Vegetative and floral development in the pistillate plant of Arceuthobium americanum (lodgepole pine dwarf mistletoe): an environmental scanning electron microscopy study of its phenology and shoot organization

Dwarf mistletoes (DM), genus Arceuthobium, are dioecious parasitic flowering plants having prolonged life cycles lasting six years, culminating with explosive discharge of the single seed from the fruit. Arceuthobium americanum Nutt. ex Engelm., the lodgepole pine dwarf mistletoe, infects lodgepole pine (Pinus contorta subsp. latifolia (Engelm.) Critchfield) in western North America, compromising the forest economy. Highly reduced flowers appear no later than two years following shoot development, with female flowers appearing and persisting for over two years. Development of the pistillate plant, including initiation of floral growth, has not been fully explored. Here, we used environmental scanning electron microscopy to demark phenological waypoints throughout the pistillate plant’s development. As successive crops of female flowers emerged every year, up to three generations of flowers/fruit could be found on a single shoot in late summer; we used these three generations to delineate specific developmental stages. Vegetative shoots could initiate growth at any time within the growing season, could assume a terminal position, could also adopt sympodial branching, but were never whorled or adventitious. Floral branches, however, could initiate adventitiously from older nodes in a whorled pattern, and could house flowers/fruits of any generation. Vegetative and floral units were structurally homogeneous, suggesting shared developmental pathways.

Fire and dwarf mistletoe (Viscaceae: Arceuthobium species) in western North America: contrasting Arceuthobium tsugense and Arceuthobium americanum

Dwarf mistletoes (Viscaceae: Arceuthobium spp.) and fire interact in important ways in the coniferous forests of western North America. Fire directly affects dwarf mistletoes by killing the host, host branch, or heating/smoking the aerial shoots and fruits. Fire is a primary determinant of dwarf mistletoe distribution on the landscape, and time since fire controls many aspects of dwarf mistletoe epidemiology. Conversely, dwarf mistletoes can influence fire by causing changes in forest composition, structure, and fuels. Prescribed fire is important for management of dwarf mistletoes, while fire suppression is thought to have increased dwarf mistletoe abundance in western forests. Two dwarf mistletoes are compared in order to illustrate fire interactions in Oregon and Washington, USA: Arceuthobium americanum Nutt. ex Engelm. (lodgepole pine dwarf mistletoe) and Arceuthobium tsugense (Rosendahl)(western hemlock dwarf mistletoe). Arceuthobium persists on the landscape where the host is not killed by fire. Arceuthobium americanum spreads directly into the regenerating Pinus contorta Dougl. ex Loud., while A. tsugense persists in refugia for 200 years or more following fire. Host successional status is a driver of fire – dwarf mistletoe interactions, but forest disturbance agents also play a role. Given the importance of these interactions to the ecology of fire-prone forests, dwarf mistletoes warrant inclusion in disturbance ecology research.

Morphology and stomatal density of developing Arceuthobium americanum (lodgepole pine dwarf mistletoe) fruit: a qualitative and quantitative analysis using environmental scanning electron microscopy

Arceuthobium americanum Nutt. ex Engelm., the lodgepole pine dwarf mistletoe, is a dioecious parasitic flowering plant infecting lodgepole pine (Pinus contorta subsp. latifolia (Engelm.) Critchfield) in the Pacific Northwest, compromising timber value by stunting the host’s growth. The plant disperses its seeds by explosive discharge, and thus understanding its reproduction is an integral step toward managing its spread. The life cycle of Arceuthobium americanum occurs over five to six years, and the fruit matures over two consecutive growing seasons, dispersing in the second. Using low-vacuum environmental scanning electron microscopy (eSEM), we examined branching architecture as well as the morphology and anatomy of fruits during their second year of development up to explosive discharge. We found that branching patterns shifted from opposite-decussate to verticillate after several years’ growth. In addition, the perianth parts (sepals), style, and stigma persisted through the season, and the fruit’s abscission zone developed slightly distal to the end of the pedicel. Stomata, found only on the sepals, were located in crypts surrounded by two subsidiary cells, and stomatal density significantly decreased during development. The decline in stomatal density along with the thick pericarp cuticle may function in retaining water inside the fruit to facilitate discharge and (or) provide a heating mechanism through reduced transpiration.

