Search

‘Dust seeds’ with an undifferentiated (organless) embryo are known to be produced by mycoheterotrophic species (MH) in nine families of angiosperms. However, aside from the numerous studies on seed germination of orchids, relatively little is known about germination in MH families. In the Ericaceae, some degree of mycoheterotrophy (full, partial or initial) and dust seeds with an undifferentiated embryo occur in all species in the three tribes of Monotropoideae, the only subfamily of Ericaceae with this combination of characters. In most species, the seed is Pityopus) or consists of few to many (30–40) cells, and the embryo is minute, consisting of as few as two cells inMonotropa. Germination in Monotropoideae is monopolar, with only the radicular pole of the embryo participating in germination. Thus, germination polarity differs from that of the dust seeds of orchids in which only the plumular pole of the embryo (protocorm) participates in germination. The dust seeds in Monotropoideae require the presence of fungi, either direct contact with a fungus or the presence of a diffusible substance therefrom, to germinate (symbiotic germination). Recently, representatives of the four genera of tribe Pyroleae have been successfully germinated asymbioticallyin vitro. We present a broad overview of dust-size seeds in angiosperms and conclude that they should be subdivided into at least two major categories.

Summary • Germination and symbiotic development of the myco-heterotrophic plant Monotropa hypopitys were studied by sequential recovery of packets of seed buried in dune slacks in relation to distance from mature M. hypopitys and presence and absence of shoots of its autotrophic coassociate Salix repens . • Fungal associates of M. hypopitys growing under S. repens in the dune slacks, and under S. caprea and Pinus sylvestris at two other locations in the UK, were identified by molecular analysis. • While the earliest stage of germination could be found in the absence both of mature M. hypopitys , and S. repens , further development was dependent upon mycorrhizal colonisation, which was most common close to these plants. Molecular analysis showed that when growing with Salix , M. hypopitys associated with the Salix -specific ectomycorrhizal fungus Tricholoma cingulatum , whereas under Pinus it was colonised by the closely related, Pinaceae-specific, T. terreum . •W e establish the first definitive chronology of development of M. hypopitys and highlight its critical dependence upon, and specificity for, locally distributed Tricholoma species that link the myco-heterotroph to its autotrophic coassociates.

The monotypic genus Eremotropa Andres (Monotropoideae, Ericaceae) is endemic to central Yunnan. It was first described by Andres in 1953, and was subsequently treated as a synonym of Monotropastrum Andres (1987); however, the phylogenetic placement of this genus has not, to date, been investigated. In this study, phylogenetic analyses were undertaken using DNA sequences from the nuclear internal transcribed spacer from all species of Monotropoideae available in GenBank. Comparative morphological studies on Eremotropa, Monotropastrum, and Monotropa L. were also carried out. The molecular phylogenetic evidence suggests that Eremotropa is sister to a clade formed by Monotropa and the remaining species of Monotropastrum, thus rendering Monotropastrum not monophyletic. Morphologically, Eremotropa differs from Monotropastrum in plant color, inflorescence type, length of the style, and size of the baccate fruit. Consequently, molecular and morphological evidence supports the resurrection of Eremotropa, which contains only one species, E. sciaphila Andres, as distinct from Monotropastrum.

Inferring interfamilial relationships within the eudicot order Ericales has remained one of the more recalcitrant problems in angiosperm phylogenetics, likely due to a rapid, ancient radiation. As a result, no comprehensive time-calibrated tree or biogeographical analysis of the order has been published. Here, we elucidate phylogenetic relationships within the order and then conduct time-dependent biogeographical and diversification analyses by using a taxon and locus-rich supermatrix approach on one-third of the extant species diversity calibrated with 23 macrofossils and two secondary calibration points. Our results corroborate previous studies and also suggest several new but poorly supported relationships. Newly suggested relationships are: (1) holoparasitic Mitrastemonaceae is sister to Lecythidaceae, (2) the clade formed by Mitrastemonaceae + Lecythidaceae is sister to Ericales excluding balsaminoids, (3) Theaceae is sister to the styracoids + sarracenioids + ericoids, and (4) subfamilial relationships with Ericaceae suggest that Arbutoideae is sister to Monotropoideae and Pyroloideae is sister to all subfamilies excluding Arbutoideae, Enkianthoideae, and Monotropoideae. Our results indicate Ericales began to diversify 110 Mya, within Indo-Malaysia and the Neotropics, with exchange between the two areas and expansion out of Indo-Malaysia becoming an important area in shaping the extant diversity of many families. Rapid cladogenesis occurred along the backbone of the order between 104 and 106 Mya. Jump dispersal is important within the order in the last 30 My, but vicariance is the most important cladogenetic driver of disjunctions at deeper levels of the phylogeny. We detect between 69 and 81 shifts in speciation rate throughout the order, the vast majority of which occurred within the last 30 My. We propose that range shifting may be responsible for older shifts in speciation rate, but more recent shifts may be better explained by morphological innovation.

