Japanese Walnut Research Articles

Introduction of rare woody plants in the Botanical Garden of the Institute of Biology of Komi Science Centre of the Ural Branch of the Russian Academy of Sciences

The Arboretum of the Botanical Garden of the Komi Institute of Biology of the Ural Branch of the Russian Academy of Sciences is one of the northernmost in Russia, which contains more than 500 taxa of woody plants, including 14 rare and endangered species of various categories and protection status. Six of them need regional protection (Pentaphylloides fruticosa, Ulmus laevis, Pinus sibirica, Tilia cordata, Cotoneaster cinnabarinus, Cotoneaster melanocarpus), the rest are in state protection (Euonymus nana, Cotoneaster cinnabarinus, Cotoneaster lucidus, Staphylea colchica, Juglans ailanthifolia, Rhododendron fauriei, Picea glehnii, Taxus baccata, Microbiota decussata) The largest number plants included in the Red Book of the Komi Republic (2019), characterized by a wide ecological amplitude, have been studied more and more. Attraction to the collection of rare woody plants of the Red Book of Russia is limited by harsh climatic conditions. Ten species of plants of state protection are represented by single specimens and are only maintained in the collection. Winter hardiness of plants is determined every season and phenological observations are carried out. Conservation and attraction of rare plant species to the collection in the conditions of the Komi Republic should become an integral part of the biodiversity conservation strategy.

Quantification and Comparison of Nutritional Components in Oni Walnut (Juglans ailanthifolia Carr.), Hime Walnut (Juglans subcordiformis Dode.), and Cultivars

Walnuts are rich sources of lipids and polyunsaturated fatty acids and are expected to promote health. There are two Japanese native walnut species: Oni walnut (Juglans ailanthifolia Carr.) and Hime walnut (Juglans subcordiformis Dode.). However, despite the fact that these Japanese native walnuts have long been consumed in local cuisine, their nutritional composition is largely unknown. This study aimed to assess the concentrations of total lipids, and fatty acid composition including polyunsaturated fatty acids, in the kernels of Oni walnut and Hime walnut. In addition, we assessed various aspects related to their nutritional and functional values, by measuring the total protein, amino acids, minerals, and total polyphenols. The concentrations of the measured compounds in the two native species were compared with those in the English walnut (Juglans regia L.), a globally recognized cultivar, and its counterpart cultivated in Japan, Shinano walnut (Juglans regia L.). The results showed that Oni walnut and Hime walnut contained significantly higher protein and minerals and lower lipid content than conventional cultivars. However, both Oni and Hime walnuts contained higher ratios of unsaturated fatty acids in total fatty acids. This study offers novel insights into the nutritional components of Oni and Hime walnuts, contributing to a deeper understanding of their nutritional value and potential applications as unique native walnut species. The findings of this study highlight the relationship between the different types of walnut species and their nutritional composition, and the value of native walnuts used in local cuisine, and will lead to new developments in functional foods from walnut species consumed around the world. It will contribute to the development of functional and processed foods by increasing the production of native walnut species, which are rich in protein, unsaturated fatty acids and minerals and by using them in local cuisines and health-promoting foods.

Interactive effects of two rodent species on the seed dispersal of Japanese walnut

The effects of seed dispersers on plant fitness (seed dispersal effectiveness, SDE) have been evaluated based on the number (quantity) and recruitment probability (quality) of dispersed seeds. Although seeds of most zoochorous species are dispersed by two or more animal species, which may interact with each other, SDE has often been studied assuming a one-plant and one-animal species system. We compared the SDE of Japanese walnut (Juglans ailanthifolia) between squirrel-only and squirrel-mouse sites in natural forests of Hokkaido, Japan, and found that the SDE from the red squirrel (Sciurus vulgaris), considered a primary seed disperser, was altered by an alternative seed disperser species, the Japanese wood mouse (Apodemus speciosus). Seed removal rates at the squirrel-mouse site were significantly higher than those at the squirrel-only site, and both dispersed seeds and seedlings were less aggregated, with a strongly repulsive relationship with adult conspecific trees at the squirrel-mouse site. Seedlings established themselves at a location with fewer medium-sized trees (< 10 cm DBH) at the squirrel-mouse site. These results suggest that the interactive effect of the rodent species affects the SDE of Japanese walnut.

