Monotropa Uniflora Research Articles

Host microtubules in the Hartig net region of ectomycorrhizas, ectendomycorrhizas, and monotropoid mycorrhizas

Various categories of mycorrhizas are recognized primarily by the structural changes that occur between fungi and roots. In all mycorrhiza categories, cytological modifications of root cells accompany the establishment of the functional symbiosis, and among these are alterations in the organization of the cytoskeleton. Using immuno labelling combined with confocal scanning laser microscopy, this study documents changes in microtubules (MTs) in root cells of ectendomycorrhizas and monotropoid mycorrhizas; in addition, ectomycorrhizas were reinvestigated to determine the effect of fungal colonization on host root cells. In Pinus banksiana L. – Laccaria bicolor (Maire) Orton ectomycorrhizas, MTs were present in epidermal and cortical cells adjacent to the Hartig net. The remaining cortical MTs had a different organization when compared with those of cortical cells of control roots. MTs were present in Hartig net hyphae. In ectendomycorrhizas formed when roots of P. banksiana were colonized by the ascomycete, Wilcoxina mikolae var. mikolae Yang & Korf, MTs were present adjacent to intracellular hyphae and host nuclei, but few cortical MTs were present. MTs were present within Hartig net and intracellular hyphae. In field-collected roots of Monotropa uniflora L., MTs were associated with fungal pegs, intracellular extensions of inner mantle hyphae within epidermal cells. The close association between MTs and fungal pegs may be related to the formation of the highly branched host-derived wall that envelops each fungal peg. The development of exchange interfaces in the three systems studied involve changes in the organization of microtubules.Key words: cytoskeleton, microtubules, Hartig net, mycorrhizas, immunolocalization, confocal microscopy.

Recovery of late-seral vascular plants in a chronosequence of post-clearcut forest stands in coastal Nova Scotia, Canada

We investigated the impacts of clearcutting on the ground vegetation of remnant late-successional coastal Acadian forests in southwestern Nova Scotia. Vegetation was sampled in 750 1-m2 quadrats established in 16 stands belonging to different recovery periods since clearcutting (3–54 years) and 9 late-successional forests (100–165 years) with no signs of significant human disturbance. Our objectives were to: i) describe the changes in species richness, diversity, and abundance of ground vegetation after clearcutting; ii) examine the responses of residual species (i.e., late-successional flora) to clearcutting; and iii) determine whether any forest species were restricted to or dependent upon the late-successional stages of stand development for maximal frequency and/or abundance. Although clearcutting had no immediate impact on overall alpha richness or diversity, the richness and diversity of residual plants declined after canopy removal and showed no evidence of recovery over 54 years of secondary succession. Consequently, compositional differences between secondary and late-seral stands persisted for many decades after clearcutting. Several understory herbs (e.g., Coptis trifolia (L.) , Oxalis montana (L.), Monotropa uniflora (L.)) were restricted to or attained their highest frequency and abundance in late-seral forests. These results suggest that the preservation of remnant old stands may be necessary for the maintenance of some residual plants in highly disturbed and fragmented forest landscapes in eastern Canada.

Monotropa uniflora: morphological and molecular assessment of mycorrhizae retrieved from sites in the sub-boreal spruce biogeoclimatic zone in central British Columbia.

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.

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.

Five Short Reviews

Chris Agee is a poet born and educated in the United States and trans planted to Ireland, where he lives and teaches in Belfast. Unlike some other Irish poets who have made the transatlantic crossing in that di rection?Brooklyn-born John Montague being the most notable among them?Agee focusses in collection on an all-encompassing empa thy with the natural world, of which he has a coherent and even clas sical vision. Although he notes in passing the imprints of human his tory and conflict in the natural surround?what poet could fail to do so in Ireland??it is the landscape itself with which he feels most at home. The first landscape is that of his native North America, specifi cally New England?one New World region whose pastoral terrain most closely resembles that of his adopted European isle?where the unretainable is nevertheless preserved, burnt in the brow of memory. And he dwells with affection on the green contours of the Irish coastlands, drawing on the Imagists, Japanese haiku, Hopkins,Yeats, and the spirits of the Celtic past for his inspiration. There are some lovely close inspections of North American flora and fauna?loons, Indian pipes, paper birches?all bound by the blood shot ground of our heritage. And there are sweeping evocations of the Old World younger than this in actual geological time: the evening peet-reek of Irish turf, of fallow deer (swift interlopers / on winter pasture), and the shifting colors of croftlands in the sea sonal cycles of the liturgical year. But the poetry doesn't quite tran scend its initial descriptive delights to reach for a deeper connection between the indwelling life of these continents and the human cul tures that overlay and inhabit them.

