Genetic architecture of resistance to aphids and mites in a willow hybrid system

Hybrid plants often differ in resistance to arthropods compared to the parental species from which they are derived. To better understand the relative contribution of genetic effects in influencing plant resistance to arthropods, we examined the genetic architecture of resistance in a willow hybrid system, Salix eriocephala, S. sericea, and their interspecific hybrids. Resistance to two arthropods, a willow leaf aphid (Chaitophorus sp.: Aphididae) and an eriophyoid mite (Aculops tetanothrix: Eriophyidae), were compared because resistance to different herbivores may be controlled by different traits and influenced by different genetic effects. We found additive and nonadditive genetic effects to be important in explaining the difference between willow species in resistance to aphids and mites. F2 hybrids exhibited low resistance to aphids, suggesting breakdown of favourable epistatic interactions that confer resistance. F2 hybrids, however, exhibited high resistance to mites, suggesting either the breakdown of interactions that affect traits used by mites in host location or the creation of favourable epistatic interactions. This study demonstrates the potential role of herbivores in affecting plant genetic structure, such that selection by herbivores can potentially lead to the creation of gene interactions that influence host resistance traits or host recognition traits used by the herbivore.     

Fitness estimation through performance comparison of F1 hybrids with their parental species Oryza rufipogon and O. sativa

BACKGROUND AND AIMS: Introgression of crop genes into populations of wild relatives has important implications for germplasm conservation as well as for the persistence of novel transgenes in wild populations. Studies of hybrid fitness can be used to evaluate the potential for introgression to occur following episodes of interspecific hybridization. METHODS: This study estimated relative fitness of interspecific hybrids through performance comparison of F(1) hybrids with their parental species, a cultivated rice (Oryza sativa) Minghui-63 and perennial common wild rice (O. rufipogon) under the cultivation conditions. KEY RESULTS: Compared with their parents, the hybrids had the lowest values of seedling survival ability, pollen viability and seed production; intermediate values of seed germination, spikelet production and flag leaf areas; and the highest values of plant height, number of tillers and panicles. The hybrids performed poorly at the stage of sexual reproduction, although they had a slightly higher hybrid vigour at the vegetative growth stage and better tillering ability than their wild parent. There were no significant differences in composite fitness across the whole life-history between the hybrids and their wild parental species. CONCLUSIONS: Rice genes, including transgenes, might persist in wild rice populations through vegetative and sexual reproduction. Further studies are needed to examine whether the extent of gene flow from rice is sufficiently significant to influence genetic diversity in wild populations of O. rufipogon, a species that has become endangered in some regions of south-east Asia.     

Maternal and paternal contributions to the fitness of hybrids between red and white mulberry (Morus, Moraceae)

The fitness of hybrids depends on the genetic disparity between parental taxa and the magnitude of their nuclear and non-nuclear contributions. To estimate the role of non-nuclear effects, we crossed red (R), white (W) and hybrid (H) mulberry in all combinations and compared the magnitude of maternal and paternal effects on offspring fitness (seed set, germination, survival and aboveground biomass) in a greenhouse environment. Variation in offspring fitness was determined largely by the identity of the maternal parent; specifically, progeny with white mothers had the highest cumulative fitness. As fathers, red, white, and hybrid mulberry had no effect on fitness, and maternal × paternal interactions were significant only for survival. Individual cross-types differed significantly for all fitness components except seed set. Offspring from hybrid crosses (W × R, H × R, H × W) often differed from at least one of the within-parent crosses (W × W, R × R) as well as from other hybrid crosses, although their fitness values never exceeded the most fit parent. Reciprocal crosses differed in only two of 15 possible parental combinations: W × H (cumulative fitness) and W × R (aboveground biomass). Overall, the strong asymmetry in magnitude of maternal and paternal effects suggests that fitness of hybrid mulberry is governed largely by non-nuclear, parental effects.     