Walnut witches'-broom disease threatens butternut restoration efforts in Indiana.

Due to the devastating effects caused by butternut canker disease (BCD), efforts to protect the endangered butternut (Juglans cinerea) tree through resistance breeding has been a primary focus of forest restoration efforts. Walnut witches'-broom (WWB) disease poses a serious threat to these restoration efforts. This study sought to confirm the presence of the WWB disease phytoplasma, 'Candidatus phytoplasma pruni,' in butternut research plantings in Indiana using molecular methods and Sanger sequencing and identify butternut families affected by the disease. We also sought to better understand the incidence of the WWB's phytoplasma in asymptomatic trees and asymptomatic branches of symptomatic trees to better direct management decisions. The results of the Sanger sequencing confirmed the presence of the WWB phytoplasma in the butternut restoration plantings, the first confirmation in Indiana based on sequencing to our knowledge, in both symptomatic and some asymptomatic trees. In addition, the WWB phytoplasma was detected in asymptomatic branches of symptomatic trees, indicating that phytoplasma infection is not necessarily localized to symptomatic tissues in a tree. Trees with positive molecular confirmation of the WWB phytoplasma consisted of 23 different butternut families and one family of Japanese walnut (J. ailantifolia), which is considered to be one of the most susceptible species to WWB disease. Based on these findings, future studies should prioritize identifying the hybridity and pedigrees of families and their susceptibility to WWB disease to aid in butternut restoration efforts.

Estimation of absolute pollen productivity based on the flower counting approach: A review

Pollen productivity estimate (PPE) is one of the parameters necessary for pollen-based quantitative reconstruction of vegetation in the past. This paper summarizes and reviews the absolute PPEs (aPPEs) that were obtained with the area-based flower counting approach since the 1970s. The data for 23 species of trees are available from Japan and for 22 species of Poaceae from Japan, Spain, and Morocco. The aPPE values vary from 1 to 60 [1012 grains·ha−1·year−1], and are comparable to those previously reported in Europe and using different methods and approaches. The mean aPPEs of temperate conifers, such as Sciadopitysverticillata, Cryptomeria japonica, and Chamaecyparis obtusa, are as high as those of some of the common broadleaved trees, Castanea mollissima, Castanopsis cuspidata, and Alnus spp. The mean aPPEs of Abies firma, Fagus crenata, and Betula platyphylla are low, and those of Pinus densiflora, Quercus spp., Juglans ailanthifolia, Carpinus tchonoskii, Aesculus turbinata, and Poaceae spp. are intermediate. The relative PPE (rPPE) values of the arboreal taxa (relative to Poaceae), calculated using the aPPEs presented here, are smaller than those obtained in Western Europe and China based on the Extended R-value model approach. Differences in the sampling and analytical methods, as well as the interregional differences in Poaceae pollen productivity, might have contributed to the discrepancies observed. Although further studies are necessary, the flower counting approach is practical and informative for species-specific estimates of absolute pollen productivity and provides critical parameters for quantitatively reconstructing past vegetation in absolute units using the rates of pollen accumulation in sediments.

Is the Japanese walnut (Juglans ailantifolia, Juglandaceae) native to Sakhalin Island?

The Japanese walnut (Juglans ailantifolia Carrière) is considered a very rare native tree species to Sakhalin Island. There is no information about J. ailantifolia records in the natural broad-leaved mixed forests of the island. The valleys of the Uglegorka and the Nadym Rivers in the Uglegorsk administrative district were noted as the main habitats areas of J. ailantifolia in several sources. We found that all walnut trees from these locations belong to artificial forest stands over 95 years old. We conclude that J. ailantifolia is a non-native species to the modern flora of Sakhalin and was introduced from Japan at the beginning of the XX century.