Mycoheterotrophic plants obtain organic carbon from associated mycorrhizal fungi, fully or partially. Angiosperms with this form of nutrition possess exceptionally small 'dust seeds' which after germination develop 'seedlings' that remain subterranean for several years, fully dependent on fungi for supply of carbon. Mycoheterotrophs which as adults have photosynthesis thus develop from full to partial mycoheterotrophy, or autotrophy, during ontogeny. Mycoheterotrophic plants may represent a gradient of variation in a parasitism-mutualism continuum, both among and within species. Previous studies on plant-fungal associations in mycoheterotrophs have focused on either germination or the adult life stages of the plant. Much less is known about the fungal associations during development of the subterranean seedlings. We investigated germination and seedling development and the diversity of fungi associated with germinating seeds and subterranean seedlings (juveniles) in five Monotropoideae (Ericaceae) species, the full mycoheterotroph Monotropa hypopitys and the putatively partial mycoheterotrophs Pyrola chlorantha, P. rotundifolia, Moneses uniflora and Chimaphila umbellata. Seedlings retrieved from seed sowing experiments in the field were used to examine diversity of fungal associates, using pyrosequencing analysis of ITS2 region for fungal identification. The investigated species varied with regard to germination, seedling development and diversity of associated fungi during juvenile ontogeny. Results suggest that fungal host specificity increases during juvenile ontogeny, most pronounced in the fully mycoheterotrophic species, but a narrowing of fungal associates was found also in two partially mycoheterotrophic species. We suggest that variation in specificity of associated fungi during seedling ontogeny in mycoheterotrophs represents ongoing evolution along a parasitism-mutualism continuum.

Рассматриваются наиболее характерные особенности растений подсемейства вертляницевые (Monotropoideae: Ericaceae): морфология, систематика, распространение, образ жизни. Из 10 родов мировой флоры на территории России оно представлено только двумя родами с тремя видами., The most characteristic features of the Indian-pipe subfamily (Monotropoideae: Ericaceae): morphology, systematics, distribution, lifestyle are considered. it is represented from the 10 genera of world flora in Russia by only two genera with three species., №203(1) (2019)

Mycorrhizal symbiosis often displays low specificity, except for mycoheterotrophic plants that obtain carbon from their mycorrhizal fungi and often have higher specificity to certain fungal taxa. Partially mycoheterotrophic (or mixotrophic, MX) plant species tend to have a larger diversity of fungal partners, e.g., in the genus Pyrola (Monotropoideae, Ericaceae). Preliminary evidence however showed that the Japanese Pyrola japonica has preference for russulacean fungi based on direct sequencing of the fungal internal transcribed spacer (ITS) region from a single site. The present study challenges this conclusion using (1) sampling of P. japonica in different Japanese regions and forest types and (2) fungal identification by ITS cloning. Plants were sampled from eight sites in three regions, in one of which the fungal community on tree ectomycorrhizal (ECM) tips surrounding P. japonica was also analyzed. In all, 1512 clone sequences were obtained successfully from 35 P. japonica plants and 137 sequences from ECM communities. These sequences were collectively divided into 74 molecular operational taxonomic units (MOTUs) (51 and 33 MOTUs, respectively). MOTUs from P. japonica involved 36 ECM taxa (96 % of all clones), and 17 of these were Russula spp. (76.2 % of all clones), which colonized 33 of the 35 sampled plants. The MOTU composition significantly differed between P. japonica and ECM tips, although shared species represented 26.3 % of the ECM tips community in abundance. This suggests that P. japonica has a preference for russulacean fungi.

Few studies of tripartite mycoheterotrophic systems have examined ecological specificity across broad geographic ranges or addressed autotrophic host specificity. Pterospora andromedea was selected as an ideal candidate to examine ecological specificity of a mycoheterotrophic system as it is widely distributed, has been shown to have high levels of symbiont specificity with Rhizopogon subgenus Amylopogon , and is found with several autotrophic hosts. Pairs of P. andromedea + Rhizopogon spp. samples were co-collected across North America and were sequenced using trnL and ITS, respectively. Bayesian phylogenetic reconstructions between the co-collected taxa were used to examine ecological specificity, and for subsequent tests for autotroph specificity. P. andromedea lineages exhibited both high specificity and relaxed specificity for fungal symbionts and autotrophic hosts across the geographic landscape under allopatric and sympatric conditions. This strong evidence for geographic mosaics of specificity in mycoheterotrophic systems is an important future consideration in determining the evolutionary ecology of mycoheterotrophs.

The mycoheterotrophic plant Pterospora andromedea is an important species in understanding the evolution and ecology of highly specialized plant-fungal symbioses. Lineages within P. andromedea have been shown to engage in highly specific interactions with obligate fungal associates found in Rhizopogon subgenus Amylopogon. Unfortunately, evidence of highly specific interactions resulted from single locus analyses and this result requires further examination. If highly specific host-symbiont selectivity is present, then the lack of compatible partners may best explain the conservation status of P. andromedea. To better evaluate and support conservation efforts for P. andromedea, more molecular loci are necessary. 115 candidate loci were screened from Illumnia MiSeq and Ion Torrent next-generation sequencing and resulted in the development of four polymorphic anonymous nuclear loci. These loci will provide the means to critically examine host-symbiont specificity for P. andromedea lineages across the endemic range and will assist conservation initiatives.