Population genetic structure of native versus naturalized sympatric shrub willows (Salix; Salicaceae)

Vegetative propagation of an introduced species can contribute significantly to its ability to spread and become naturalized, potentially in competition with native species. This study focused on the naturalization of a willow shrub, Salix purpurea, which was introduced to the United States from Europe and is commonly sympatric with the native shrub willow, S. eriocephala. Both species are capable of vegetative and sexual reproduction, but little is known about their relative frequency, nor the impact of clonal propagation on population-level genetic diversity. We analyzed genotypes at several microsatellite loci in 993 individuals belonging to 30 subpopulations of S. eriocephala and 28 subpopulations of S. purpurea in areas of sympatry across three watersheds to compare their genetic diversity and genetic structure. Our results revealed six subpopulations of S. purpurea containing plants with identical multilocus genotypes, while clonal individuals were rare among S. eriocephala populations. These species are dioecious with relatively high levels of heterozygosity, but S. eriocephala had much higher allelic diversity and genotypic diversity than did S. purpurea. These results strongly suggest that vegetative propagation has contributed to the naturalization of S. purpurea and has resulted in higher levels of genetic differentiation among S. purpurea populations than among native S. eriocephala populations.     

Ecologically dependent and intrinsic genetic signatures of postzygotic isolation between sympatric host races of the leaf beetle Lochmaea capreae

The fitness of hybrids might be compromised as a result of intrinsic isolation and/or because they fall between ecological niches due to their intermediate phenotypes (“extrinsic isolation”). Here, we present data from several crosses (parental crosses, F1, F2, and backcrosses) between the two host races of Lochmaea capreae on willow and birch to test for extrinsic isolation, intrinsic isolation, and environmentally dependent genetic incompatibilities. We employed a reciprocal transplant design in which offspring were raised on either host plant and their survival was recorded until adulthood. We also applied joint‐scaling analysis to determine the genetic architecture of hybrid inviability. The relative fitness of the backcrosses switched between environments; furthermore, the additive genetic–environment interaction was detected as the strongest effect in our analysis. These results provide strong evidence that divergent natural selection has played a central role in the evolution of hybrid dysfunction between host races. Joint‐scaling analysis detected significant negative epistatic effects that are most evident in the poor performance of F2‐hybrids on willow, indicating signs of intrinsic isolation. We did not find any evidence that genetic incompatibilities are manifested independently of environmental conditions. Our findings suggest the outcome of natural hybridization between these host races is mainly affected by extrinsic isolation and a weak contribution of intrinsic isolation.     

Lifetime fitness in two generations of Ipomopsis hybrids

Various models purporting to explain natural hybrid zones make different assumptions about the fitness of hybrids. One class of models assumes that hybrids have intrinsically low fitness due to genetic incompatibilities, whereas other models allow hybrid fitness to vary across natural environments. We used the intrinsic rate of increase to assess lifetime fitness of hybrids between two species of montane plants Ipomopsis aggregata and Ipomopsis tenuituba planted as seed into multiple field environments. Because fitness is predicted to depend upon genetic composition of the hybrids, we included F1 hybrids, F2 hybrids, and backcrosses in our field tests. The F2 hybrids had female fitness as high, or higher, than expected under an additive model of fitness. These results run counter to any model of hybrid zone dynamics that relies solely on intrinsic nuclear genetic incompatibilities. Instead, we found that selection was environmentally dependent. In this hybrid zone, cytoplasmic effects and genotype-by-environment interactions appear more important in lowering hybrid fitness than do intrinsic genomic incompatibilities between nuclear genes.     

Nine-year reciprocal transplant experiment in the gardens of the basin and mountain big sagebrush (Artemisia tridentata: Asteraceae) hybrid zone of Salt Creek Canyon: the importance of multiple-year tracking of f itness

We have studied reciprocal transplant gardens involving the hybrid zone between basin and mountain big sagebrush ( Artemisia tridentata ) in Salt Creek Canyon, Utah, for 9 years. Previously, we showed that the parental taxa and hybrids had superior reproductive and vegetative performance in their native garden. These earlier data supported the Bounded Hybrid Superiority model. Now, after 9 years, we find that the mountain seed source plants have greater relative fitness than middle hybrid zone seed source plants in the middle hybrid zone garden. These results may be due to plant density and climatic factors more conducive to mountain seed source growth than that of either basin or middle hybrid zone seed source plants. On the other hand, these fitness estimates do not take into account the timing of reproduction, which together with the age-specific survival rate, can profoundly affect lifetime fitness. The intrinsic rate of increase ( r ) takes both of these factors into account, providing another estimate of fitness. Middle hybrid zone seed source plants had the greatest rate of increase in both the middle hybrid zone and mountain gardens and a greater rate of increase than either parent in the basin garden. This is most likely due to the greater reproductive performance of middle hybrid zone plants earlier in life than either parental taxon. These results partly support the Bounded Hybrid Superiority model and show the importance of long- term studies of hybrid fitness.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 86 , 213–225.     