Relationship between diet and occurrence around human settlements in Asiatic black bears

Increased occurrences of bears in human settlements in search of food have led to a high incidence of human–bear interactions in Japan; therefore, a better understanding of bear occurrence in relation to bears' diet is required to establish effective management practices. In this study, we investigated the composition of the diet of Asiatic black bears (Ursus thibetanus) and the number of bear sightings in Shirakawa Village, Gifu Prefecture, Japan, during pre- and hyperphagic periods (Aug–Dec) from 2008 to 2016. In years with low numbers of sightings, bears consumed natural foods in natural areas, such as fruits of Japanese bird cherry (Prunus grayana), dogwood (Swida controversa), oak (Quercus spp.), and beech (Fagus crenata), which constituted the main part of the bears' diet in our study area. In years with a medium number of sightings, bears consumed both natural and anthropogenic foods in or near human habitations, such as Japanese chestnut (Castanea crenata) and kaki (Diospyros kaki). In the high-sighting year of 2014, bears exhibited a particularly strong preference for Japanese walnut (Juglans mandshurica) and kaki. The number of bear sightings was negatively related to the consumption rates of Japanese bird cherry, dogwood, oak, and beech, and positively related to the consumption rates of Japanese walnut, Japanese chestnut, and kaki. These findings suggest that consumption of fleshy fruits in late summer or hard mast in autumn in or near human settlements has a large effect on the risk of bear–human interactions.

Cold and heat tolerances of hybrids for restoration of the endangered Juglans cinerea L

• Key message Hybrids had overall intermediate cold and heat tolerances compared to their progenitors, Juglans cinerea and Juglans ailantifolia . Differences in hybrids were small compared to J. cinerea though, indicating that when considering extreme temperature tolerances, hybrids might be usesd in some restoration circumstances. However, hybrids might exceed their cold tolerance in especially cold areas, such as J. cinerea’s northern range limits. • Context Hybridization could incorporate traits for surviving detrimental global changes. Juglans cinerea, an endangered North American tree species, can hybridize with non-native Juglans ailantifolia Carr. Evidence indicates their hybrids could hold resistance to the fungal disease threatening J. cinerea. Consequently, the hybrids are being evaluated for restoration use, but to be effective, they must survive in J. cinerea’s distribution. An ecophysiological evaluation could contribute to predicting potential hybrid utilization areas. • Aims To provide a relative comparison of the cold and heat tolerances among J. cinerea provenances and between J. cinerea, J. ailantifolia, and their hybrids. • Methods In the cold test, twigs were subjected to five freeze treatments and resulting damage was estimated using electrolyte leakage. In the heat test, leaflets were subjected to six hot water bath treatments and damage was estimated using chlorophyll fluorescence. • Results Within J. cinerea, trees from colder areas exhibited less cold damage than those from warmer areas. Differences in heat damage did not occur among hardiness zones. Juglans cinerea exhibited greatest cold tolerance, J. ailantifolia exhibited greatest heat tolerance, and hybrids were intermediate. • Conclusion Differences in the cold and heat tolerances of J. cinerea and its hybrids were overall minimal, although greater in cold tolerance.

Breeding research of genus Juglans representatives on the territory of EBDFC "Veseli Bokovenki"