Pradhan P. 2015. Potential distribution of Monotropa uniflora as a surrogate for range of Monotropoideae (Ericaceae) in South Asia. Biodiversitas 16: 109-115. Monotropoideae is a mycoheterotrophic subfamily of Ericaceae. Its members are highly specific to a particular fungal family, which has attributed to the rarity and limited distribution of Monotropoideae. In the past two decades, there are considerable developments in understanding their biology and biogeography, among which,the distribution of Monotropa uniflora L. and M. hypopitys L. has been extensively studied. In this contribution, Ecological Niche Modeling of M. uniflora has been conducted to test its earlier proposed distribution in South Asia, to test the spatial scale of the said proposal, to test its potential distribution as a surrogate for range of Monotropoideae in South Asia and to prioritize conservation areas for M. uniflora in the region. The model was built with five occurrence details of the rare plant M. uniflora in Western and Eastern Himalaya, in relation to 19 bioclimatic explanatory variables, performed in MaxEnt. The results show the good performance of the model with the training AUC of 0.994. 1,50,316 square Km. of suitable areas have been predicted for the growth of M. uniflora (IHS ≥0.5) in South Asia, many areas of which is in line with earlier distributional reports. The bioclimatic variables are able to predict and suitably justify the spatial distribution of M. uniflora. The predicted range of the species could be established for potential distribution of other Asian Monotropoids like Monotropastrum and Cheilotheca. Key words: Ecological Niche Modeling, Himalaya, MaxEnt, Mycoheterotrophy, Russulaceae

Most recent treatments of Monotropsis recognize a single species of achlorophyllous, mycoheterotrophic herbs endemic to the southeastern U. S. A. (M. odorata), although four species have been described. This study reevaluates these proposed taxa using a comprehensive approach to species delimitation analyzing variation in morphology, phenology, geography, nuclear DNA, and chloroplast DNA. Principal components analysis of morphometric data reveals two clusters that are geographically distinct. These groups correspond to clades resolved with nuclear (ITS/26S and Xdh) and plastid (rpl32-trnL) DNA that are furthermore distinct phenologically. These data support recognition of M. reynoldsiae, a Florida endemic and M. odorata, an Appalachian endemic. Monotropsis lehmaniae, proposed based on an autumn flowering period and supposed floral differences, is shown to comprise individuals that are not yet in anthesis, and should not be recognized. Cryptophila pudica, named based on its disjunct geographical d...

Studies of the Monotropoideae (monotropes; Ericaceae), a monophyletic group of non-photosynthetic, mycoheterotrophic, and often rare or endangered plants, have been limited by the inability to propagate them. Monotropes associate with specific fungal hosts, and the only previously known method of seed germination was induction by host fungi or closely related fungi. In order to overcome very low monotrope seed germination rates and to facilitate further study and conservation efforts, we developed a method using gibberellic acid (GA) to induce asymbiotic germination. Pterospora andromedea Nutt. (Monotropoideae, Ericaceae) and Sarcodes sanguinea Torr. (Monotropoideae, Ericaceae) seeds from California were exposed to their fungal symbiont or to agarose infused with 0–1 mM GA and then scored for germination. Continuous exposure to 0.5 mM GA for two months induced 75% P. andromedea germination, compared to only 21% with its host fungus, Rhizopogon salebrosus A.H. Sm. (Basidiomycota). Even short GA exposure (one or three days) significantly enhanced germination (69% and 90%, respectively). The highest germination rate was observed with exposure to 0.5 mM GA for three or 14 d. The closely related S. sanguinea required a three-month exposure to GA and even then produced far lower germination rates (∼1%). Nevertheless, this is the only known method of inducing monotrope germination without the presence of a specific fungal symbiont. In the case of P. andromedea, exogenous GA stimulates germination at rates far higher than that achieved with its fungal symbiont. Application of GA to induce monotrope germination may be used to examine the early stages of mycoheterotroph development, to improve assays for seed viability, and potentially to aid conservation efforts.

Imbalances in phylogenetic diversity could be the result of variable diversification rates, differing limits on diversity, or a combination of the two. We propose an approach to distinguish between rates and limits as the primary cause of phylogenetic imbalance, using parasitic plants as a model. With sister-taxon comparisons, we show that parasitic plant lineages are typically much less diverse than their autotrophic sisters. We then use age estimates for taxa used in the sister-taxon comparisons to test for correlations between clade age and clade diversity. We find that parasitic plant diversity is not significantly correlated with the age of the lineage, whereas there is a strong positive correlation between the age and diversity of nonparasitic sister lineages. The Ericaceae sister pair Monotropoideae (parasitic) and Arbutoideae (autotrophic) is sufficiently well sampled at the species level to allow more parametric comparisons of diversification patterns. Model fitting for this group supports ecological limitation in Monotropoideae and unconstrained diversification in Arbutoideae. Thus, differences in diversity between parasitic plants and their autotrophic sisters might be caused by a combination of ecological limitation and exponential diversification. A combination of sister-taxon comparisons of diversity and age, coupled with model fitting of well-sampled phylogenies of focal taxa, provides a powerful test of likely causes of asymmetry in the diversity of lineages.