Drawings

^Drawings Elizabeth Layton Literature and Medicine 13, no. 1 (Spring 1994) 47-59 © 1994 by The Johns Hopkins University Press 48 DRAWINGS Figure 1. On the Death of a Child. Colored pencil on paper, 28 χ 22 inches, 1977. "No matter how old your child is when it dies, you tend to think of it as a baby." Elizabeth Layton 49 Figure 2. Pushing up the Daisies. Colored pencil on paper, 28 χ 22 inches, 1978. "This is my gravestone ... some child has pulled some daisies and laid them there on the grave. See, she is winking... she knows what's after this world. It's a hopeful picture." 50 DRAWINGS Figure 3. Pulling the Plug. Colored pencil on paper, 30 χ 22 inches, 1987. "Symbolic of the many means of keeping a body functioning. Restraint is a necessity. The symbols are mainly the machines and plug. ... I put a cockroach in symbolizing danger.. . her skin is the color of air, quite transparent." Elizabeth Layton 51 Figure 4. Traditional (Natural) Dying (Tender Loving Care). Colored pencil on paper, 30 χ 22 inches, 1987. "Intensive caring or only caring [is] what I was trying to say... time has run out, the bell tolls, candle is almost burned out, patient has become nothing (bed).... In the traditional way of dying, we spoon-fed a little ice, not enough to sustain life but sufficient to comfort the body and nourish the soul...." 52 DRAWINGS Figure 5. The Magic Gate to the Cosmos. Colored pencil on paper, 41 χ 29 inches, 1987. "She goes through the magic gate to the Cosmos, the pain of death almost unnoticed as she recognizes her welcome. In the world she is leaving behind are flowers, weeks, beauty, ugliness, cruelty, love... . She is far enough into the gate that she can tell it's not going to be a bad place. You can tell by the light in her eyes___" Elizabeth Layton 53 Figure 6. Void. Colored pencil on paper, 28 χ 22 inches, 1978. "I had this feeling that I was out in the middle of nowhere... absolutely nothing ... and it was such a lost feeling. I was lit up by a white light, but I didn't light up the black." 54 DRAWINGS Figure 7. Stroke. Colored pencil on paper, 28 χ 22 inches, 1978. "This is an out-of-body experience at a time of stress. With no feeling in half her body, she looks devastated. Yet, there is hope in the flowers and butterflies of her robe." Figure 8. Hansel and Gretel Lost in the Woods. Colored pencil on paper, 22 χ 28 inches, 1987. "Hansel and Gretel are lost in the woods, at the candy house in the realm of the wicked witch. In the upper Figure 9. Tfa's Motherless Child No. 4, "Her Bath." Colored pencil on paper, 22 χ 30 ^ inches, 1987. "Some Residents like their bath. Some don't. All seem to feel better ™ afterwards!" W er Sr > ζ Γι Figure 11. Buttons. Colored pencil on paper, 22 χ 30 inches, 1982. "Her strength is in her DrinciDles. m S1 r1 Figure 12. Indian Pipes. Colored pencil on paper, 22 χ 30 inches, 1984. "... an old Indian, as his time to die drew near, went out, or was put out, from the tepees to the mercy of the elements.... Here the old chief still sharpens his arrowhead. The old squaw knits on booties for coming generations' never-ending needs. Death zeroes in on the old people. Not something black and ugly but a silvery crystal softness." ...

Gynoecial Pathway for Pollen Tube Growth in the Genus Monotropa

The gynoecial micromorphologies of Monotropa uniflora and M. hypopitys are compared to characterize the gynoecial pathway for pollen tube growth. The receptive surface of the concave stigma is restricted to a papillate rim coated with an exudate. An internal transmitting tissue in the stigma joins the papillae with an external transmitting tissue system formed by the lining of the fluted stylar canal that may limit pollen tube growth to specific ovule-bearing placentae. Consequently, pollen tube growth is facilitated in the vicinity of the ovular micropyles.

POSTFERTILIZATION CHANGES IN OVULES OF MONOTROPA UNIFLORA L. (MONOTROPACEAE)

Monotropa uniflora is an achlorophyllous angiosperm consisting of a mycorrhizae‐dependent root system that produces floriferous, aerial shoots. Each of the numerous, minute ovules is anatropous, unitegmic, and contains a Polygonum type female gametophyte. Following double fertilization, a lipid‐rich, cellular endosperm develops in association with both chalazal and micropylar haustoria. The vacuolate zygote elongates prior to a cytoplasmically unequal division resulting in a small terminal cell subtended by a larger, vacuolate basal cell. The basal cell eventually degenerates, isolating the terminal cell which is completely surrounded by endosperm. The terminal cell undergoes a cytoplasmically equal transverse division resulting in a two‐celled embryo embedded in endosperm. In final stages of seed maturation, lipids decrease and reserve proteins increase in the cytoplasm of both the endosperm and embryo. The morphological reduction of the mature embryo may be associated with a specialized mode of nutrition.

