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Premise Difficulties in comparing extremely divergent features in fully mycoheterotrophic plants with those in closely related chlorophyllous plants have complicated attempts to reveal the evolutionary patterns and processes of fully mycoheterotrophic plants. Albino mutants of partially mycoheterotrophic plants, generally observed in Orchidaceae, have provided an ideal model for investigating the evolution of mycoheterotrophy within similar genetic backgrounds. In 2018, we found a putative albino population of Pyrola (Ericaceae). Here we aimed to reveal the identity of the albino pyroloid and confirm its fully mycoheterotrophic status. Methods To reveal the putative albino pyroloid's identity, we examined its morphology and sequenced its chloroplast DNA. In addition, we assessed the trophic status of the putative albino pyroloid by analyzing chlorophyll fluorescence, chlorophyll concentration, and natural 13 C and 15 N abundances. Results We identified albino individuals as P. japonica-otherwise a partially mycoheterotrophic species. We confirmed their albino status by their considerably lower chlorophyll fluorescence and concentrations than those of sympatrically occurring chlorophyllous plants. 13 C abundance in the albino individuals was significantly higher than in the green individuals of P. japonica. Conclusions This first report of albino mutants from partially mycoheterotrophic species in angiosperms other than orchids will play a valuable role in further studies focused on mycoheterotrophy. For instance, their δ13 C and δ15 N values represent a reference for fully mycoheterotrophic plants in Pyrola. Our findings also indicate the strong dependence of some leafy Pyrola species on fungal C during their entire life cycle.

The Central Asian species of the winter moth genus Phthorarcha Meyrick, 1892 are revised and some specimens of the type series of Phthorarcha primigena Staudinger, 1895 are separated and described as Phthorarcha haberhaueri, sp. nov. These poorly known moths and their male and female genitalia are described and illustrated and discrimination of the genera Phthorarcha, Alsophiloides Inoue and Chimaphila Nakajima & Wang is analysed.

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.

A phytochemical study of the whole plants of Chimaphila japonica Miq. led to the isolation of 23 compounds, including ten triterpenoids (1–10), six flavonoids (11–16), two sterols (17 and 18), two quinonoids (19 and 20), one saccharide derivative (21), one phenolic glycoside (22), and one megastigmane glycoside (23). The structures of these isolated compounds were identified using NMR spectroscopy (1H and 13C) by comparison with previously reported data. All compounds, except 19 and 22, were reported from C. japonica for the first time. Among them, 16 compounds (1–4, 6–9, 12, 13, 15, 16, 18, 20, 21, and 23) were reported from genus Chimaphila for the first time, while compounds 12, 16, and 23 were isolated from the Ericaceae family for the first time. The chemotaxonomic significance of the isolates was also discussed.

Background and Aims: Tertiary relict and Arctic/circumboreal distributions are two major patterns of Northern Hemisphere intercontinental disjunctions with very different histories. Each has been well researched, but members of one biome have generally not been incorporated the biogeographic analyses of the other, and links or transitions between these twoΞ biomes have rarely been addressed. Methods: Phylogenies were generated based on cpDNA and nuclear ITS, using Bayesian and ML methods. A time-calibrated phylogeny was generated using BEAST. Ancestral Area Reconstruction (AAR) was inferred using both Statistical Dispersal-Vicariance Analysis (S-DIVA) and dispersal-extinction-cladogenesis (DEC) model. Key Results: The Chimaphila crown group was estimated to have originated in the early Miocene. The lineages of C. umbellata diverged early, but its present circumboreal distribution was not achieved until around the middle Pliocene or later. Sister to this is a clade of four species with Tertiary relict distribution. Among these, two expansions occurred from North America to Asia, probably via the Bering Land Bridge generating its current disjunctions. Conclusions: Our data concurs with a few other studies, indicating that the circumboreal woodland biome has an older origin than most true arctic–alpine taxa, having gradually recruited taxa since the early Oligocene. For the origin of Asia – North America disjunctions in Chimaphila, an ‘out-of-America’ migration was supported. It is not clear in which direction Pyroloideae lineages moved between Tertiary relict disjunctions and Arctic/circumboreal distributions; each biome might have recruited species from the other.