We provide a preliminary report of the mycobionts found within four Monotropoideae (Ericaceae) species from China: Monotropa uniflora, Hypopitys monotropa, Monotropastrum humile and Monotropastrum sciaphilum (a rare endemic species never previously studied for mycorrhizae). Such achlorophyllous Monotropoideae plants obtain their carbohydrates from mycorrhizal fungi linking them to surrounding trees, on which these fungi form ectomycorrhizae. Since Monotropoideae were rarely studied in continental Asia, the root systems of the four species sampled in Yunnan were examined using morphological and molecular methods. All the roots of these four species exhibit a typical monotropoid mycorrhizal morphology, including a fungal mantle, a Hartig net and hyphal pegs. In M. uniflora and M. humile mycorrhizae, cystidia typical of Russula symbionts covered the fungal mantle. ITS barcoding revealed that Russulales were the most frequent colonizers in all species, but Hypopitys monotropa displayed various additional mycorrhizal taxa. Moreover, a few additional ectomycorrhizal and saprotrophic Basidiomycota taxa were identified in the three other species, challenging that these four Monotropoideae species are as strictly fungal specific as the other Monotropoideae species hitherto studied. Moreover, a comparison with accompanying fungus sporocarps revealed that the fruiting fungal community significantly differed from that associated with the Monotropoideae roots, so that a clear fungal preference was evident. Finally, four fungal species were found on more than one Monotropoideae species: this contrasted with previous reports of sympatrically growing mycoheterotrophic plants, which did not reveal any overlap. This again challenges the idea of strict fungal specificity.

The perennial evergreen herb, Chimaphila japonica is found exclusively in East Asian temperate coniferous or sometimes in deciduous forests. By using the Fast Isolation by Amplified Fragment Length Polymorphism (AFLP) of Sequences Containing repeats (FIASCO) protocol, 20 microsatellite primer sets were identified in two wild populations. Of these primers, 16 displayed polymorphisms and 4 were monomorphic. The number of alleles per locus ranged from one to six among populations, values for expected and observed heterozygosities ranged from 0.000 to 0.848 and from 0.000 to 1.000, respectively. The new SSR markers will be useful in obtaining estimates of population-level genetic diversity and in phylogeographic studies of C. japonica.

The relations between Monotropa hypopitys and four members of Pyrolaceae collected in Bulgaria were investigated using five ISSR markers. The genetic differences given by each used primer were illustrated graphically. The selective ability of each primer was discussed according to the obtained results. The phylogenetic relationships were illustrated using a resulting dendrogram constructed with the data from all polymorphic variants. Monotropa was separated in a single clade only by one marker and displays highest genetic similarity with Orthilia secunda. Our results suggested that Monotropoideae may belong to Pyrolaceae, together with Pyroloideae. Both groups represent two mycoheterotropic lines dependent on the nutrition, which determines their distribution. Monotropa is the terminal result of the gradual passage from authotrophy to mycorrhizal parasitism. In spite of the fact that Monotropaceae is known as a paraphyletic family, it showed a close similarity to Orthilia. The data supported the p...

Pterospora andromedea, a mycoheterotroph, has been shown to form obligate symbioses with only three species of Rhizopogon in section Amylopogon: R. salebrosus, R. arctostaphyli and an undescribed molecular taxon. Sarcodes sanguinea, another my coheterotroph in Ericaceae, and sister taxon to Pterospora andromedea, has been found to form symbioses with two species of Rhizopogon in section Amylopogon: R. ellenae and R. subpurpurascens. To date no overlap has been recorded between these two achlorophyllous plants and their associated mycobionts. Tissue from Pterospora andromedea rootballs and Rhizopogon spp. basidiocarps were collected from the Greater Yellowstone Ecosystem. The mycobionts were identified using sequence analysis of the ITS locus and compared with sequences of Rhizopogon spp. section Amylopogon from GenBank. Sequences of two additional loci, ATP6 and RPB2 were also generated and analyzed. In addition to Rhizopogon salebrosus, Pterospora andromedea was found for the first time in association with a fourth mycobiont, Rhizopogon ellenae, a known associate of Sarcodes sanguinea. The discovery of a new symbiont may provide evidence for an undiscovered lineage of Pterospora andromedea inhabiting the Greater Yellowstone Ecosystem. In addition, overlap in obligate mycobionts between closely related mycoheterotrophs provides interesting new information on the phylogenetic history and coevolution of the mycoheterotrophs in the Monotropoideae (Ericaceae).

