy occurs in some green forest orchids, phylogenetically close to MH species, that gain their C via a combination of MH and photosynthesis (mixotrophy). In orchid evolution, mixotrophy evolved in shaded habitats and preceded MH nutrition. By generalizing and applying this to Ericaceae, we hypothesized that green forest species phylogenetically close to MHs are mixotrophic. Using stable C isotope analysis with fungi, autotrophic, mixotrophic and MH plants as comparisons, we found the first quantitative evidence for substantial fungi-mediated mixotrophy in the Pyroleae, common ericaceous shrubs from boreal forests close to the MH Monotropoideae. Orthilia secunda, Pyrola chlorantha, Pyrola rotundifolia and Chimaphila umbellata acquired between 10.3 and 67.5% of their C from fungi. High N and 15N contents also suggest that Pyroleae nutrition partly rely on fungi. Examination of root fungal internal transcribed spacer sequences at one site revealed that 39 species of mostly endophytic or ectomycorrhizal fungi, including abundant Tricholoma spp., were associated with O. secunda, P. chlorantha and C. umbellata. These fungi, particularly ectomycorrhizal associates, could thus link mixotrophic Pyroleae spp. to surrounding trees, allowing the C flows deduced from isotopic evidence. These data suggest that we need to reconsider ecological roles of understorey plants, which could influence the dynamics and composition of forest communities.

From the water-soluble constituents of the whole herb of Pyrola rotundifolia (Pyrolaceae), one novel phenolic glycoside dimer, pyrolaside A (1), and one novel phenolic glycoside trimer, pyrolaside B (2), together with two known phenolic glycosides homoarbutin (3) and isohomoarbutin (4), were isolated. The structures were elucidated by spectroscopic analysis and confirmed with chemical degradation. In vitro tests for antimicrobial activity showed pyrolaside B (2) to possess significant activity against two Gram-positive organisms, Staphylococcus aureus and Micrococcus luteus.

This paper presents a review of some of the fungi that parasitize hosts in the Pyrolaceae. Lophodermium pyrolae Parmelee on Pyrola spp. and Strasseria nigra Dearn. on Pyrola spp. are described as new. Mycosphaerella chimaphilae (Ell. & Ev.) Hoehn. is considered to be synonymous with Mycosphaerella chimaphilina (Pk. in Sacc.) House. Ovularia pyrolae Trel. is transferred to the genus Colletotrichum and new hosts and range extensions are listed for other fungi.

As part of a general survey of the flavonoids of Pyrolaceae, the flavonoids of Pyrola virens and P. chlorantha were investigated. Eight flavonol glycosides based upon kaempferol, quercetin and rhamnetin were identified from each of the two species. Two of the glycosides, rhamnetin 3,3′,4′-tri- O -glucoside and rhamnetin 3- O -arabinoside-3′,4′-di- O -glucoside are previously unreported and further, represent an unusual pattern of glycosylation. The similarity of flavonoids and the presence of the unusual substitution pattern supports a conspecific status for the two taxa.

Pollen morphology of the Pyrolaceae and Monotropaceae reinforces the view that the Pyrolaceae and Ericaceae are closely related and that the Monotropaceae should be treated as a distinct family. It further supports the separation of Moneses uniflora and Orthilia secunda from the genus Pyrola, and in the Monotropaceae it suggests a realignment of species within the Hypopitys-Monotropa complex. The Pyrolaceae as aligned by Schultze-Motel (1964) constitute a diverse group consisting of two subfamilies Pyroloideae and Monotropoideae, the latter largely saprophytic. Others have reduced one or both taxa to subdivisions of the Ericaceae, either as the tribe Pyroleae (Bentham & Hooker, 1873), or as the subf. Monotropoideae with the Pyroleae included in the subf. Arbutoideae (Copeland, 1941, 1947). A palynological study was made to determine if pollen morphology, a relatively unused attribute, might better characterize the affinities of the taxa involved. Erdtman (1952), largely following the systematic treatment of Drude (1889), described the pollen of 15 species representing eight genera. According to him, species of Pyrola L. have pollen united in tetrads with individual grains 3-colporate; pollen of Chimaphila umbellata Nutt. is in tetrads with apertures not always sharply delimited; pollen of Moneses uniflora (L.) Gray also is united in tetrads in which individual grains are 3-colp(oroid) ate; and Ramischia secunda (L.) Garcke [=Orthilia secunda (L.) House] has single grains which are (2-) 3colporate. In the Monotropoideae, Erdtman described pollen of Monotropa hypophegea Wallr. as (2-) 3-colporate, Pleuricospora fimbriolata Gray as (3-) 4colporoidate, Pterospora andromedea Nutt. as 4 (-5)-colporoidate, and Sarcodes sanguinea Torrey as 4 (-5)-colpate. Erdtman utilized Copeland's (1938) description of Allotropa virgata Torrey & Gray which was noted as three-grooved. Copeland (1934, 1935, 1937, 1938, 1939, 1941, 1947), in an extensive anatomical and systematic study of the tribe Pyroleae and subf. Monotropoideae, included a brief description of pollen of various genera. In his treatment of the Pyroleae (1947), he recognized four genera with the pollen grains of Ramischia Opiz (=Orthilia Raf.) solitary and tricolpate, those of Chimaphila Pursh in easily disrupted tetrads, those of Pyrola united in tetrads with the wall of each individual grain "marked by three half-grooves, continued as half grooves on the three as