DNA barcoding has been widely used as a tool for species discrimination in closely related plants and at more coarse taxonomic levels, but there have been few reports of such a tool being applied to taxa in "morphological stasis". In this study, we test the performance of core barcodes (rbcL and matK) and two additional candidate barcodes [psbA-trnH and nuclear ribosomal internal transcribed spacer (ITS)] for the widely distributed, northern temperate monophyletic genus Chimaphila, which exhibits morphological conservatism. In addition to ITS, slowly evolving rbcL and matK independently demonstrate a high resolving ability among Chimaphila species, indicative of the old age of disjunction. We tentatively attribute morphological stasis in Chimaphila to convergence under similar habitat conditions and complex, stable relationships with surrounding autotrophic trees via common mycorrhizal symbioses.

SPE IPM UB; International audience; In temperate forests, some green plants, namely pyroloids (Pyroleae, Ericaceae) and some orchids, independently evolved a mode of nutrition mixing photosynthates and carbon gained from their mycorrhizal fungi (mixotrophy). Fungal carbon is more enriched in 13C than photosynthates, allowing estimation of the proportion of carbon acquired heterotrophically from fungi in plant biomass. Based on 13C enrichment, mixotrophic orchids have previously been shown to increase shoot autotrophy level over the growth season and with environmental light availability. But little is known about the plasticity of use of photosynthetic versus fungal carbon in pyroloids. Methods : Plasticity of mixotrophy with leaf age or light level (estimated from canopy openness) was investigated in pyroloids from three Estonian boreal forests. Bulk leaf 13C enrichment of five pyroloid species was compared with that of control autotrophic plants along temporal series (over one growth season) and environmental light gradients (n=405 samples). Key Results : Mixotrophic 13C enrichment was detected at studied sites for Pyrola chlorantha and Orthilia secunda (except at one site for the latter), but not for Chimaphila umbellata, Pyrola rotundifolia and Moneses uniflora. Enrichment with 13C did not vary over the growth season or between leaves from current and previous years. Finally, although one co-occurring mixotrophic orchid showed 13C depletion with increasing light availability, as expected for mixotrophs, all pyroloids responded identically to autotrophic control plants along light gradients. Conclusions : A phylogenetic trend previously observed is further supported: mixotrophy is rarely supported by 13C enrichment in the Chimaphila + Moneses clade, whereas it is frequent in the Pyrola + Orthilia clade. Moreover, pyroloid mixotrophy does not respond plastically to ageing or to light level. This contrasts with the usual view of a convergent evolution with orchids, and casts doubt on the way pyroloids use the carbon gained from their mycorrhizal fungi, especially to replace photosynthetic carbon

Pyroleae (Ericaceae) consist of four genera, all of which are distributed widely in temperate coniferous or sometimes deciduous forests of the Northern Hemisphere. To investigate the phylogenetic relationships among these genera and to explore the evolution of the characteristics of the subfamily, we conducted maximum parsimony and Bayesian analyses with nrDNA ITS and three cpDNA intergenic spacers (atpB-rbcL, trnS-trnG and trnL-trnF). The results from cpDNA and combined cpDNA + ITS data sets strongly support the monophyly of Pyroleae as well as a sister relationship between Pyrola and Moneses-Chimaphila, with Orthilia as the basal lineage. The sister-group relationship between Moneses and Chimaphila is supported by a set of synapomorphies, e.g., single flower, colpate pollen, five bundles in the style, straight fruiting pedicel orientation, complete capsule dehiscence, and the basic chromosome number, x = 13. The Moneses-Chimaphila-Pyrola clade is supported by at least one homologous character of pollen in tetrads. Conflicts associated with the phylogenetic position of Orthilia may imply a hybrid origin for it, and therefore further study is needed.

A survey of the seed morphology of 24 species and all of the four genera, Pyrola, Orthilia, Moneses, and Chimaphila, of the subfamily Pyroloideae of the Ericaceae showed that the testa and the endosperm dimensions, and especially the pitting patterns on the inner periclinal walls of the testa cells, are significant taxonomically at or below the generic level and even at the infraspecific level in some species. Seeds of Moneses are clearly distinguished from those of the other three genera in having transversely elongated large pits (pitting type 5) on the inner periclinal walls. Chimaphila species are uniform in having smooth or slightly pitted inner periclinal walls (pitting types commonly 1-2). Thus, the seed morphology of Moneses is clearly different from that of Chimaphila. Seed morphology of Pyrola shows considerable variability and no seed characters define the genus. Orthilia seeds do not have any peculiar characteristics and are more similar to those of Chimaphila than Pyrola according to principa...