Dust seeds are the smallest existing seeds in angiosperms. This paper summarizes taxonomic distribution, phylogeny, ontogeny, morphology, and recruitment behavior of dust seeds, concluding with a g ...

We explored the diversity of mycorrhizal fungi associated with Monotropastrum humile in the central part of Japan's main island. We collected 103 M. humile individuals from 12 sites with various forest types. We analyzed the DNA sequences of the internal transcribed spacer region from fungal and plant nuclear ribosomal DNAs to assess the genetic diversity of the fungi associated with M. humile roots and to position the plant with respect to known Monotropoideae groups, respectively. The plants formed a monophyletic clade with other members of M. humile but were separated from M. humile var. glaberrimum and other monotropes (97% bootstrap support). Of the 50 fungal phylotypes, 49 had best matches with the Russulales, and the other had highest similarity with the Thelephoraceae. Our phylogenetic analysis suggests that M. humile roots have a highly specialized association with fungal partners in the Russulaceae. Moreover, a few fungal phylotypes from the M. humile roots had positions neighboring those from Monotropa uniflora roots. These results indicated that the genetic diversity of mycorrhizal fungi of M. humile was highly specific to the Russulaceae, but with high diversity within that family, and that the fungi associated with M. humile differ from those associated with M. uniflora.

All members of the Monotropoideae (Ericaceae), including the species, Allotropa virgata and Pleuricospora fimbriolata, are mycoheterotrophs dependent on associated symbiotic fungi and autotrophic plants for their carbon needs. Although the fungal symbionts have been identified for A. virgata and P. fimbriolata, structural details of the fungal-root interactions are lacking. The objective of this study was, therefore, to determine the structural features of these plant root-fungus associations. Root systems of these two species did not develop dense clusters of mycorrhizal roots typical of some monotropoid species, but rather, the underground system was composed of elongated rhizomes with first- and second-order mycorrhizal adventitious roots. Both species developed mantle features typical of monotropoid mycorrhizas, although for A. virgata, mantle development was intermittent along the length of each root. Hartig net hyphae were restricted to the host epidermal cell layer, and fungal pegs formed either along the tangential walls (P. fimbriolata) or radial walls (A. virgata) of epidermal cells. Plant-derived wall ingrowths were associated with each fungal peg, and these resembled transfer cells found in other systems. Although the diffuse nature of the roots of these two plants differs from some members in the Monotropoideae, the structural features place them along with other members of the Monotropoideae in the "monotropoid" category of mycorrhizas.

Genera in the tribe Pyroleae (subfamily Monotropoideae, family Ericaceae) occur as understory plants in northern temperate zones where some form major components of ecosystems. Most have been poorly studied in terms of their association with symbiotic fungi. In this study, colonization patterns of mycorrhizal roots of five members of the Pyroleae ( Pyrola asarifolia Michx., Pyrola chlorantha Sw., Orthilia secunda (L.) House, Chimaphila umbellata (L.) W. Bart., Moneses uniflora (L.) Gray) were explored. Root samples were processed for light, fluorescence, and laser scanning confocal, scanning electron, and transmission electron microscopy, as well as for immunocytochemistry. Roots of all species had enlarged epidermal cells containing hyphal complexes, Hartig nets confined to the epidermis, and mantles. Epidermal cells were penetrated by hyphae originating from the Hartig net at more than one site either along the inner tangential wall or radial walls. The outer tangential wall of epidermal cells of all species, except M. uniflora, was thicker than radial and inner tangential walls and consisted of two layers, the outer containing nonesterified pectins that were labeled with JIM 5 antibodies. Radial walls and inner tangential walls did not label, but cortical cell walls did. Intracellular hyphal complexes developed initially around centrally positioned, enlarged epidermal cell nuclei and, through branching, occupied most of the cell volume. Senescence and degradation of the complexes followed. The fungal species in these symbiotic associations may be important functionally in nutrient exchange, as well as in contributing to broader linkages with other hosts in these plant communities.

Taxonomic treatment of the achlorophyllous monotropoid plant Monotropastrum humile is still unclear and confusing because of the lack of detailed morphological analyses and molecular phylogeny. In particular, the taxonomic status of a glabrous variety, M. humile var. glaberrimum, is under debate. Our detailed examination of the morphological characteristics of living plants revealed that M. humile var. glaberrimum can be easily distinguished from the putative conspecific taxon M. humile var. humile by characteristics not previously recognized, namely the shape and color of the floral disc. Most morphological features characterizing Cheilotheca were also found in M. humile var. glaberrimum. Moreover, there was considerable nucleotide differentiation in the internal transcribed spacer (ITS)2 sequences of M. humile var. humile and var. glaberrimum. Molecular analysis of the phylogenetic relationship of M. humile var. humile, var. glaberrimum, and other monotropoids using ITS2 sequences showed that two varieties of M. humile formed a monophyletic clade with a member of a different genus, Monotropa L., but obvious phylogenetic relationships among these three taxa were not obtained. Thus we conclude that Monotropastrum humile var. glaberrimum should be treated as a distinct species. However, the generic affiliation of M. humile var. glaberrimum could not be determined because of its intermediate character state combination and the insufficient characterization of related species. We strongly suggest that Monotropastrum as a whole needs re-evaluation.