Aim. To summarize the results on the breeding of the genus Juglans representatives within the territory of the Experimental and Breeding Dendrological Forest Center "Veseli Bokovenki" and to outline the prospects for future research. Results and Discussion. The current state, research results and prospects of using collections of breeding forms of the genus Juglans representatives at the Experimental Breeding Dendrological Forest Center (EBDFC) "Veseli Bokovenki" are analyzed. The 2016 survey showed that 7 out of 10 experimental objects derived from the genus Juglans representatives over a 120-year period have been preserved and are in a satisfactory condition within the territory of the Center. They are all in the context of the gene pool value. The Persian walnut (Juglans regia L.) breeding history within the territory of EBDFC "Veseli Bokovenki" started with the creation of the Big and Small Kutsivsky gardens by M.L.Davydov in 1900. Planned scientific research into the Persian walnut breeding started here in the 1930s. In different periods of time, under the guidance of A.P.Yermolenko, F.L.Shchepotiev and P.P.Badalov, selection of drought-resistant and winter-hardy forms, interspecies hybridization and studies of reproduction and physiology of the genus Juglans representatives were carried out. Apomictic fruits of the Japanese walnut (Juglans ailantifolia Carriere), heartnut (J. ailantifolia Carr. ssp. cordiformis Rehd), Manchurian walnut (Juglans mandshurica Maxim) and white walnut (Juglans cinerea L.) were obtained. The breeding results were summarized in chapters of the books "Nut Cultivation" and "Nuts". Recently, studies have focused on reproductive biology of breeding forms. The dichogamy type and laterality degree of the 80 best forms presented on the mother-graft plantation have been determined. The vast majority of the studied Persian walnut breeding forms are protandrous. Forms giving good yields in the harsh steppe conditions have been identified. Conclusions. All objects need preserving and wider using as a basis for breeding research. The priority objectives of further studies are phenotypic and genotypic diversity, reproductive biology and adaptability of the best forms of the Persian walnut and interspecies hybrids of the genus Juglans, inheritance of economically valuable traits, identification of more valuable forms and their involvement in subsequent breeding. The best cultivars need to be popularized, propagated vegetatively and introduced into nut industry, forestry, forest melioration and gardening.

Save Our Species: A Blueprint for Restoring Butternut (Juglans cinerea) across Eastern North America

Abstract Butternut is a relatively uncommon hardwood tree native to eastern North America. The species’ abundance has declined over the past 50 years, primarily because of an invasive pathogen (Ophiognomonia clavigignenti-juglandacearum [Oc-j]) and loss of suitable habitat for regeneration. Although genetic diversity of butternut is highest along the southern range edge, genetic diversity rangewide is fairly high, except in small and isolated populations. Although there is little evidence for even moderate resistance in native butternut, hybrids with Japanese walnut, a closely related species, display enough tolerance to infection to persist on the landscape and bear abundant nut crops year after year. Cryostorage of native embryogenic axes has yielded promising initial results as a strategy for gene conservation, but additional action is needed to conserve the remaining native gene pool. We describe a strategy for canker-resistance breeding in butternut using naturally occurring hybrids, hybrids in research orchards, and sources of native trees from as many regions as possible. Forest managers are encouraged to find surviving trees and collect seed for planting in suitable habitat to develop actionable knowledge that will enable the restoration of butternut with enough resistance to be self-sustaining on the landscape.

Hybrid Breeding for Restoration of Threatened Forest Trees: Evidence for Incorporating Disease Tolerance in Juglans cinerea.

Hybridization is a potential tool for incorporating stress tolerance in plants, particularly to pests and diseases, in support of restoration and conservation efforts. Butternut (Juglans cinerea) is a species for which hybridization has only recently begun being explored. This North American hardwood tree is threatened due to Ophiognomonia clavigignenti-juglandacearum (Ocj), the causal fungus of butternut canker disease (BCD), first observed in 1967. Observational evidence in some wild J. cinerea populations indicates that naturalized hybrids of J. cinerea with Japanese walnut (Juglans ailantifolia) may be more tolerant to BCD than non-admixed J. cinerea, but this has not been formally tested in a controlled trial. We aimed to examine potential BCD tolerance within and between J. cinerea and J. cinerea × J. ailantifolia hybrids and to determine if there is a difference in canker growth between BCD fungal isolates. Five-year-old J. cinerea and hybrid trees were inoculated with two Ocj fungal isolates collected from natural infections found in two different sites in Indiana, United States, and a blank control (agar only). Measurements of both artificially induced and naturally occurring cankers were taken at 8-, 12-, 20-, 24-, and 32-month post-inoculation. Differences in canker presence/absence and size were observed by fungal isolate, which could help explain some of the differences in BCD severity seen between J. cinerea populations. Smaller and fewer cankers and greater genetic gains were seen in hybrid families, demonstrating that hybrids warrant further evaluation as a possible breeding tool for developing BCD-resistant J. cinerea trees.

Bird droppings on chestnut leaves or sawfly larvae: DNA barcodes verify the occurrence of the archaic Megaxyela togashii (Hymenoptera, Xyelidae) in Hokkaido, Japan.

