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.

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.

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 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.

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.

Previous reports of sequences of Sebacinales (basal Hymenomycetes) from ericoid mycorrhizas raised the question as to whether Sebacinales are common mycorrhizal associates of Ericaceae, which are usually considered to associate with ascomycetes. Here, we sampled 239 mycorrhizas from 36 ericoid mycorrhizal species across the world (Vaccinioideae and Ericoideae) and 361 mycorrhizas from four species of basal Ericaceae lineages (Arbutoideae and Monotropoideae) that do not form ericoid mycorrhizas, but ectendomycorrhizas. Sebacinales were detected using sebacinoidspecific primers for nuclear 28S ribosomal DNA, and some samples were investigated by transmission electron microscopy (TEM). Diverging Sebacinales sequences were recovered from 76 ericoid mycorrhizas, all belonging to Sebacinales clade B. Indeed, some intracellular hyphal coils had ultrastructural TEM features expected for Sebacinales, and occurred in living cells. Sebacinales belonging to clade A were found on 13 investigated roots of the basal Ericaceae, and TEM revealed typical ectendomycorrhizal structures. Basal Ericaceae lineages thus form ectendomycorrhizas with clade A Sebacinales, a clade that also harbours ectomycorrhizal fungi. This further supports the proposition that Ericaceae ectendomycorrhizas involve ectomycorrhizal fungal taxa. When ericoid mycorrhizas evolved secondarily in Ericaceae, a shift of mycobionts occurred to ascomycetes and clade B Sebacinales, hitherto not described as ericoid mycorrhizal fungi. Fil: Selosse, Mark André. Centre d’Ecologie Fonctionnelle et Evolutive ; Francia Fil: Setaro, Sabrina. Universität Tübingen. Botanisches Institut; Alemania Fil: Glatard, Florent. Centre d’Ecologie Fonctionnelle et Evolutive; Francia Fil: Richard, Franck. Centre d’Ecologie Fonctionnelle et Evolutive; Francia Fil: Urcelay, Roberto Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Weiss, Michael. Universität Tübingen. Botanisches Institut; Alemania

Pityopus californicus (Eastw.) H. F. Copel., a monotypic member of the Monotropoideae in the family Ericaceae, is a myco-heterotrophic species with distribution limited to the Pacific Northwest of the USA. Young embryos of P. californicus developed mycorrhizal associations in seed packets that had been buried for up to 681 days, suggesting that seeds of P. californicus may require the presence of a fungus to achieve germination. Samples of nongerminated seeds and early stages in embryo and root development were subsequently processed for light microscopy, histochemistry, and transmission electron microscopy (TEM). Nongerminated seeds possessed a thick testa, lacked a shoot and root meristem, and consisted of an embryo with large parenchymatous cells containing protein bodies and starch grains as storage reserves. In the earliest developmental stage (seed coat still attached), fungal hyphae were present on the testa surface and between the testa and embryo. This stage was followed by embryo elongation, the organization of a root apical meristem, and the development of a well-developed fungal mantle surrounding the elongated embryo. At least two morphotypes were identified based on structural characteristics of the mantle. One of these, with ascomycetous septa, had Cenococcum-like features. Late-stage embryo/early root development revealed a typical mantle and Hartig net, with fungal pegs penetrating the outer tangential walls of epidermal cells. Transfer cell-like deposits of wall material, similar to those described in Monotropa spp., enclosed fungal pegs. The development of a Hartig net and fungal pegs suggests that nutrient exchange interfaces are required for seedling development.

Plant species in the subfamily Monotropoideae are mycoheterotrophs; they obtain fixed carbon from photosynthetic plants via a shared mycorrhizal network. Previous findings show mycoheterotrophic plants exhibit a high level of specificity to their mycorrhizal fungi. In this study we explore the association of mycorrhizal fungi and Monotropa uniflora (Monotropoideae: Ericaceae) in eastern North America. We collected M. uniflora roots and nearby basidiomycete sporocarps from four sites within a 100 km2 area in eastern Massachusetts. We analyzed DNA sequences of the internal transcribed spacer region (ITS) from the fungal nuclear ribosomal gene to assess the genetic diversity of fungi associating with M. uniflora roots. In this analysis we included 20 ITS sequences from Russula sporocarps collected nearby, 44 sequences of Russula or Lactarius species from GenBank and 12 GenBank sequences of fungi isolated from M. uniflora roots in previous studies. We found that all 56 sampled M. uniflora mycorrhizal fungi were members of the Russulaceae, confirming previous research. The analysis showed that most of the diversity of mycorrhizal fungi spreads across the genus Russula. ITS sequences of the mycorrhizal fungi consisted of 20 different phylotypes: 18 of the genus Russula and two of Lactarius, based on GenBank searches. Of the sampled plants, 57% associated with only three of the 20 mycorrhizal fungi detected in roots, and of the 25 sporocarp phylotypes collected three, were associated with M. uniflora. Furthermore the results indicate that the number of different fungal phylotypes associating with M. uniflora of eastern North America is higher than that of western North America but patterns of fungal species abundance might be similar between mycorrhizae from the two locations.