Achlorophyllous monotropoid plants (Monotropoideae, Ericaceae) are epiparasites that obtain all of their carbon from their host plants via connections with mycorrhizal fungi. The mycorrhizal fungi of the epiparasitic monotropoid Monotropastrum humile var. glaberrima were identified based on mitochondrial, large ribosomal DNA sequences, and were compared with those of another variety, M. humile var. humile. The fungi that inhabit M. humile var. glaberrimum belong to the Thelephoraceae, whereas that of M. humile var. humile is a member of the Russulaceae. Two explanations are possible for this phenomenon: a misunderstanding of the taxonomic position of M. humile var. glaberrimum, or a change in the fungal partner within the Monotropastrum.

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular "fungal pegs" within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.

Summary • Over 400 species of achlorophyllous vascular plants are thought to obtain all C from symbiotic fungi. Consequently, they are termed ‘myco-heterotrophic.’ However, direct evidence of myco-heterotrophy in these plants is limited. • During an investigation of the patterns of N and C stable isotopes of various ecosystem pools in two old-growth conifer forests, we sampled six species of mycoheterotrophic achlorophyllous plants to determine the ability of stable isotope ratios to provide evidence of myco-heterotrophy and host-specificity within these symbioses. • Dual-isotope signatures of the myco-heterotrophic plants differed from those of all other pools. They were most similar to the signatures of ectomycorrhizal fungi, and least like those of green plants. δ 15 N values of the myco-heterotrophic plants correlated strongly and positively with those of putative mycobionts. • Used in conjunction with other techniques, N and C stable isotope ratios can be used to demonstrate myco-heterotrophy and host-specificity in these plants when other ecosystem pools are well characterized. They also appear promising for estimating the degree of heterotrophy in photosynthetic, partially myco-heterotrophic plants.

A new classification of Ericaceae is presented based on phylogenetic analyses of nuclear and chloroplast DNA sequence data, morphology, anatomy, and embryology. Eight subfamilies and 20 tribes are recognized. In this classification Epacridaceae are included as Styphelioideae and Empetraceae as tribe Empetreae within the Ericoideae. The herbaceous taxa previously recognized as Pyrolaceae and Monotropaceae by some authors are also included within Ericaceae, in the subfamily Monotropoideae. A key, morphological descriptions, and representative images are provided for all named groups. Two new combinations inKalmia (K. buxifolia andK. procumbens) are made, and three new taxa are described: Oligarrheneae, Richeeae, and Cosmelieae (all within Styphelioideae).

The Monotropoideae (Ericaceae) are non-photosynthetic angiosperms that obtain fixed carbon from basidiomycete ectomycorrhizal fungi. In previous work, we showed that each plant species is associated with a single genus or a set of closely related genera of ectomycorrhizal fungi. Here we show that the level of specificity is much higher. We used a molecular phylogenetic approach to contrast specificity patterns among eight plant lineages and three fungal genera. We relied on fungal nuclear internal transcribed spacer (nrITS) sequence data obtained from 161 basidiocarps and 85 monotropoid roots representing 286 sampled plants screened using restriction length polymorphisms. From the phylogenetic placement of fungal symbionts in fungal phylograms, we found that three basal (Sarcodes, Pterospora, Pleuricospora) and one derived lineage (Allotropa) of plants target narrow clades of closely related species groups of fungi, and four derived lineages (Monotropa hypopithys species group, Pityopus) target more distant species groups. Within most plant lineages, geography and photobiont association constrain specificity. Specificity extended further in Pterospora andromedea, in which sequence haplotypes at the plastid trn L-F region of 73 plants were significantly associated with different fungal species groups even in sympatry. These results indicate that both the macro- and microevolution of the Monotropoideae are tightly coupled to their mycorrhizal symbionts.

Monotropastrum sciaphilum (Ericaceae), a mycoheterotrophic member of subfamily Monotropoideae, was rediscovered at its type locality in Yunnan Province, China, 91 years after it was first collected. The type locality is the only locality from which it is known. Field observations in 2007–2010 indicated that inflorescences emerge from the soil between late April and early September. The restricted distribution of M. sciaphilum recommends its listing in the IUCN red book.

• Premise of the study: Pterospora andromedea (Ericaceae) is a mycoheterotrophic plant endemic to North America with a disjunct distribution. Eastern populations are in decline compared to western populations. Microsatellite loci will allow comparison of genetic diversity in endangered to nonthreatened populations. • Methods and Results: Illumina MiSeq sequencing resulted in development of 12 polymorphic microsatellite loci from 63 perfect microsatellite loci tested. One polymorphic locus was obtained from a traditional enrichment method. These 13 loci were screened across two western and two eastern populations. For western and eastern populations, respectively, number of alleles ranged from one to 10 and one to four, and observed heterozygosity ranged from 0.000 to 0.389 and 0.000 to 0.143. • Conclusions: These are the first microsatellite loci developed for Pterospora. They will be useful in conservation efforts of the eastern populations and for examination of population genetic parameters at different geographic scales and comparison with mycorrhizal fungal hosts.