Fitness of natural willow hybrids in a pioneer mosaic hybrid zone

Hybrid fitness is an important parameter to predict the evolutionary consequences of a hybridization event and to characterize hybrid zones. We studied fitness parameters of F₁ and later‐generation hybrids between the lowland species Salix purpurea and the alpine S. helvetica that have recently emerged during colonization of an alpine glacier forefield. Fruit production (number of capsules per catkin and fruit set) did not differ between hybrids and parents, but the number of seeds per capsule of F₁ hybrids was slightly lower than that of later‐generation hybrids and of the parents. Germination rates and seedling growth were tested on three substrates (pH 4.5, 7.0, and 8.0). Germination rates of seeds collected from F₁ hybrids were lower on acid and neutral substrates, but equal at pH 8.0 compared to all other groups, while the seeds from later‐generation hybrids performed as well as the parents on all three substrates. In seedling growth, the colonizer S. purpurea performed better than all other taxa on all three substrates, while hybrids resembled the subalpine species S. helvetica. Results suggest that endogenous selection acts against F₁ hybrids, but favors fitter genotypes in later‐generation hybrids. Exogenous selection via soil pH appears to be weak during seedling establishment. The pioneer vegetation on the glacier forefield may offer sufficient niche space for hybrid seedlings. Owing to the relatively high fitness of the hybrids and the scattered distribution of hybrids and parental individuals on the glacier forefield, this hybrid zone can be assigned to a mosaic model, probably facilitating gene flow and introgression between the parental species. As establishment of the hybrid zone appears to be linked to a colonization process, we propose to call it a pioneer mosaic hybrid zone.     

Seasonal abundance of Tuberolachnus salignus and its effect on flowering of host willows of varying susceptibility

The giant willow aphid Tuberolachnus salignus Gmelin is a large phloem-feeding insect which colonizes the stems of willow trees. This aphid is a new invasive species in New Zealand and there is limited knowledge of its inter-annual population patterns and the damage it can cause to willow hosts. Our study investigated the T. salignus population dynamics and its effects on the flowering parameters of fifteen willow species and hybrids in a field trial. The aphid population levels were found to vary among the willow species and hybrids. Based on the aphid population levels, the willow species and hybrids were classified as resistant, moderately resistant, susceptible or highly susceptible. T. salignus infestation had no effect on the flowering of resistant and moderately resistant willows, but significantly delayed the flowering time, extended the flowering duration, and decreased the catkin length in susceptible species and hybrids. Interestingly, aphid infestation was found to increase the catkin number and total floral output of some willow species and hybrids. It can be concluded that aphid populations and their effects on flowering are host-specific, with large differences between resistant and susceptible host plants.     

Genotype-by-environment interaction and the fitness of plant hybrids in the wild

Natural hybrid zones between related species illustrate processes that contribute to genetic differentiation and species formation. A common viewpoint is that hybrids are essentially unfit, but they exist in a stable tension zone where selection against them is balanced by gene flow between the parent species. An alternative idea is that selection depends on the environment, for example, by favoring opposite traits in the two parental habitats or favoring hybrids within a bounded region. To determine whether selection of hybrids is environment dependent, we crossed plants of naturally hybridizing Ipomopsis aggregata and I. tenuituba in the Colorado Rocky Mountains and reciprocally planted the seed offspring into a suite of natural environments across the hybrid zone. All types of crosses produced similar numbers and weights of seeds. However, survival of the offspring after 5 years differed markedly among cross types. On average, the F1 hybrids had survival and growth rates as high as the average for their parents. But hybrid survival depended strongly on the direction of a cross, that is, on which species served as the maternal parent. This fitness difference between reciprocal hybrids appeared only in the parental environments, suggesting cytonuclear gene interactions that are environment specific. These results indicate that complex genotype-by-environment interactions can contribute to the evolutionary outcome of hybridization.     

Do exotic generalist predators alter host plant preference of a native willow beetle?