We made a molecular phylogenetic analysis using mitochondrial cytochrome oxidase subunit 1 (COI) gene sequences for ten unidentified Megaxyela larvae from Hokkaido, Honshu and Shikoku, Japan, and 15 identified adults of four Megaxyela, one Macroxyela and three Xyela species. It revealed that all larvae belonged to M. togashii Shinohara, 1992, which showed rather large intraspecific genetic variability even among the individuals from the same population. This is the first distribution record of M. togashii from Hokkaido. Megaxyela togashii is a univoltine species with a very short larval feeding period, only nine days in one rearing experiment from egg to larval maturation. The larva is a solitary, external leaf-feeder on Juglans ailanthifolia, resting curled around the central leaf vein at the apex of a leaflet, and may resemble the excrement of birds. The prepupa overwinters in an earthen cell whose wall is made only of soil, neither parchment-like nor containing fiber. The mature larva is described and several life traits are discussed.

Vegetative and Adaptive Traits Predict Different Outcomes for Restoration Using Hybrids.

Hybridization has been implicated as a driver of speciation, extinction, and invasiveness, but can also provide resistant breeding stock following epidemics. However, evaluating the appropriateness of hybrids for use in restoration programs is difficult. Past the F1 generation, the proportion of a progenitor’s genome can vary widely, as can the combinations of parental genomes. Detailed genetic analysis can reveal this information, but cannot expose phenotypic alterations due to heterosis, transgressive traits, or changes in metabolism or development. In addition, because evolution is often driven by extreme individuals, decisions based on phenotypic averages of hybrid classes may have unintended results. We demonstrate a strategy to evaluate hybrids for use in restoration by visualizing hybrid phenotypes across selected groups of traits relative to both progenitor species. Specifically, we used discriminant analysis to differentiate among butternut (Juglans cinerea L.), black walnut (J. nigra L.), and Japanese walnut (J. ailantifolia Carr. var. cordiformis) using vegetative characters and then with functional adaptive traits associated with seedling performance. When projected onto the progenitor trait space, naturally occurring hybrids (J. × bixbyi Rehd.) between butternut and Japanese walnut showed introgression toward Japanese walnut at vegetative characters but exhibited a hybrid swarm at functional traits. Both results indicate that hybrids have morphological and ecological phenotypes that distinguish them from butternut, demonstrating a lack of ecological equivalency that should not be carried into restoration breeding efforts. Despite these discrepancies, some hybrids were projected into the space occupied by butternut seedlings’ 95% confidence ellipse, signifying that some hybrids were similar at the measured traits. Determining how to consistently identify these individuals is imperative for future breeding and species restoration efforts involving hybrids. Discriminant analysis provides a useful technique to visualize past selection mechanisms and current variation in hybrid populations, especially when key ecological traits that distinguish progenitors are unknown. Furthermore, discriminant analysis affords a tool to assess ecological equivalency of hybrid populations and breeding program efforts to select for certain traits and monitor the amount of variability of those traits, relative to progenitors.

航空機LiDARの反射強度を用いた単位照射領域における樹種別の樹冠葉面占有率把握に関する検討

On this study, it was discuss about possibility of extraction of the leaf area density difference between tree species, such as Japanese walnut, White willow and Robinia pseudoacasia, from LiDAR intensity information. It was calculated canopy leaf area ratio in order to grasp the tree species difference. The canopy leaf area ratio was calculated by effective irradiation area versus unit irradiation area using a reflectance intensity of the airborne LiDAR. The canopy leaf area ratio was a difference in leaf area of a single leaf. Possibility of tree species identification was suggested by difference of the canopy leaf area ratio.

Phylogeographic breaks within Asian butternuts indicate the existence of a phytogeographic divide in East Asia.