Postfertilization Changes in Ovules of Monotropa uniflora L. (Monotropaceae)

Monotropa uniflora is an achlorophyllous angiosperm consisting of a mycorrhizae-dependent root system that produces floriferous, aerial shoots. Each of the numerous, minute ovules is anatropous, unitegmic, and contains a Polygonum type female gametophyte. Following double fertilization, a lipid-rich, cellular endosperm develops in association with both chalazal and micropylar haustoria. The vacuolate zygote elongates prior to a cytoplasmically unequal division resulting in a small terminal cell subtended by a larger, vacuolate basal cell. The basal cell eventually degenerates, isolating the terminal cell which is completely surrounded by endosperm. The terminal cell undergoes a cytoplasmically equal transverse division resulting in a two-celled embryo embedded in endosperm. In final stages of seed maturation, lipids decrease and reserve proteins increase in the cytoplasm of both the endosperm and embryo. The morphological reduction of the mature embryo may be associated with a specialized mode of nutrition.

Fungal calcium oxalate in mycorrhizae of Monotropa uniflora

Monotropa uniflora is an achlorophyllous angiosperm that is obligately mycotrophic. The "monotropoid" mycorrhizae it forms resemble ectomycorrhizae but are distinguished by elaborations of the epidermal cell walls that surround intruding fungal hyphae. Monotropoid mycorrhizae collected from blooming plants in late summer contained calcium oxalate crystals between mantle hyphae. The crystals appeared to form in association with hyphal walls and grew into a matrix outside the hyphae. Production of calcium oxalate by M. uniflora's mycobiont seems to be a coordinated metabolic process rather than a random precipitation event. The significance of calcium translocation and isolation as calcium oxalate to this mycorrhizal fungus is unclear, but the presence of extensive crystal deposits during and after flowering of the host plant suggests a possible link with the nutrient transfer occurring at that time. Mycorrhizal regulation of calcium may affect the availability of mineral nutrients to the associated Monotropa plants. Key words: Monotropa uniflora, mycorrhiza, calcium oxalate, ectomycorrhiza.

Ghosts of Summer's Woods

GHOSTS OF SUMMER'S WOODS / Jack Sanders THERE'S SOMETHING EERIE about seeing a cluster of Indian pipes, heads downturned, on a warm summer day. Ghostly and greenless, they remind one more of mushrooms than of the herbs they are. Their white flesh is unexpected and freakish, especially when you realize that they are wUdflowers and not some oddly formed fungus. Because of their unusual appearance, Indians pipes are, once seen, never forgotten. I can remember finding them as a child on Nantucket Island and being told their name. That, the daisy, black-eyed susan, and the violet were probably the only wildflowers I could name till well into adulthood. Indian pipes are white or bluish-white (rarely pink or red), almost leafless plants bearing a single five-petaled flower that, when young, faces the earth. The shape of the plant resembles a clay pipe whose stem has been stuck in the earth, and the flower is not unlike the bowl. This albino of the flowering plant world is, surprisingly, somewhat closely related to the dogwoods, heaths, and even to the evergreen laurels and rhododendrons. But to look at its almost leafless white stalk and its plain white fleshy flower, one would be hard pressed to imagine similarities with those of chlorophyllous plants. The Indian pipe is a member of a tiny clan of only three or four species. Only it and pinesap are found in North America; another inhabits such far-away places as Japan and the Himalayas—where our own Indian pipe may also be found. Another, called bird's nest, is found in Britain and Europe. Indian pipes are one of our few native wildflowers that might be called transcontinental, for they may be found from Maine to Florida, and from Washington to California, right down to and into Mexico. They also range across southern Canada and into Alaska. In most areas they appear in mid-summer. Scientists call it Monotropa uniflora, meaning "once-turned" and "single-flowered." "Once-turned" refers to the fact that the flowers, which face the ground early in their life, turn straight upward once they begin producing seeds (though you'd think the plant would be straight first and bent second, in order to spiU out the seeds). Monotropa is in turn a member ofthe wintergreen family (Pyrolaceae), a small clan of only ten genera in North American, their chief habitat. 84 · The Missouri Review They include the equally unusual pine-drops and beech-drops (which, not surprisingly, live near pine and beech trees). The Saprophytic Plant The Indian pipe is strange not only in appearance, but in habit. It's a saprophyte (from the Greek, "rotten plant"), living chiefly on the decaying roots of other plants, particularly trees. Indian pipes are most often found near a dead stump in deep woods, although they will sometimes pop up in a lawn near dead tree roots. They favor beech woods, but will live in others, and it is said that the best time to find them is after a heavy, soaking midsummer rain. Some botanists believe that the roots, working in symbiotic conjunction with certain soil fungi such as mushrooms, also obtain food from live tree roots, which would make the plant a parasite as well as a saprophyte.1 If this is true, the Indian pipe certainly has a small enough appetite that it can do no harm to large trees, most of whose roots are well beyond its reach. Since the plant obtains all the nutrients it needs from other plants, it requires neither leaves nor chlorophyll, the factories and the chemical employed by most plants in using sunlight to create carbohydrates for food. In the long process of evolution, it lost both—with only vestiges of leaves remaining in the form of white, scale-like appendages on the stems. The plant can't be picked for display—not that anyone would want it as a decoration—because its flesh turns black when cut or even bruised. It also oozes a clear, gelatinous substance when picked or wounded. Such unattractive characteristics have earned the Indian pipe some unflattering names, like ghost flower, corpse plant, and American iceplant. Another is pretty: fairy...