1 Selection can favour herbivores that choose host plants benefitting their offspring either by enhancing growth rates or by increasing larval defences against native predators. For exotic predator species that feed on herbivores, their success with invading new habitats may depend upon overcoming defences used by native prey. Whether exotic predators can alter herbivore host choice has remained unexamined. Therefore, we compared the efficacy of larval defence by Chrysomela knabi (a native beetle species) that had fed on two native willow hosts: Salix sericea (a phenolic glycoside (PG)-rich species) and Salix eriocephala (a PG-poor species), when attacked by exotic generalist predators. In addition, the preference and performance of C. knabi on S. sericea and S. eriocephala was examined.
2  Chrysomela knabi preferred and performed better on S. sericea. In a common garden, adult C. knabi were nine-fold more common and oviposited five-fold more frequently on S. sericea than on S. eriocephala . In the laboratory, adult feeding preference on leaf discs and survival rates of larvae were both greater on S. sericea , and time to pupation was shorter.
3  Chrysomela knabi larvae produced significantly more salicylaldehyde when fed S. sericea leaves than when fed S. eriocephala leaves. Additionally, those larvae with greater salicylaldehyde had reduced predation by two exotic generalist predators, Harmonia axyridis larvae and juvenile Tenodera aridifolia sinensis .
4 The results obtained in the present study suggest that selection favoured the preference of C. knabi for PG-rich willow plants because larvae grew and survived better and that selection by common exotic generalist predators would reinforce this preference.     

Cuticular wax composition of Salix varieties in relation to biomass productivity

The leaf cuticular waxes of six Salix clones (one Salix miyabeana, one Salix dasyclados, one Salix eriocephala, two Salix purpurea, and one interspecific hybrid of Salix eriocephala x interior) with different biomass productivities were characterized by gas chromatography-mass spectrometry. Total wax content ranged from 6.3 to 16.8 microg cm(-2), and two distinct patterns of wax were measured. The wax from leaves of S. dasyclados 'SV1' differed from all other clones and was dominated by fatty acids (42%), high concentrations of n-alkanes (25%) and n-alcohols (28%), with low n-aldehyde content (4%). All other clones produced cuticular wax dominated by n-alcohols (32-51%), particularly 1-hexacosanol, with fatty acids (14-37%) and n-aldehydes (19-26%) present in lower abundances. Clones of Salix grown under identical environmental conditions produce noticeably different amounts of cuticular wax. In contrast to previous studies of Salix, total wax content was independent of biomass productivity, measured as basal area, suggesting that wax production is not directly linked with woody biomass production by shrub willows under these site conditions.     

The strength of reproductive isolation in two hybridizing food-deceptive orchid species

Reproductive isolation is of fundamental importance for maintaining species boundaries in sympatry. In orchids, the wide variety of pollination systems and highly diverse floral traits have traditionally suggested a prominent role for pollinator isolation, and thus for prezygotic isolation, as an effective barrier to gene flow among species. Here, we examined the nature of reproductive isolation between Anacamptis morio and Anacamptis papilionacea, two sister species of Mediterranean food-deceptive orchids, in two natural hybrid zones. Comparative analyses of the two hybrid zones that are located on soils with volcanic origin and have different and well-dated ages consistently revealed that all hybrid individuals were morphologically and genetically intermediate between the parental species, but had strongly reduced fitness. Molecular analyses based on nuclear ITS1 and (amplified fragment length polymorphism) AFLP markers clearly showed that all examined hybrids were F1 hybrids, and that no introgression occurred between parental species. The maternally inherited plastid DNA markers indicated that hybridization between A. morio and A. papilionacea was bidirectional, as confirmed by the molecular analysis of seed families. The genetic architecture of the two hybrid zones suggests that the two parental species easily and frequently hybridize in sympatry as a consequence of partial pollinator overlap but that strong postzygotic barriers reduce hybrid fitness and prevent gene introgression. These results corroborate that chromosomal divergence is instrumental for reproductive isolation between these food-deceptive orchids and suggest that hybridization is of limited importance for their diversification.     