East Asia has been hypothesized to be subdivided into two distinct northern and southern areas, separated by a band of dry climate that was far more severe in the early Tertiary but still exists today. However, this biogeographic hypothesis has rarely been tested using a molecular phylogeographic approach. We genotyped 70 populations throughout the distributional range of Asian butternuts (Juglans section Cardiocaryon) using eight chloroplast DNA regions, one single-copy nuclear gene, and 17 nuclear microsatellite loci, supplemented with paleodistribution modeling of the major genetic clades. The genetic data consistently identified two clades, one northern, comprising Juglans mandshurica and Juglans ailantifolia, and one southern, comprising Juglans cathayensis. The two clades diverged through climate-induced vicariance of an ancestral northern range during the mid-Miocene and remained mostly separate thereafter, with geographical isolation of the Japanese Islands and refugial isolation or secondary contacts in the late Pleistocene producing further subdivision within the northern clade. But beyond all that, we also discovered a role of environmental adaptation in maintaining and/or reinforcing the north-south divergence. Asian butternuts offer a strong case for the existence of a biogeographic divide between the northern and southern parts of East Asia during the Neogene and into the Pleistocene.

Luther Burbank’s Contributions to Walnuts

Luther Burbank began making controlled crosses between walnut species in the late 19th century after hearing about a “supposed natural European hybrid walnut.” He crossed Juglans hindsii (northern California black walnut) × J. regia (Persian walnut) and produced progeny that he named ‘Paradox’ because of its extremely fast growth and other “anomalies.” He also crossed two American species, J. hindsii × J. nigra (eastern black walnut), producing ‘Royal’ walnut progeny that were fast-growing and prolific nut producers. A third interspecific hybrid was a cross between J. ailantifolia (Japanese walnut) × J. regia that resulted in extremely vigorous progeny but was not named. He observed segregation in the F2 populations and described giants and dwarfs as reversions to ancestral forms. Luther Burbank also made selections for walnut scion cultivars and was especially interested in thin-shelled nuts. He collected seeds from a J. regia growing in San Francisco because it produced regularly and had very high-quality nuts with relatively thin but poorly sealed shells. He selected one of its seedlings as ‘Santa Rosa Soft-Shell’ and described it as bearing large crops of nuts that were nearly white with thin shells and delicious white meat. Burbank’s contributions to the walnut industry endure to this day, especially through the widespread use of seedling and clonal ‘Paradox’ walnut rootstocks.

Butternut (Juglans cinerea) health, hybridization, and recruitment in the northeastern United States

Butternut (Juglans cinerea L.) trees are being extirpated from their natural range by an epidemic caused by a fungal pathogen. Widespread mortality is reminiscent of past epidemics on American chestnut (Castanea dentata (Marsh.) Borkh.) and American elm (Ulmus americana L.). Butternut has remained relatively understudied, resulting in unsampled areas and gaps in our understanding of this forest epidemic and the future outlook of this species in North America. The previously unsampled area consisting of the northeastern United States was surveyed for the presence of J. cinerea, and several population health metrics were recorded, including recruitment, disease pressure, and hybridization. A total of 252 butternut trees were sampled. Analysis indicates that there is insufficient J. cinerea recruitment to maintain population sizes. Further compounding low recruitment, butternut saplings demonstrate elevated levels of disease impact from the fungal pathogen Ophiognomonia clavigignenti-juglandacearum Broders &amp; Boland. Natural hybridization of butternut with introduced congenics such as Juglans ailantifolia Carrière is strongly associated with lower disease impact. Hybrid trees displayed an average of 2.4 cankers per tree compared with 4.5 cankers for nonhybrid butternut. Further niche and resistance studies are required to assess whether butternut hybrids can replace butternut in a natural setting. It still remains uncertain whether tree size or habitat affect disease impact; however, smaller trees, often residing in riparian habitats, were found to have a greater number of cankers. The data presented here, combined with past studies, provide critical information for use in butternut management strategy plans.