Premise of the study: Pterospora andromedea (Ericaceae) is a mycoheterotrophic plant endemic to North America with a disjunct distribution. Eastern populations are in decline compared to western populations. Microsatellite loci will allow comparison of genetic diversity in endangered to nonthreatened populations. Methods and Results: Illumina MiSeq sequencing resulted in development of 12 polymorphic microsatellite loci from 63 perfect microsatellite loci tested. One polymorphic locus was obtained from a traditional enrichment method. These 13 loci were screened across two western and two eastern populations. For western and eastern populations, respectively, number of alleles ranged from one to 10 and one to four, and observed heterozygosity ranged from 0.000 to 0.389 and 0.000 to 0.143. Conclusions: These are the first microsatellite loci developed for Pterospora. They will be useful in conservation efforts of the eastern populations and for examination of population genetic parameters at different geographic scales and comparison with mycorrhizal fungal hosts.

We have sampled the mycorrhizal roots of 76 snow plants (Sarcodes sanguinea, Monotropoideae, Ericaceae) in two areas of the Sierra Nevada of California that are ;180 km apart. To identify the fungal symbionts associated with these plants, we first analyzed restriction fragment length polymorphisms (RFLPs) of the internal transcribed spacer region (ITS) of the fungal nuclear ribosomal repeat. Fungal ITS-RFLPs were successfully produced from 57 of the 76 plants sampled, and all symbionts shared the same DNA fragment pattern. The morphology of S. sanguinea mycorrhizae was consistent with that expected from a Rhizopogon species in section Amylopogon. To confirm and refine this identification, a total of six fungal ITS sequences were determined fromS. sanguinea mycorrhizae. These sequences were analyzed together with eight existing and eight newly determined ITS sequences from Rhizopogon section Amylopogon. The newly determined sequences include an ITS sequence from the fungal symbiont of pine drops (Pterospora andromedea, Monotropoideae, Ericaceae), a plant that was previously reported to be exclusively associated with the Rhizopogon subcaerulescens group. When these sequences were analyzed together, the Sarcodes symbionts grouped tightly with several collections of R. ellenae including the holotype, one collection of R. idahoensis, and one collection of R. semireticulatus. A different lineage comprised collections of R. subgelatinosus, R. subcaerulescens,another collection of R. semireticulatus, and the Pterospora symbiont. We conclude that S. sanguinea associates exclusively with a single species in the R. ellenae species complex throughout our sampling range. These results indicate a much higher level of specificity in S. sanguinea than was previously reported and confirm the emerging pattern that nonphotosynthetic, monotropoid plants generally associate very specifically with a narrow range of ectomycorrhizal fungi.

summary Sarcodes sanguinea and Pterospora andromedea (Ericaceae, Monotropoideae) are nonphotosynthetic mycoheterotrophic plants. Recent studies have shown that the roots of the adult plants are always associated with closely related but exclusive sets of Rhizopogon species (Basidiomycota, Boletales) from section Amylopogon. We have isolated Rhizopogon species that were associated with the adult plants and used them to germinate seeds under gnotobiotic conditions. All Rhizopogon species isolated from either plant species were capable of stimulating seed germination in both Sarcodes and Pterospora. Under the primary conditions used, germination varied from 9 to 73% in the case of Sarcodes and 0 to 13% in that of Pterospora. The single Rhizopogon strain that failed to elicit germination in Pterospora under these conditions did stimulate germination under slightly dierent conditions. By contrast, seeds failed to germinate on all media which lacked these Rhizopogon species, or in the presence of six other genera of basidiomycetes. Seed germination could be stimulated either through cellophane or at the edge of fungal colonies without direct fungus‐seed contact. These results suggest that a diusible or volatile compound that is unique to Rhizopogon stimulates germination of these plant seeds. Seed lots of Sarcodes from two successive years had similar germination levels. Sarcodes seeds that had overwintered under natural conditions were also stimulated to germinate. These results demonstrate the potential for long-term dormancy. We suggest that a combination of dormancy and the use of specific germination cues might increase the opportunities of these plants for recruitment. In addition, the specific germination response explains at least a part of the specialized associations observed in the adult plants. Nevertheless, the seeds respond to a slightly broader range of Rhizopogon species than has been observed to be associated with the adult plants; thus other factors must also be involved with specificity under natural conditions.

MONOTROPES (Monotropoideae, Ericaceae) are achlorophyllous, epiparasitic plants that receive all of their fixed carbon from green plants through a common ectomycorrhizal association rather than by a direct parasitic connection (Fig. 1)1,2. Using molecular identification methods we show that some monotropes are highly specific in their fungal associations and at least one species, Pterosporo andromedea is specialized on a single species group within the genus Rhizopogon. Phylogenetic analysis of the Monotropoideae shows that specialization has been derived through narrowing of fungal associations within the lineage containing P. andromedea. High specificity is contrary to past predictions for the Monotropoideae and for plant communities in general, raising many questions about the roles of mycorrhizal specificity in ecosystem function.