Vole preference for Salix caprea, S. repens, and their F1, F2, and backcross hybrids

In recent years, the influence of plant hybridization on plant–animal interactions has become an important issue; however, these studies have mainly focused on insects or slugs and to a large degree ignored mammalian herbivores despite their importance in many ecosystems. This study aims to determine the preference of voles for pure Salix caprea , S. repens , and their F1, F2, and backcross hybrids to evaluate whether voles select against hybrids in natural willow populations. To address this, we conducted two field studies and a cafeteria experiment in the laboratory with bank voles ( Clethrionomys glareolus ). We found no strong indications of reduced resistance in any of the hybrid taxa. Voles damaged more S. repens than S. caprea individuals. There was a general decreasing trend of utilization from pure S. repens to pure S. caprea , hybrid taxa being intermediate between the parents. Thus, voles seemingly do not have a more detrimental effect on hybrid fitness than on the fitness of pure individuals and probably will not select against hybrids in these willows populations.     

Inbreeding and outbreeding depression in Stylidium hispidum: implications for mixing seed sources for ecological restoration

The benefits of composite rather than local seed provenances for ecological restoration have recently been argued, largely on the basis of maximizing evolutionary potential. However, these arguments have downplayed the potentially negative consequences of outbreeding depression once mixed provenances interbreed. In this study, we compared intraspecific F1 hybrid performance and molecular marker differentiation among four populations of Stylidium hispidum, a species endemic to Southwestern Australia. Multivariate ordination of 134 AFLP markers analyzed genetic structure and detected two clusters of paired sites that diverged significantly for marker variation along a latitudinal boundary. To test for outbreeding depression and to determine the consequences of molecular population divergence for hybrid fitness, we conducted controlled pollinations and studied germination and survival for three cross categories (within‐population crosses, short‐ and long‐distance F1 hybrids) for paired sites distributed within and between the two genetically differentiated regions. We found evidence of outbreeding depression in long‐distance hybrids (111–124 km), and inbreeding depression among progeny of within‐population crosses, relative to short‐distance (3–10 km) hybrids, suggesting an intermediate optimal outcrossing distance in this species. These results are discussed in light of the evolutionary consequences of mixing seed sources for biodiversity restoration.     

Genotype, soil type, and locale effects on reciprocal transplant vigor, endophyte growth, and microbial functional diversity of a narrow sagebrush hybrid zone in Salt Creek Canyon, Utah

When addressing the nature of ecological adaptation and environmental factors limiting population ranges and contributing to speciation, it is important to consider not only the plant's genotype and its response to the environment, but also any close interactions that it has with other organisms, specifically, symbiotic microorganisms. To investigate this, soils and seedlings were reciprocally transplanted into common gardens of the big sagebrush hybrid zone in Salt Creek Canyon, Utah, to determine location and edaphic effects on the fitness of parental and hybrid plants. Endophytic symbionts and functional microbial diversity of indigenous and transplanted soils and sagebrush plants were also examined. Strong selection occurred against the parental genotypes in the middle hybrid zone garden in middle hybrid zone soil; F(1) hybrids had the highest fitness under these conditions. Neither of the parental genotypes had superior fitness in their indigenous soils and habitats; rather F(1) hybrids with the nonindigenous maternal parent were superiorly fit. Significant garden-by-soil type interactions indicate adaptation of both plant and soil microorganisms to their indigenous soils and habitats, most notably in the middle hybrid zone garden in middle hybrid zone soil. Contrasting performances of F(1) hybrids suggest asymmetrical gene flow with mountain, rather than basin, big sagebrush acting as the maternal parent. We showed that the microbial community impacted the performance of parental and hybrid plants in different soils, likely limiting the ranges of the different genotypes.     

Protein storage and root:shoot reallocation provide tolerance to damage in a hybrid willow system

To determine the mechanistic basis of tolerance, we evaluated six candidate traits for tolerance to damage using F₂ interspecific hybrids in a willow hybrid system. A distinction was made between reproductive tolerance and biomass tolerance; reproductive tolerance was designated as a plant’s proportional change in catkin production following damage, while biomass tolerance referred to a plant’s proportional change in biomass (i.e., regrowth) following damage. F₂ hybrids were generated to increase variation and independence among candidate traits. Using three clonally identical individuals, pre-damage candidate traits for tolerance to damage (root:shoot ratio, total nonstructural carbohydrate, and total available protein) and post-damage candidate traits (relative root:shoot ratio, phenolic ratio, and specific leaf area ratio) were measured. The range of variation for these six candidate traits was broad. Biomass was significantly increased two years after 50% shoot length removal, and catkin production was not significantly reduced when damaged, suggesting that F₂ hybrids had great biomass tolerance and reproductive tolerance. Based on multiple regression methods, increased reproductive tolerance was associated with increased protein storage and decreased relative root:shoot ratio (reduced root allocation after damage). In addition, a positive relationship between biomass tolerance and condensed tannins was detected, and both traits were associated with increased reproductive tolerance. These four factors explained 57% of the variance in the reproductive tolerance of F₂ hybrids, but biomass tolerance explained the majority of the variance in reproductive tolerance. Changes in plant architecture in response to plant damage may be the underlying mechanism that explains biomass tolerance.     

