Numerous fast growing and highly competitive exotic crops are being selected for production of renewable bioenergy. Tolerance of poor growing conditions with minimal inputs are ideal characteristics for bioenergy feedstocks, but have attracted concern for their potential to become invasive. Miscanthus × giganteus is one of the most promising bioenergy crops in the US, but grower adoption is hindered by high establishment costs due to sterility. Newly developed fertile tetraploid M. × giganteus may streamline cultivation while reducing establishment costs. However, fertile seed dramatically increases the potential propagule pressure, and thus probability of off-site plant establishment. To empirically evaluate the invasive potential of fertile M. × giganteus in the Southeastern US, we compared fitness and spread potential relative to ten grass species comprising 19 accessions under both high and low levels of competition and disturbance. We chose species known to be invasive in the US (positive controls: Arundo donax, naturalized M. sinensis, M. sacchariflorus, Phalaris arundinacea, Sorghum halepense) and non-invasive (negative controls; Andropogon gerardii, ornamental M. sinensis, Panicum virgatum, Sorghum bicolor, Saccharum spp.). This novel design allows us to make relative comparisons of risk among species with varying invasiveness. After three years of establishment and growth in Blacksburg, Virginia, neither aboveground disturbance nor interspecific weed competition influenced fitness for fertile M. × giganteus or our positive and negative control groups. Fertile M. × giganteus produced 346% and 283% greater aboveground biomass than our positive and negative species, respectively. However, fertile M. × giganteus produced 74% fewer inflorescences m^-2 than our positive controls and 7% and 51% fewer spikelets inflorescence^-1 than the positive and negative control species. After 18 months of growth, we observed the vegetative and seedling spread of three of our positive control species (S. halepense, P. arundinacea, and M. sacchariflorus) outside the cultivated plot into receiving areas of both high and low competition. After 24 months of growth, numerous species were observed outside the cultivated plot including fertile M. × giganteus and 50% of negative control species. Notably, in three years sterile M. × giganteus ‘Illinois’ and Arundo donax never moved from the cultivated plot. The addition of fertile seed appears to increase the potential for offsite movement, but within the geographic confines of our empirical evaluation, fertile M. × giganteus seedlings are more similar to native P. virgatum and were not nearly as fast growing or as competitive as our positive control S. halepense. The use of numerous species providing relative comparisons allow us to draw important conclusions which may help prepare for widespread commercialization, while providing novel methodology for ecological risk assessment of new species.

Biofuel, giant miscanthus, habitat susceptibility, invasibility

There is a global push towards renewable biomass based energy (Yauan et al. 2008), and large statured perennial grasses hold the most promise as dedicated energy crops. Candidate feedstocks are ideal because of their perennial growth habit, rapid growth and high annual biomass production, low management and input requirements following establishment, and relatively low pest pressure. However, it is this desirable set of agronomic characteristics which has been the major source of concern for their potential to contribute to the invasiveness of numerous bioenergy crops (Lewandowski et al. 2003; Raghu et al. 2006). Barney and DiTomaso (2010) diagram the “thin green line” between many agronomic weeds, introduced and even subsidized in some cases, for purposes such as forage or erosion control, and the relatively benign crops vitally important to our economy and food supply. However, identifying which side of this line new crops fall is challenging at best.

Spatial demographic models (Matlaga and Davis 2013) and weed risk assessments (Barney and DiTomaso 2008; Davis et al. 2010; Gordon et al. 2011) do offer predictions regarding the ability of novel species to establish and spread. However, in reality, empirical data from in situ field trials, supporting conclusions of invasiveness or long-term sustainability, do not exist. Several candidate bioenergy crops have a history of invasiveness, which is a robust predictor of future invasive potential (Dawson et al. 2009; Gordon et al. 2008). For example, Arundo donax L. is a documented noxious weed of riparian habitats in the southeastern United States (Bell 1997; Katibah 1984). Despite this label, no peer-reviewed data exists evaluating the ability of A. donax to spread from a cultivated field. Two miscanthus species in particular have been widely grown in the United States for horticultural use: Miscanthus sinensis Andersson is listed as potentially invasive, but not prohibited in Connecticut (Council 2013), and is known to form extensive infestations after spreading from older or abandoned ornamental plantings (Dougherty et al. 2014; Miller 2003); and M. sacchariflorus (Maxim.) Franch is on the Massachusetts prohibited plant list (Resources 2014), and has repeatedly escaped from cultivation, particularly in the Midwest (Bonin et al. 2014). However, at this time only limited studies have begun to examine the invasive potential of bioenergy crops in the context of a managed agricultural cropping system (see Barney et al. 2012; Matlaga et al. 2012b; Quinn et al. 2011).