Pollen morphology and its taxonomic significance of the monotropoideae (Ericaceae)

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.

Chromosomes of Monotropa uniflora

Anther tissue and the apical tissue of adventitious root buds of Monotropa uniflora were squashed using standard techniques. The chromosome number of 48 is the same as that of M. hypopithys.

SEED MORPHOLOGY OF MONOTROPA UNIFLORA L. (ERICACEAE)

Mature seeds of Monotropa uniflora L., an achlorophyllous mycotrophic perennial, underwent imbibition and were processed for study using modern histological and histochemical techniques. The seeds ranged from 0.6‐0.8 mm in length and 0.12‐0.15 mm in width and exhibited integumentary winglike structures at either end. As in other members of the Ericaceae, seeds are unitegmic, tenuinucellate, albuminous, and form both micropylar and chalazal endosperm haustoria. A two‐celled reduced embryo was observed in all seeds, except one where the embryo was three‐celled. Protein granules in the thick walled endosperm were found to contain an aniline blue‐fluorescent material that may be a calloselike carbohydrate. The limited amount of seed food reserves and the retardation of embryo differentiation may reflect specialized germination requirements.

STUDIES OF THE MONOTROPOIDEAE (ERICACEAE). FLORAL NECTARIES: ANATOMY AND FUNCTION IN POLLINATION ECOLOGY

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.

Sterols and fatty acids of some non-photosynthetic angiosperms

Sterols and fatty acids were extracted and identified from three parasitic angiosperms, Cuscuta campestris, Monotropa uniflora and M. hypopitys. Each plant contained the typical 16 and 18-carbon fatty acids of angiosperms, but the partially-photosynthetic Cuscuta contained much larger quantities of linolenic acid than the non-green Monotropa species which had smaller amounts of linolenic acid characteristic of non-photosynthetic tissue. Sterol quantity was three times higher in Cuscuta than in the Monotropa species. Sitosterol was the major sterol in all species with smaller amounts of campesterol and cholesterol.

Development of the Generative Cell Wall in Monotropa uniflora L. Pollen

During an ultrastructural survey of an achlorophyllus dicotyledonous plant, Monotropa uniflora L., a stage of pollen development was encountered which suggests a relationship between the activity of the rough endoplasmic reticulum and the development of the generative cell wall.

MONOTROPA UNIFLORA: ULTRASTRUCTURAL DETAILS OF ITS MYCORRHIZAL HABIT

A microscopic survey of the achlorophylus plant Monotropa unijlora 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.

Stem and Root Anatomy of Monotropa uniflora

HIRCE, E. G., and A. F. FINOCCHIO. (St. Bonaventure University, St. Bonaventure, New York 14778). Stem and root anatomy of Monotropa uniflora. Bull. Torrey Bot. Club 99: 89-94. 1972.-Quantitative studies of the vascular tissue of Monotropa uniflora L. reveal that the amount of xylem is significantly greater than the amount of phloem. In the stem, vascular tissue occurs in eustelic arrangement. An ontogenetic sequence from annular protoxylem tracheids to scalariform-reticulate metaxylem vessels is observed. Insoluble polysaecharides are stored in the cortical regions of the middle and upper stem during July. The root system is composed of first, second and third order roots uniformly covered by a dense mycorrhizal sheath that is neither strictly ectotrophic nor endotrophic. Mycelial penetration is greatest in second order roots. The stelar arrangement of the root varies: complex first order roots are typically hexarch; second order roots, tetrarch or triarch; third order roots, protostelic. Trachearv elemeints are scalariform-reticulate tracheids and vessel members.