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.

Fungal-induced seed germination is a phenomenon characteristic of mycorrhizal plants that produce dust-like seeds with only minimal nutritional reserves. In such systems, fungi trigger germination and/or subsidize development. We studied mycorrhizal germination in relation to mycorrhizal specificity in the Monotropoideae, a lineage of dust-seeded non-photosynthetic plants that are dependent upon ectomycorrhizal fungi of forest trees. A total of 1695 seed packets, each containing two to five compartments with seeds from different sources, were buried for up to 2 years near known ectomycorrhizal fungi in six different native forest locations. Upon harvest, seedlings were analysed by cultivation-independent molecular methods to identify their mycorrhizal fungi. We report that (i) germination is only induced by the same fungus that associates with mature plants or by closely related congeners; (ii) seedlings associated with the latter fungi develop less than those associated with maternal fungal species in most settings; and (iii) exceptions to this pattern occur in allopatric settings, where novel plant-fungal associations can result in the greatest seedling development. We interpret these results as evidence of performance trade-offs between breadth of host range and rate of development. We propose that in conjunction with host-derived germination cues, performance trade-offs can explain the extreme mycorrhizal specificity observed at maturity. The allopatric exceptions support the idea that performance trade-offs may be based on a coevolutionary arms race and that host range can be broadened most readily when naive fungal hosts are encountered in novel settings.

Plant species in the subfamily Monotropoideae are achlorophyllous and have developed a complex mode of nutrition, receiving photosynthates from neighboring trees via shared fungi. To explore the mycorrhizal associations of Monotropa uniflora in central British Columbia (B.C.), plants were sampled from three sites: a Betula-dominated site and two sites with a mixture of conifer and hardwood trees. Fifteen M. uniflora root-clusters were sampled (five per site) and the mycorrhizal diversity was assessed using morphological and molecular (PCR-RFLP analysis and DNA sequencing) methods. Both methods showed that root-clusters (often comprising several hundred mycorrhizal tips) belonging to the same plant appeared to involve fungus monocultures in the family Russulaceae. All mycorrhizae exhibited typical Russula morphology and had mantle cystidia. Two root-clusters, one each from sites 1 and 3, lacked one of the two types of cystidia present on all other root-clusters. PCR-RFLP analysis resulted in three fragment patterns for the 15 root clusters. One molecular fragment pattern included the two root-clusters displaying the single cystidium type plus an additional root-cluster with both cystidia types. DNA sequencing of a portion of the ITS2 region of the ribosomal DNA suggests that the three variants represent different species; two of the variants clustered with the hypogeous fungi Martellia and Gymnomyces. The study provides increased evidence of low diversity and high specificity in the Monotropa-fungus relationship and suggests that M. uniflora associates uniquely with fungi in the family Russulaceae in central B.C.

The Monotropoideae (Ericaceae) are nonphotosynthetic plants that obtain fixed carbon from their fungal mycorrhizal associates. To infer the evolutionary history of this symbiosis we identified both the plant and fungal lineages involved using a molecular phylogenetic approach to screen 331 plants, representing 10 of the 12 described species. For five species no prior molecular data were available; for three species we confirmed prior studies which used limited samples; for five species all previous reports are in conflict with our results, which are supported by sequence analysis of multiple samples and are consistent with the phylogenetic patterns of host plants. The phylogenetic patterns observed indicate that: (i) each of the 13 plant phylogenetic lineages identified is specialized to a different genus or species group within five families of ectomycorrhizal Basidiomycetes; (ii) mycorrhizal specificity is correlated with phylogeny; (iii) in sympatry, there is no overlap in mature plant fungal symbionts even if the fungi and the plants are closely related; and (iv) there are geographical patterns to specificity.

A survey of the nonphotosynthetic plant Monotropastrum humile was conducted to determine its mycorrhizal status and characterize the fungal structures observed. Thirteen populations and 40 individuals were collected from six forest types, including coniferous and broadleaf trees, in central Japan. The nearly spherical root system of M. humile intertwines with the root systems of neighboring trees, and individual roots were branched up to third-order structure, forming monopodial-pinnate or monopodial-pyramidal morphologies. In addition to the formation of a fungal mantle and Hartig net in association with the epidermis, fungal penetration pegs consistently were observed around and within the epidermal cells. These structures indicate that the mycorrhizal status of M. humile is of the monotropoid type.

