Pterospora Research Articles

Largely invariant communities of bacterial endophytes in the nonphotosynthetic mycoheterotrophic plant Pterospora andromedea.

Mycoheterotrophic plants rely on fungi to obtain their carbon requirements. Recent experiments demonstrated the presence of endophytic bacteria associated with mycoheterotrophs. Although mycoheterotrophs show high specificity for their fungal partners, it is not known whether they also show high specificity for associated bacteria or whether the bacteria have a definite function in the symbiosis. Two 16S rRNA sequencing experiments were designed to explore endophytic microbial community composition and function in root ball fractions of the mycoheterotroph Pterospora andromedea (Ericaceae), and rhizosphere soil and control soil 5 m away from each plant. One experiment compared microbial assemblages in fractions of six plants to those in rhizosphere and control soil samples. Another experiment documented bacterial endophyte diversity in root balls of 97 plants from across North America. Soil samples were similar in bacterial community structure but were significantly more diverse and less consistently structured than were bacterial communities within root balls. The proportion of endophytic bacterial species varied slightly but not their community composition despite differences in P. andromedea lineage, geography, conifer species, and fungi. Predictive metagenomic profiling of the endophytes in P. andromedea-only root ball fractions showed many of the bacterial endophytes likely function in N-metabolism and N-fixation. Our results document a consistent and largely invariant community of endophytic bacteria in P. andromedea across biotic and abiotic environmental conditions at a continental scale. It is unknown what role these bacteria may play in the quad-partite symbiotic network centered on P. andromedea; however, the predictive metagenomic profiling suggests a possible function in N-metabolism or N-fixation. Discovery of a ubiquitous community of endophytic bacteria with a putative function centered on N-metabolism or N-fixation could have a previously unrecognized impact on understanding of mycoheterotroph ecophysiology.

Databasing and georeferencing historical collections to discover potential sites for rare and endangered plants of New York, U.S.A.

Over 400 years of natural history inventories of preserved specimens and vouchers in museums and research institutions are now important repositories of invaluable information and permanent records documenting life on earth. Plant collections, in particular, play roles in understanding climate change, conservation modeling, molecular and medicinal studies, invasive-species management, and biodiversity assessments. Researchers reexamine these collections in order to establish conservation priorities for threatened and endangered taxa. Herbarium specimens, spanning many decades, can provide data on a natural population’s geographical range and its shift over time. Although historical specimens provide valuable information about past distributions, label information may lack geographic coordinates or precise locations, or may have descriptions that are too vague or confusing to be useful. Here we present a case study for plant specimens collected by Thomas F. Lucy in the late 1800’s in Chemung County, New York. The goal of this research was to determine if Lucy’s label localities could be improved using historical land use and topographic maps, and georeferenced using Google Earth©. Lucy documented 134 plant families for the Chemung County area: 106 species of flowering plants (ca. 982 taxa, excluding hybrids), 12 fern and fern allies (48 taxa), 13 mosses (21 taxa), and one species each for lichens, liverworts, and algae. Nine taxa are noteworthy for the state of New York. Fourteen taxa are listed as endangered, threatened, or unknown: Chaerophyllum procumbens (Apiaceae), Arabis shortii (Brassicaceae), Cacalia suaveoleus and Lactuca floridana (Compositae), Carex retroflexa (Cyperaceae), Hydrangea arborescens (Hydrangeaceae), Allium cernuum and Chamaelirium luteum (Liliaceae), Liparis lilifolia and Platanthera hookeri (Orchidaceae), Polygonum erectum (Polygonaceae), Lysimachia quadrifolia (Primulaceae), Geum virginianum (Rosaceae) and Vitis vulpine (Vitaceae). Nine other plants are recommended for protection: Panax quinquefolius (Araliaceae), Rudbeckia fulgida (Compositae), Carex conjuncta (Cyperaceae), Pterospora andromedea (Ericaceae), Aesculus grabra (Hippocastanaceae), Epilobium ciliatum (Onagraceae), Cypripedium arietinum (Orchidaceae), and Calamagrostis porteri and Panicum scoparium (Poaceae).