Drought and flood stress tolerance of butternut (Juglans cinerea) and naturally occurring hybrids: implications for restoration

Efforts to restore species threatened by introduced pathogens often include breeding resistance into susceptible native species from resistant exotics, but this is a lengthy and expensive process. Naturally occurring hybrids between susceptible and resistant species may accelerate this process if they fulfill the desired species’ ecological function. To explore this possibility, we exposed the following seedlings to drought and flood treatments in a controlled environment: Juglans cinerea L. (butternut), which has been devastated by an exotic pathogen; Juglans ailantifolia Carr. var. cordiformis (heartnut); Juglans × bixbyi Rehd. [ailanthifolia × cinerea] (buartnut), multigenerational hybrids between butternut and heartnut; and Juglans nigra L. (black walnut). There was a strong taxa × treatment interaction. Butternut had a negative response to flood treatments, expressed by reduced leaf area (LA), photosynthetic assimilation (A), and chlorophyll fluorescence (Fv/Fm). Heartnut had a negative response to drought, expressed by lower LA and A. Hybrid A and LA were reduced in response to both treatments. Results indicate that hybrid drought and flood tolerance may limit their overall ability to completely occupy the ecological niche formerly filled by butternut. However, the strong dichotomous response of progenitors to moisture stress suggests that hybrids could be efficiently screened for J. cinerea character in conjunction with disease resistance.

Biotic and abiotic factors affecting the genetic structure and diversity of butternut in the southern Appalachian Mountains, USA

The abundance of butternut (Juglans cinerea L.) trees has severely declined rangewide over the past 50 years. An important factor in the decline is butternut canker, a disease caused by the fungus Ophiognomonia clavigigenti-juglandacearum, which has left the remaining butternuts isolated and sparsely distributed. To manage the remaining populations effectively, information regarding how butternut’s population genetic structure is affected by environmental and historical factors is needed. In this study, we assessed genetic structure and diversity of 161 butternut trees from 19 adjacent watersheds in the southern portion of butternut’s range using 12 microsatellite markers. We assessed the genetic diversity and genetic differentiation among trees grouped at various spatial scales. Our goal was to use historical abundance and land use data for these watersheds, which are now all a part of the Great Smoky Mountains National Park (GSMNP), to understand the ecological and evolutionary forces that challenge the conservation and management of butternut. In general, butternuts within the 19 neighboring watersheds were all part of one continuous population, with gene flow throughout. Significant genetic differentiation was detected between some groups of trees, but the differentiation was quite small and may not represent an ecologically significant distinction. The mean heterozygosity in all watersheds remained high, despite extensive mortality. Overall, genetic diversity and rare alleles were evenly distributed across all watersheds, with some variability in subpopulations containing butternut-Japanese walnut hybrids (Juglans x bixbyi or buarts). These results indicate that management of this species should focus on protection from future hybridization with Japanese walnut, promotion of regeneration, and persistence of all remaining butternut trees, which still retain high levels of genetic diversity.

RAPD derived markers for separating Manchurian walnut (Juglans mandshurica) and Japanese walnut (J. ailantifolia) from close congeners

AbstractRandom amplified polymorphic DNA (RAPD) was used as a molecular tool to evaluate genomes of butternut (Juglans cinerea), Japanese walnut (Juglans ailantifolia), black walnut (Juglans nigra), Persian walnut (Juglans regia), Manchurian walnut (Juglans mandshurica), and an interspecific hybrid (J. ailantifolia × J. cinerea) for species‐specific markers. Forty decamer RAPD primers generated 668 RAPD amplicons that were used to characterize bulked DNA pools of butternut, Japanese walnut Persian walnut, Manchurian walnut, and buartnut hybrids and to identify genomic regions found only in Japanese walnut or Manchurian walnut. The amplicons ranged in size from 150 to 2068 bp; sixty‐five were polymorphic (9.7%) among the six taxa evaluated. Thirty‐eight amplicons were unique to Japanese walnut and buartnut hybrids but absent in butternut. The RAPD markers we used were not sufficiently polymorphic to characterize intraspecific variability within Japanese walnut, butternut, or their hybrids. However, they can be used to identify hybrids based on the presence of introgressed genomic fragments inherited from Japanese walnut. They may also be useful for distinguishing whether Japanese walnut or Manchurian walnut was the parent of a hybrid lineage. The results confirmed that some hybrids can be distinguished by their morphology, and genetic similarity based on RAPDs appears to reflect known pedigree information. These RAPD markers may prove useful for conservation of butternut, an endangered North American species. These results also may prove useful for the molecular identification of species and species hybrids within Juglans.