The small subfamily Pyroloideae, comprising four genera, is unusual in Ericaceae because of its nearly herbaceous habit, largely free petals, and tendencies toward leaflessness. A surprising number of hypotheses exist with respect to the relationships among these genera. Parsimony analysis of 23 morpholog- ical characters gave a pattern that is largely congruent with that obtained from analysis of ITS sequences. Two sister clades of monophyletic/monospecific genera are resolved-Chimaphila + Moneses and Orthilia + Pyrola. Relationships among three species of Chimaphila are not clearly resolved. Some structure is present within Pyrola, with two strongly supported clades (P picta + PR aphylla + PR chlorantha and P elliptica + P minor). The placement of P minor is unsuspected; most past classifications have segregated it from other Pyrola due to its short, straight style (as opposed to a longer, curved style). Developmental study reveals that the style terminates elongation early, so that in fact it is not clear which style orientation is present. The overall bud-like nature of the flower in P minor further suggests a juvenile morphology relative to other Pyrola. The developmental information, in combination with its position on the cladogram, indicates a pae- domorphic origin for the floral morphology of P minor.

Chimaphilin is a yellow naphthoquinone which occurs naturally in various chimaphila and Pyrola species. In Chimaphila umbellata (winter green) and C. maculata, it is a major constituent. Folk medicine recommends the leaves of Chimaphila species as a topical application to treat skin diseases. Since 1887, winter green is claimed to have caused dermatitis and to have been responsible for "idiosyncrasy". Experimental sensitization using the open epicutaneous as well as Freund's complete adjuvant technique has now revealed that chimaphilin is a moderate contact sensitizer.

A reevaluation of the generic limits within the Pyroloideae is presented, taking into consideration the more recent evidence of chromosome number within the group. Chromosome number is correlated with more obvious morphological features, many of which have been the prime basis for the delimitation of generic boundaries in earlier treatments. Brief consideration is given also to the implications of data obtained from a thin-layer chromatographic analysis of the subfamily.Three generic segregates from within the original broad concept of the genus Pyrola are recognized. The diploid chromosome numbers which have been reported for Orthilia secunda (L.) House (2n = 38) and Moneses uniflora (L.) Gray and Chimaphila spp. (2n = 26) in conjunction with discrete differences in inflorescence types and floral modifications among these taxa serve as useful characteristics on which to base their recognition as genera distinct from Pyrola s.str. (2n = 46).

Four hundred and eighty plants in northern California from 27 taxa in the Ericaceae and 5 in the Pyrolaceae were surveyed for presence of ectomycorrhizae, arbutoid, or ericoid mycorrhizae between September 1977 and April 1978. Of these, 337 in the Ericaceae and 8 in the Pyrolaceae had one or more types of mycorrhizae.At least 88% of the plants of larger shrubs or trees (Arbutus menziesii or Arctostaphylos spp.) were mycorrhizal in various locations in northern California. Of the plants in the remaining twenty-two taxa that were mycorrhizal, 67–100% had ectomycorrhizae or arbutoid mycorrhizae. Twenty-three taxa of ericaceous or pyrolaceous plants in the genera Cassiope, Gaultheria, Kalmia, Ledum, Leucothoe, Phyllodoce, Rhododendron, Vaccinium, Pyrola, and Chimaphila were also mycorrhizal. The type and abundance of mycorrhizae appear to depend on as yet undefined ecological factors.Ectomycorrhizae (including the Cenococcum type) or ericoid mycorrhizae are reported for the first time in Arbutus, most Arctostaphylos spp., Gaultheria shallon, Kalmia polifolia, Ledum glandulosum var. columbianum, Leucothoe davisiae, Rhododendron macrophyllum, Vaccinium arbuscula, V. ovatum, V. scoparium, Chimaphila menziesii, and Pyrola picta f. picta.

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