Development of Anonymous Nuclear Loci for Pterospora andromedea (Monotropoideae) Using Illumina and Ion Torrent Sequencing Data

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.

Increased phylogenetic resolution within the ecologically important Rhizopogon subgenus Amylopogon using 10 anonymous nuclear loci

ABSTRACTRhizopogon species are ecologically significant ectomycorrhizal fungi in conifer ecosystems. The importance of this system merits the development and utilization of a more robust set of molecular markers specifically designed to evaluate their evolutionary ecology. Anonymous nuclear loci (ANL) were developed for R. subgenus Amylopogon. Members of this subgenus occur throughout the United States and are exclusive fungal symbionts associated with Pterospora andromedea, a threatened mycoheterotrophic plant endemic to disjunct eastern and western regions of North America. Candidate ANL were developed from 454 shotgun pyrosequencing and assessed for positive amplification across targeted species, sequencing success, and recovery of phylogenetically informative sites. Ten ANL were successfully developed and were subsequently used to sequence representative taxa, herbaria holotype and paratype specimens in R. subgenus Amylopogon. Phylogenetic reconstructions were performed on individual and concatenated data sets by Bayesian inference and maximum likelihood methods. Phylogenetic analyses of these 10 ANL were compared with a phylogeny traditionally constructed using the universal fungal barcode nuc rDNA ITS1-5.8S-ITS2 region (ITS). The resulting ANL phylogeny was consistent with most of the species designations delineated by ITS. However, the ANL phylogeny provided much greater phylogenetic resolution, yielding new evidence for cryptic species within previously defined species of R. subgenus Amylopogon. Additionally, the rooted ANL phylogeny provided an alternate topology to the ITS phylogeny, which inferred a novel set of evolutionary relationships not identified in prior phylogenetic studies.

Variability of ecological and autotrophic host specificity in a mycoheterotrophic system: Pterospora andromedea and associated fungal and conifer hosts

Abstract 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.

Characterization of microsatellite markers for pinedrops, Pterospora andromedea (Ericaceae), from Illumina MiSeq sequencing.

• 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.

Rhizopogon kretzerae sp. nov.: the rare fungal symbiont in the tripartite system with Pterospora andromedea and Pinus strobus

The first species of Rhizopogon subgenus Amylopogon identified from eastern North America is described here as Rhizopogon kretzerae Grubisha, Dowie, & Mill. sp. nov. In nature, R. kretzerae has been identified only from DNA sequences of the nuclear ribosomal internal transcribed spacer region (ITS) from mycorrhizal roots of Pinus strobus L. and Pterospora andromedea Nutt. Phylogenetic analyses and nucleotide divergence estimates of Rhizopogon ITS sequences from Pterospora roots from Quebec, Michigan, New York, and New Hampshire combined with ITS sequences of Rhizopogon subgenus Amylopogon species type collections suggest R. kretzerae is a sister-group to Rhizopogon salebrosus A.H. Sm. An absence of shared mutations, two informative sequence gaps, and 12 fixed nucleotide differences differentiate R. kretzerae and R. salebrosus. Furthermore, ITS sequences of 40 Pterospora root samples from five populations in the province of Quebec provided the first record of the distribution of R. kretzerae, a potentially rare to endangered species, in eastern Canada.

Microsatellite primers for the fungi Rhizopogon kretzerae and R. salebrosus (Rhizopogonaceae) from 454 shotgun pyrosequencing.