The Pyrola picta species complex (Pyroleae: Monotropoideae: Ericaceae) is thought to be composed of three morphological taxa within a single species, Pyrola picta. All taxa typically inhabit mature coniferous or Fagaceous forests of North America west of the Rocky Mountains from British Columbia to Baja California, Mexico between ca. 250 and 3,000 m in elevation. Taxa within P. picta are distinguished from each other on the basis of leaf morphology and the degree to which they employ mycoheterotrophy rather than photosynthesis, but considerable variation has been documented in both of these features, confounding diagnosis. For this study Pyrola picta, P. picta f. aphylla, and P. picta ssp. dentata were collected from populations throughout their range in western North America and examined for genetic differences to determine whether they indeed constitute a single polymorphic species or, alternatively, multiple distinct species. Multiple individuals per population were described morphologically a...

Mycotrophism occurs in two ericaceous subfamilies, the Monotropoideae and the Pyroloideae. However, three aspects of the evolution of these subfamilies remain equivocal; 1) morphological, biochemical, and molecular analyses have failed to establish that the Monotropoideae is monophylectic; 2) the relationships among members of the Monotropoideae remain unresolved; 3) it is unclear whether the Pyroloideae should be included as a subfamily within the Ericaceae or treated as a separate family. To address these topics a phylogeny was constructed using a portion of the 28s ribosomal RNA gene from 22 members of the Ericaceae. Results indicate that the present Monotropoideae is polyphyletic; one species, Monotropsis ordata, is more closely related to members of the Vaccinioideae than to the rest of the Monotropoideae. This arrangement is supported by consideration of mycorrhizal type, consequently multiple origins of mycotrophic parasitism must be assumed. The relationships depicted within the Monotropoideae are nearly congruent with those depicted in Copeland’s (1941) phylogeny, and morphological or biochemical characters are identified which support the relationships depicted by the molecular tree which do not agree with the morphological tree. In addition, the data indicate, although not strongly, that the Pyroloideae should be included within the Ericaceae.

Studies of mycoheterotrophs, defined as plants that obtain carbon resources from associated mycorrhizal fungi, have fundamentally contributed to our understanding of the importance and complexity of symbiotic ecological interactions. However, to date, the reproductive ecology of these organisms remains empirically understudied, with existing literature presenting hypotheses about traits including a generalist pollination syndrome and autogamous self-pollination. To address this gap in our knowledge of the reproductive ecology of mycoheterotrophic plants, we comparatively analyzed three species of two monotropoid genera, Monotropa and Monotropsis. During three consecutive years of field observations and manipulations of four populations of Monotropa uniflora, seven of M. hypopitys (both red and yellow color forms), and two of Monotropsis odorata, we investigated flowering phenology, pollination ecology, breeding system, floral herbivory, and reproductive effort and output. Contrary to previous predictions, our results revealed that taxa are largely outcross-pollinated and specialized toward Bombus pollinators. Additionally, species differ in breeding system, timing and duration of reproductive development, fluctuations in reproductive effort and output, and fitness impacts of herbivory. This study is the first thorough investigation of the reproductive ecology of mycoheterotrophic species and provides insight into possible limitations in reproductive traits imposed by a mycoheterotrophic life history.

The myco-heterotroph Monotropa hypopitys is a perennial, circumboreally distributed herb of significant importance in studies of nonphotosynthetic plant biology. To address a deficiency in our knowledge of myco-heterotroph population genetics, 11 microsatellite markers were developed using a cost-effective, nonradioactive protocol. Multiplex reactions revealed polymorphism in the red and yellow colour forms of M. hypopitys with an average of 2.69 alleles per primer. Many primers additionally amplified in the congener Monotropa uniflora and five other closely related genera. This is the first report of microsatellite primer development and amplification in the Monotropoideae (Ericaceae).

Despite more than 100 years of detailed analysis of morphology and macromolecules, the phylogenetic origin of the Monotropoideae remains unclear. In this study partial sequences from the 28S rRNA gene were used to test two alternative hypotheses: (i) that the Monotropoideae share a most recent common ancestor with the Arbutoideae in the Ericaceae or (ii) that the Monotropoideae is the end result of a gradual transition from autotrophism to mycotrophic parasitism in the Pyrolaceae. Our results support the hypothesis that the Monotropoideae and the Arbutoideae are a monophyletic group and that the Monotropoideae should be included in the Ericaceae rather than the Pyrolaceae. The Monotropoid species Pleuricospora fimbriolata could not be placed within either the Monotropoideae or the Arbutoideae with any degree of confidence, leaving open the possibility that the Monotropoideae is polyphyletic. Key words: Monotropoideae, Ericaceae, 28S rRNA gene, PCR, molecular systematics.