Plants, fungi, and freeloaders: examining temporal changes in the “taxonomic richness” of endophytic fungi in the dwarf mistletoe Arceuthobium americanum over its growing season

Arceuthobium americanum Nutt. ex Engelm. (lodgepole pine dwarf mistletoe) is a dioecious plant parasitizing conifers in North America. Here, we documented changes in the “taxonomic richness” of culturable endophytic fungi in male and female A. americanum over the growing season. Endophytic fungi were isolated from vegetative stems of surface-sterilized aerial shoots from male and female A. americanum collected weekly from April to September. Isolated fungi were characterized macroscopically, generating a database of 48 morphologically unique forms that likely represented individual taxa. A random sample of endophytic fungi was sequenced to determine identity based on ITS rDNA. Richness increased throughout the growing season similarly in the stems of both sexes, suggesting that increased developmental age or time in the environment facilitates an increase in endophyte richness: male and female endophyte communities were 91% similar based on Sorensen’s coefficient. Sequenced endophytes came from many diverse taxa, including Serpula, Alternaria, and Tremella, which may function as mutualistic symbionts within the mistletoe. An increase in fungal richness throughout the growing season has been observed in all flowering plants examined to date, although our observation is the first for the genus and for parasitic plants in general, and has also contributed to knowledge of fungal diversity in dioecious plants.

Thermogenesis-triggered seed dispersal in dwarf mistletoe

Lodgepole pine dwarf mistletoe (DM), Arceuthobium americanum, is a parasitic flowering plant and forest pathogen in North America. Seed dispersal in DM occurs by explosive discharge. Notably, slight warming of ripe DM fruit in the laboratory can trigger explosions. Previously, we showed that alternative oxidase, a protein involved in endogenous heat production (thermogenesis) in plants, is present in DM fruit. These observations have led us to investigate if thermogenesis induces discharge. Here, infrared thermographs reveal that ripe DM fruits display an anomalous increase in surface temperature by an average of 2.1±0.8 °C over an average time of 103±29 s (n=9, 95% confidence interval) before dehiscence. Furthermore, both non-isothermal and isothermal modulated differential scanning calorimetry consistently show an exothermic event (~1 J g−1) in the non-reversible heat flow just prior to discharge. These results support thermogenesis-triggered seed discharge, never before observed in any plant.

Anther and pollen development in the lodgepole pine dwarf mistletoe (Arceuthobium americanum) staminate flower

The lodgepole pine dwarf mistletoe, Arceuthobium americanum Nutt. ex Engelm., is a parasitic angiosperm that infects conifers in western Canadian forests. While production of viable pollen in anthers is critical to dwarf mistletoe reproduction, the few existing reports that examine staminate development in Arceuthobium are often incomplete or conflicting. The objective of this work was to investigate the developmental anatomy of anther and pollen of A. americanum using modern microscopy. We found that the microsporangium was toroidal from the outset and gave rise to a central peg-shaped sterile “columella” early in anther development. The endothecium was absent, the epidermis persisted as an “exothecium” fulfilling the role of the endothecium, and a primary parietal layer generated a secretory tapetum and middle layer. Thus, we suggest that a new category of anther wall development, the Arceuthobium type, be created. Microsporogenesis produced tetrahedral microspores via simultaneous cytokinesis and involved callose wall formation. Microgametogenesis resulted in round and atypical generative and vegetative nuclei. Additionally, the heterocolpate, echinate pollen grains, which were shed at the two-celled stage, were seen for the first time in their native state with environmental scanning electron microscopy. This work contributes to the understanding of A. americanum, the genus Arceuthobium, and angiosperms as a whole.