A palynological survey, including LM, SEM and TEM is presented for eight genera, nine species and 76 samples of the Monotropoideae which is composed of 10 genera and 13 species. On the basis of the aperture number and shape, the following six pollen types are recognized: 1) 3-colp(oroid) ate—Allotropa, 2) 2-colpor(oid)ate—Monotropsis andHemitomes, 3) 4-colpor(oid)ate—Pterospora, Sarcodes andPleuricospora, 4) 2- and 3-colpor(oid)ate—Monotropa hypopitys, 5) 3-colporate and-porate —Monotropa uniflora, 6) 3- to 4- porate—Monotropastrum humile. Relationships among taxa within the Monotropoideae are illustrated on the palynological characters including the aperture type, exine sculpture and structure. Both pollen grains with two and four apertures have evolved independently from pollen grains with three apertures, according to the infraspecific variation of aperture numbers and the usual occurrence of three-aperturate pollen grains in the Ericaceae. 3-colp(oroid)ate pollen ofAllotropa is the most primitive and occupies an isolated position in the subfamily, on the other hand 3- to 4-porate pollen ofMonotropastrum humile is most advanced in the subfamily, probably even in the Ericaceae. A reduced sexine inMonotropastrum humile appears to be a specialized pollen character. Infraspecific geographical difference in palynological characters is revealed in two species ofMonotropa for the first time.M. hypopitys is basically characterized by pollen grains with two apertures in the New World vs. three apertures in the Old World.M. uniflora is basically characterized by 3-porate pollen in the New World vs. 3-colporate in the Old World. In both species pollen grains with more primitive characters usually occur in the Old World.

SUMMARY The ultrastructural differentiation of mycorrhizas of the achlorophyllous plant Monotropa hypopitys was examined in relation to the stages of growth of the shoot. Plants were collected at regular intervals during the growing season and their roots were immediately excised and prepared for scanning and transmission electron microscopy. In addition to the presence of a fungal sheath and Hartig net of similar structure to those seen in ectomycorrhizas, the outer cortical cells frequently contain characteristic fungal intrusions. While not breaching the host cell wall, these intrusions stimulate its invagination. The resulting structures, analogous to the transfer cells found in regions of intensive solute mobilization and transport, are produced in greatest numbers during the period of most rapid shoot extension. The relationship between their structure and possible function is discussed. The structure of mycorrhizas in Monotropa hypopitys is sufficiently distinct from that of arbutoid mycorrhizas, to warrant the creation of a new category which we term ‘monotropoid’.

(1985). Mycorrhizal Associations of Five Species of Monotropoideae in Oregon. Mycologia: Vol. 77, No. 3, pp. 499-502.

The morphological and vascular characteristics of the nectaries of the species of the Monotropoideae (Ericaceae) reflect the morphology and declination of the flowers. These, in turn, are related to the pollination systems of the species. The nectaries of members of the Monotropoideae exhibit a range in form from slender, elongate projections, to short, stout ones, or to low ridges between the staminal bases. ANATOMICAL FEATURES may be expected to reflect particular aspects of the functional morphology of a flower. The necessity of functional interpretations of features of floral morphology has been pointed out by Carlquist (1969). Previous studies of floral nectaries have included few, if any, correlations with functional morphology. Knuth (1906-1909) discussed the floral nectaries of several plant groups in relation to their pollination mechanisms. Elias, Rozich, and Newcombe (1975) have related the functional relationships of the foliar and floral nectaries of Turnera ulmifolia (Turneraceae). Floral nectaries have been classified according to their location in the flower, (Fahn, 1953; Bonnier, 1879), their mode of secretion, (Behrens, 1879), and their vasculature, (Frei, 1955). Brown (1938) noted that nectaries seem to have arisen independently in several plant groups and have undergone modifications in those groups. Brown also pointed out that some families, like the Ericaceae, had various types of nectaries while others were fairly uniform in the type of nectary they possessed. Brown's illustrations of the nectaries depict sectional views of the flowers, but his descriptions do not relate to the pollination systems of the species under consideration. Correlations among production of nectar, stigmatic secretions, and the development of reproductive organs have been reported (Shuel, 1961). Puri (1951) has reviewed the literature concerning the many definitions and descriptions of nectaries. For the purposes of this study the term nectary denotes the floral nectary since no extrafloral nectaries have been found among members of the Monotropoideae. These nectaries are those structures or areas of the flowers involved in the production and secretion of nectar. 1Received for publication 3 May 1976. Research supported, in part, by National Science Foundation Grant GB 31648. Thanks are extended to Dr. R. Snelling, L.A. County Museum of Natural History for his identifications of insects. The loan of photographic equipment from Dr. S. Carlquist and his reading of the manuscript are appreciated. Since the nectar-producing areas of these species are not contiguous, each separate site of localized nectar secretion will be considered a nectary. Size and spatial arrangement of the nectaries is intimately related to the morphology of the corolla and reproductive organs of the flowers, so descriptions of these floral features will be included where pertinent. Nectar is almost exclusively a phloem exudate according to Frey-Wyssling (1955). He noted the preponderance of phloem in the vascular traces which supply the nectaries. Esau (1969) mentions a close positive relationship between the sugar concentration of a nectary and the amount of phloem present in the vascular supply to the nectary. The Ericaceae is a large, widely distributed family of angiosperms. It is composed of five subfamilies (Ericoideae, Monotropoideae, Pyroloideae, Rhododendroideae, and Vaccinioideae) whose interrelationships have been discussed elsewhere (Wallace, 1975a). The taxonomy and distribution of the members of the Montropoideae have also been covered (Wallace, 1 975b). Twelve achlorophyllous, mycoparasitic species comprise the Monotropoideae. Annual floral axes, the only aboveground portions of these species, arise from the perennial root masses. Populations of these species are never very dense; usually only a few floral axes are found in a given area. The axes are often inconspicuous, in spite of the fact that they may be brightly colored. The floral axes range in height from 2 cm, though usually taller, (e.g., Hemitomes, Pleuricospora, Pityopus), to 50 cm (e.g., Allotropa, Sarcodes) , to the tallest species, Pterospora, whose floral axes may be as high as 1.5 m. The number of flowers per inflorescence ranges from one (e.g., Monotropa uniflora, Monotropastrum humile, and irregularly in other species), to several (e.g., Hemitomes, Pityopus, usually), to many (e.g., Sarcodes, Pterospora). Members of the Ericaceae are usually considered to possess nectaries, although Henderson (1919) did not find nectaries in Pterospora