Genetic variation and self-incompatibility within and outside a Rorippa hybrid zone (Brassicaceae)

Rorippa amphibia and R. sylvestris are both self-incompatible, perennial species. In northern Germany they show a geographic pattern of interspecific gene flow: R. amphibia and R. sylvestris form a hybrid zone at the river Elbe but do not hybridise elsewhere in northern Germany. In the present paper we analyse the relationship between molecular diversity, seed set and seed germination within and outside the hybrid zone. In both species we observed high genetic variation and high seed set in the hybrid zone, and low genetic variation and low seed set outside the hybrid zone. In R. amphibia, high genetic variation is correlated with high seed set, but with low germination rates of the seeds. The results of an isolation experiment show that self-incompatibility is maintained in the parental species and their hybrids. The impact of genetic self-incompatibility on hybrid zone formation and hybrid fitness is discussed.     

No effect of transgene and strong wild parent effects on seed dormancy in crop–wild hybrids of rice: implications for transgene persistence in wild populations

Soil seed banks act as a gene pool for local plant species and, as such, can buffer local populations, especially those experiencing challenging environmental conditions. Seed dormancy has important implications to dynamics of soil seed banks. Therefore, estimating the seed dormancy of transgenic crop–wild hybrids could shed light on the persistence of transgenes in wild‐plant soil seed banks. Individuals from eight populations of wild rice Oryza rufipogon were crossed with those of three insect‐resistant transgenic rice lines. Selfed (F2–F4) and backcrossed populations (BC1, BC1F2 and BC1F3) were then made from the hybrids. Seed germination was tested under three treatments: (a) normal; (b) overwintering in soil; and (c) one‐week heat‐shocking. The effects of transgene, wild parent and hybrid generation on hybrid seed germination were examined. No significant effect of insect‐resistant transgenes (Bt and CpTI) was detected on the seed dormancy of crop–wild hybrids, while a significant wild parent effect was found. The seeds of advanced generation hybrids have higher germination percentages and lower dormancy than do those of F1 and BC1 generations. The study showed that the dormancy of hybrid seeds was determined mainly by their genetic backgrounds. All hybrid seeds have higher germination percentages and lower dormancy (and, consequently, a poorer overwintering ability), compared with wild seeds, and reduce dormancy would contribute to a fitness disadvantage, compared with wild types. Therefore, such seeds might form part of naturally occurring soil seed banks, through which crop genes would persist in wild populations.     

Biomass Harvest from Natural Willow Rings around Prairie Wetlands

The study followed the harvest of natural willow from three wetlands using a prototype modified agricultural round baler nicknamed a Bio-Baler. The study reports fuel characteristics and combustion testing of biomass harvested from natural willow rings. Composition of native willow species in the harvested willow rings was determined. We specifically measured regrowth of the biomass and number of regenerated stems per stump 1 year after harvest to determine how different willow species responded to mechanical biomass cutting with the Bio-Baler. The results of combustion testing for the natural willow were essentially similar to those with “conventional” wood chips or planted willows. The ash content was approximately 1.65%, slightly lower than for planted willow plantations. The calorific value of the natural willow was 19.6 MJ kg^?1 (dry basis) similar to what is expected for wood and planted willow. Four Salix species (Salix bebbiana Sarg., Salix petiolaris Sm., Salix eriocephala Michx., and Salix discolor Muhl) were identified in the willow rings. Stem biomass increased for all species except S. bebbiana after willow was harvested with the Bio-Baler. Overall, willow regeneration was not affected by mechanical harvesting compared to hand cutting. Regenerated stem density was 93 stems per square meter for mechanically harvested stumps compared to 105 stems per square meter for hand-pruned stumps. Based on the results, biomass harvested from natural willow rings has acceptable fuel characteristics when compared to purpose-grown willows and mechanical harvest with a Bio-Baler does not have a negative effect on willow regeneration.