Crop breeding and improvement will be imperative to improve quality, increase yield, and reduce pest pressure (Gressel 2008). The sterile triploid ‘Illinois’ variety of Miscanthus × giganteus J.M. Greef & Deuter ex Hodkinson & Renvoize has emerged as one of the most promising bioenergy crops in the US and Europe, but planting is expensive and requires specialized equipment (Lewandowski et al. 2003). Despite the explicit inclusion of M. × giganteus in the Massachusetts prohibited plant list as a progeny of M. sacchariflorus (Resources 2014), qualitative weed risk assessments have suggested that the sterile cultivar is of low risk for invasiveness (Barney and DiTomaso 2008). Newly developed fertile lines of tetraploid M. × giganteus may streamline cultivation by reducing labor and establishment costs (Sacks et al. 2013); but the addition of fertile seed has the potential to dramatically increase propagule pressure to surrounding habitats, and must be evaluated for its influence on invasiveness.

Despite the vigilant approach with bioenergy crops in regards to invasiveness, the majority of introduced species have neutral ecological consequences and many provide a direct benefit to society (Barney and DiTomaso 2010; Davis 2003). However, the intentional cultivation and transport of exotic bioenergy crops over a vast geographic range would bypass the early environmental filters of introduction and colonization (Barney et al. 2012), as well as the geographical, environmental and reproductive barriers to spread (Richardson and Blanchard 2011; Smith and Barney 2014b). Both vegetative and seed propagules from bioenergy crops will be exposed to a diversity of landscapes along the biofuel supply chain (cultivated field to refinery). Therefore, susceptibility to invasion will need to be evaluated across numerous geographies and habitat types (Smith and Barney 2014b).

Since Herbert Baker (1965; 1974) put forth the theory that a set of 12 defining characteristics could identify the ‘ideal weed’, the importance of traits has been widely debated (Thompson and Davis 2011; van Kleunen et al. 2010). Bioenergy crops have been selected for a suite of agronomic traits making them ideal candidates for cultivation (Lewandowski et al. 2003), but this may also serve as the crux for their potential to become invasive (Raghu et al. 2006). For these reasons, it is imperative that we reflect that no single species is invasive in every location it inhabits. For example, populations of Sorghum halepense L. are particularly devastating in the southeastern United States, earning a reputation as one of the world’s worst weeds (Holm et al. 1977). Yet in its northern range, S. halepense populations are rarely regarded as detrimental and despite its perennial growth many populations do not overwinter as rhizomes (Warwick et al. 1984), illustrating that both invasiveness and habitat susceptibility vary (Smith and Barney 2014b).

Here we use a comparative framework to relativize the invasive potential of newly developed fertile tetraploid M. × giganteus. We compare fertile M. × giganteus against ten grass species, comprising 19 accessions, in four environments. We selected the ten grass species to allow a comparison against species that are known invaders in the US (positive controls), and species that are generally considered not to be invasive (negative controls). This design allows us to make important relative comparisons of risk, for candidate bioenergy crops, along a spectrum of invasiveness. We impose both competition and aboveground disturbance treatments to capture a range of conditions which bioenergy crops may encounter in or adjacent to the cultivated field. These treatments allow us to determine conditions that facilitate invasive spread and determine susceptible environments for establishment of nascent populations. This relative methodology was recently tested and proved critical in accurately interpreting the probability of fertile tetraploid M. × giganteus establishment in a diversity of habitats across the southeastern United States (Smith and Barney 2014b). The objective of this study is to compare the growth and spread potential of fertile M. × giganteus to known invasive and noninvasive control groupings. Specifically, we aim to: (1) evaluate the invasive potential of fertile M. × giganteus, during the first three years of establishment and growth, in comparison with 10 species (19 total accessions) of known invasive and non-invasive species in relation to their population dynamics, competitive ability, local recruitment and spread potential; (2) evaluate the performance of each bioenergy crop in response to various levels of competition and disturbance by assessing survival and performance; and (3) quantify seed production as a novel propagule source for M. × giganteus and compare across our invasiveness diversity panel.

All taxa in our study established under all treatment conditions, and all fertile crops produced offspring, with the exception of the two Saccharum spp., which were only grown for two years. Despite enhanced traits for cold tolerance, these cultivars may have been well beyond their suitable geographic range in Blacksburg, VA (Barney and DiTomaso 2011) because they were the only species in this study to decrease in biomass (1300% Mg ha^-1) and culm number (380%), as well as fail to produce inflorescences; however, the lack of inflorescences production was not unexpected as several abiotic factors are responsible for inhibiting and inducing tasseling, such as temperature and photoperiodism (LaBorde 2007).