• Premise of the study: Rhizopogon kretzerae and R. salebrosus (Rhizopogonaceae) are ectomycorrhizal fungi symbiotic with pines and the mycoheterotrophic plant Pterospora andromedea (Ericaceae). Microsatellite loci will allow population genetic study of fungal hosts to P. andromedea.• Methods and Results: Shotgun pyrosequencing of R. kretzerae DNA resulted in primer development of 23 perfect microsatellite loci and screened across two populations each for R. kretzerae and R. salebrosus. Twelve loci were polymorphic in R. kretzerae populations, and 11 loci cross-amplified in R. salebrosus populations. For R. kretzerae and R. salebrosus, number of alleles was one to eight and one to nine, respectively, and observed heterozygosity ranged from 0.00–0.57 and 0.00–0.70, respectively.• Conclusions: These are the first microsatellite loci developed for any species within Rhizopogon subgenus Amylopogon. These microsatellite loci will be used in conservation genetic studies of rare to endangered eastern populations and to compare plant and fungal population genetic structure at different hierarchical levels.

Gibberellic Acid Induces Asymbiotic Germination of the Obligate MycoheterotrophPterospora andromedea(Ericaceae)

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.

Is rarity of pinedrops (Pterospora andromedea) in eastern North America linked to rarity of its unique fungal symbiont?

Like other myco-heterotrophic plants, Pterospora andromedea (pinedrops) is dependent upon its specific fungal symbionts for survival. The rarity of pinedrops fungal symbiont was investigated in the eastern United States where pinedrops are rare. Wild populations of eastern pinedrops were sampled, and the plant haplotypes and fungal symbionts were characterized with molecular techniques; these data were compared to those from the West with phylogenetic analyses. The frequency of the fungal symbiont in eastern white pine forests was assessed using a laboratory soil bioassay and in situ pinedrops seed baiting. Only one plant haplotype and fungal symbiont was detected. The plant haplotype was not unique to the East. The fungal symbiont appears to be a new species within the genus Rhizopogon, closely related to the western symbionts. This fungal species was not frequent in soils with or without pinedrops, but was less frequent in the latter and in comparison to the fungal symbionts in western forests. Seed baiting resulted in few germinants, suggesting that mycelial networks produced by the eastern fungal symbiont were rare. Results suggest that eastern pinedrops rarity is influenced by the distribution and rarity of its fungal symbiont.

Identity, genetic lineages and putative hybrids of an obligate mycobiont associated with the mycoheterotrophic plant Pterospora andromedea in the south-central Rocky Mountains

Mycoheterotrophic plants are nonphotosynthetic and must form a relationship with a mutualistic fungus and an autotrophic host to acquire carbon. In this study, identity of fungal associates of Pterospora andromedea in the south-central Rocky Mountains was determined. Although P. andromedea has been found to associate with at least three Rhizopogon species groups throughout its range in North America, it was found to exclusively associate with the Rhizopogon salebrosus species group in the Rocky Mountains. In addition, the genetic structure of R. salebrosus was examined. The data support the presence of two genetic lineages within the R. salebrosus species group that exhibit strong biogeographic distribution linked to historic conifer association with Pinus contorta and Pinus ponderosa . A zone of secondary contact between the two lineages was also identified along with a number of putative hybrids. ► In this study, all mycobionts of Pterospora andromedea were Rhizopogon salebrosus . ► Two separate lineages were identified by network analyses in ITS and RPB2 . ► The separate lineages associated with either Pinus ponderosa or P. contorta . ► A third group comprised of the putative hybrids was also identified.

Mycobiont overlap between two mycoheterotrophic genera of Monotropoideae (Pterospora andromedea and Sarcodes sanguinea) found in the Greater Yellowstone Ecosystem

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).

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea

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.

Fine-level mycorrhizal specificity in the Monotropoideae (Ericaceae): specificity for fungal species groups.

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.

Regional specialization of Sarcodes sanguinea (Ericaceae) on a single fungal symbiont from the Rhizopogon ellenae (Rhizopogonaceae) species complex

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 from S. 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.

In vitro germination of nonphotosynthetic, myco‐heterotrophic plants stimulated by fungi isolated from the adult plants

Sarcodes sanguinea and Pterospora andromedea (Ericaceae, Monotropoideae) are nonphotosynthetic myco‐heterotrophic 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 different 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 diffusible 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.