A microscopic survey of the achlorophylus plant Monotropa uniflora L. reveals an ectendotrophic mycorrhizal fungus with dolipore septa associated with the roots. Special transfer regions are found in the root cell wall ensheathing all but the ruptured tip of the fungal intrusion. MONO)TROPA HAS long fascinated biologists because its waxy white appearance indicates that it is unusual among flowering plants. Its absence of chlorophyll (less than 0.001 mg per gram fresh weight) and therefore its inability to produce metabolic materials by the photosynthetic fixation of CO2 has led many experimenters to investigate the mysteries of its rather novel existence. The historical details as well as the descriptive and physiological literature has been admirably re

Leaves of 344 species of the Ericaceae and of 37 species in three related families have been surveyed for flavonoids and simple phenols. The yellow flavonol gossypetin was found in 158 ericaceous species and is of chemotaxonomic interest within the Rhododendroideae (where it occurs in 11 of 19 genera) and in the Vaccinioideae (in 3 of 22 genera). 5-0-methylflavonols appear in 81 species, again mainly in the Rhododendroideae (eight genera); whereas 3,5-di-O-methylquercetin, caryatin, is present only in Bejaria, Phyllodoce and Rhododendron. Dihydroflavonols. common in Rhododendron, show a sporadic distribution elsewhere in the family. Of the simpler phenols surveyed, gentisic acid is common but methyl salicylate is present mainly in Gaultheria. Hydroquinone appears in ten related genera, methylhydroquinone in three and orcinol in seven. The chemical evidence generally supports Stevens' recent classification of the family, particularly his inclusion of Epigaea in the Rhododendroideae and his transfer of Diplarche from the Diapensiaceae to the Ericaceae. There is also chemical evidence to support the separation of Calluna and Cassiope, the inclusion of the Aibutoideae within the Vaccinioideae and the retention of the Pyroloideae and Monotropoideae within the Ericaceae. In addition, chemistry indicates that Phyllodoce should be placed nearer to the Rhodoreae than in Stevens' system and that Cassiope and Harrimanella, placed together in the Cassiopeae by Stevens, are distinctly different.

Flavonoid glycosides are either lacking or comprise simple profiles in five species representing four genera of the mycotrophic, achlorophyllous Monotropoideae of the Ericaceae. Monotropa hypopitys and Pterospora andromedea afforded no flavonoids while the profiles of M. uniflora Hemitomes conjestum and Sarcodes sanguinea cornprise monoglycosides and digylcosides of kaempferol and quereetin, although not all taxa have all compounds. Hemitomes also possess isorhamnetin 3- O -rutinoside The red colour of Sarcodes arises from a pelargonidin xyloside. The observed flavonoid profiles are more similar to the simple profiles reported for members of the Pyroloideae than they are to those of the Ericaceae sens str.