Reassessment of phylogenetic relationships of some members of the Monotropoideae based on partial 28S ribosomal RNA gene sequencing

2In 1992, 1994, and 1996, I published papers describing the evolution of the Monotropoideae and Ericaceae (Cullings and Bruns 1992; Cullings 1994; Cullings 1996) using 28S ribosomal RNA (rRNA) gene sequences (Fig. 1). The phylogenetic analysis presented in these manuscripts depicted a polyphyletic Monotropa, and an unexpectedly close relationship between Monotropa hypopithys and Pterospora andromedea. At the time, there were no other molecular treatments of the Monotropoideae for comparison, and morphological and biochemical characters were identified that supported all relationships. In 1997, Kathleen Kron gave a presentation and published an abstract (Kron and Johnson 1997) that depicted monotropoid relationships based on 18S rRNA gene and internal transcribed spacer (ITS) sequences. Kron and Johnson’s data differed from my own in two primary respects. First, the genus Monotropa, while still polyphyletic, was placed not with Pterospora andromedeaand Sarcodes sanguinea, but on the large clade containing Allotropa virgata, Hemitomes congestum, Pityopus californicus, and Monotropa uniflora. Second, Pityopus californicus, rather than being placed with Pleuricospora fimbriolataas in the 28S tree, was depicted as sharing a most recent common ancestor with H. congestum. Based on this information, I re-examined my phylogeny. I obtained four additional M. hypopithys individuals from a second population of this species in Yellowstone Park; both neighbor-joining and cladistic (parsimony) analysis of sequences from the additional four individuals from the second population of M. hypopithys depict a phylogeny in keeping with Dr. Kron’s combined 18S/ITS data (Fig. 1). This casts further doubt on my original data; however I have reason to believe the data were correct. The original two individuals used in Cullings and Bruns (1992) (also from Yellowstone) had been sequenced several times using DNA obtained from at least five independent polymerase chain reaction (PCR) amplifications using both terminal and internal, and univer

Pigment composition of putatively achlorophyllous angiosperms

Chlorophyll and carotenoid pigment composition was determined for ten species of putatively achlorophyllous angiosperms using high-performance liquid chromatography. Four families were represented:Lennoaceae (Pholisma arenarium);Monotropaceae (Allotropa virgata, Monotropa uniflora, Pterospora andromedea, Sarcodes sanguinea); Orobanchaceae (Epifagus virginiana, Orobanche cooperi, O. uniflora);Orchidaceae (Cephalanthera austinae, Corallorhiza maculata). Chlorophylla was detected in all taxa, but chlorophyllb was only detected inCorallorhiza maculata. The relative amount of chlorophyll and chlorophyll-related pigments in these plants is greatly reduced compared to fully autotrophic angiosperms.

Flavonoids in some monotropoideae

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.

ULTRASTRUCTURE OF PTEROSPORA ANDROMEDEA NUTTALL AND SARCODES SANGUINEA TORREY MYCORRHIZAS

SUMMARYMycorrhizas of Pterospora andromedea Nuttall and Sarcodes sanguinea Torrey were examined with light and electron microscopes. Both plants were found to have a multi‐layered fungal sheath covering the surface of their roots and a Hartig net surrounding the epidermal cells but not penetrating the underlying cortex. Dolipore septa are present in the fungi of both plants. Individual determinate hyphae enter each epidermal cell through the radial wall which is oriented toward the tip of the root. As they penetrate, the epidermal cell wall appears to invaginate forming a fungal peg. The wall of the peg produces numerous protuberances similar to those found in transfer cells. In the combined fungal‐epidermal cell wall of the fungal peg the two walls do not remain distinct and it is impossible to be certain if both continue to the tip. Eventually, the tip of the fungal peg appears to open and form a mebranous sac, delimited by the epidermal cell plasmalemma, containing granular material with fibrillar inclusions which are approximately the size of microtubules. The relationship of the contents of the membranous sac to the contents of the fungal peg is discussed.