Though all of the perennial species in our study have the ability to spread vegetatively, local spread was equivocal. Despite the three year clonal expansion of sterile M. × giganteus ‘Illinois’ (0.23 m² plant^-1 increase in area) and A. donax (0.28 m² plant^-1 increase in area), this did not contribute to nascent plants outside the cultivated plot. Unlike the culms of most species in our study, which die back at the end of each growing season, the culms of A. donax remain dormant during the winter months (Saltonstall et al. 2010). Arundo donax produced 33 ± 1 nodes culm^-1; the majority of which produced new axial shoots each growing season. Despite overwhelming evidence of A. donax clonal spread in warm riparian or coastal freshwaters of the southwestern United States (Bell 1997; Quinn and Holt 2008; Seawright et al. 2009); the numerous culms we observed, bending to the ground at the perimeter of our plots, failed to root and produce new ramets; so called layering (Boland 2006). It has been suggested that the probability of a plant becoming invasive increases with the ability to reproduce vegetatively (Kolar and Lodge 2001). The only species in our trial for which vegetative reproduction appeared to contribute to invasive spread were S. halepense, M. sacchariflorus, and P. arundinacea (all positive controls), all of which began to spread from the planted plots in the second growing season. Conversely, Pyšek and Richardson (2007) argue that while vegetative traits may benefit persistence, the ability to spread may be hindered by vegetative reproduction, especially if seed production is limited or absent. In agreement with our results, all of the caespitose grasses in our study failed to spread vegetatively beyond the borders of the planted plot under our growing conditions.

The production of fertile seed enhanced the ability of many species to spread, but only locally. Sorghum halepense was the only accession to have large numbers of first season inflorescences (Figure 2B), and so it is not surprising that this was the only species to generate seedling volunteers in the second year. In contrast, most taxa increased inflorescence production in the second season, extending the range of species detected as seedlings in the receiving areas in the third season. With the exception of ‘PowerCane’, inflorescence numbers continued to increase from the second to the third growing season, suggesting that propagule pressure in the receiving plots did not reach a plateau during the experiment. While other factors such as habitat invasibility and timing are essential to invasive success (Barney and Whitlow 2008), this dramatic increase in propagule pressure facilitated establishment of ‘PowerCane’ seedlings in both the Lc and Hc receiving area, though recruitment varied dramatically between the habitats. Inflorescence production and therefore the total number of spikelets plot^-1 in our three invasive groups increased between year one and year three. Even small increases in propagule pressure can result in a substantial increase in invasion pressure even in inhospitable environments (Davis 2009), which likely contributed to the observed lag.

The more individuals released into an environment the higher the probability that some propagules will endure environmental barriers and overcome stochastic biotic and abiotic factors (Blackburn et al. 2009). The number of introduced individuals, therefore, has a substantial influence on establishment success (Lockwood et al. 2009). Seeds of A. gerardii, P. virgatum and P. arundinacea are known to have variable dormancy and potentially low seedling vigor (Beckman et al. 1993; Lewandowski et al. 2003; Parrish and Fike 2005; Smart et al. 2003), which agreed with our observations in the establishment year. Miscanthus sacchariflorus (positive control) has been reported to have low seed set, ~746 viable seeds mature plant^-1, with population growth predominantly due to vegetative spread (Madeja et al. 2012). Despite these assumptions, 1% of tested M. sacchariflorus seeds did germinate, which was greater than seed germinability of ‘PowerCane’ (0.3%) and M. sinensis NC (0.6%). Previous research has shown ornamental cultivars of M. sinensis are extremely variable, ranging from 190,000 seeds plant^-1 to 3,100 in ‘Gracillimus’, 785 in ‘Dixieland’ and 0 filled spikelets plant^-1 in ‘Cabaret’ (negative controls) (Madeja et al. 2012; Meyer and Tchida 1999). Results from our seed germination testing support these conclusions in which no ornamental M. sinensis seeds germinated; however, we observed high variability (31% to 0.6%) in germinability for naturalized M. sinensis accessions (Figure 3). Dougherty et al. (2014) showed that ~44% of seed from weedy accessions of M. sinensis germinated in a laboratory setting. Previous research indicates that both inflorescence and spikelet production in almost all M. sinensis cultivars are positively correlated with plant hardiness zone (Madeja et al. 2012; Meyer and Tchida 1999; Wilson and Knox 2006); indicating that invasion pressure for these species may vary with latitude.

After three years of growth, all naturalized accessions of M. sinensis produced a greater number of inflorescences m^-2 and more spikelets inflorescence^-1 than ‘PowerCane’ (Figure 3). In this study, ‘PowerCane’ produced ~1.3 billion spikelets ha^-1 in the third growing season, which would yield 3.9 million viable seed ha^-1 given the 0.3% germination rate. It is possible that we saw greater numbers of seed produced than in a production field; like P. virgatum (Martinez-Reyna and Vogel 2002), Miscanthus spp. are self-incompatible (Hirayoshi et al. 1955). The genetic diversity among the accessions and species in our study, ideal for outcrossing species, may have led to inflated seed production (Madeja et al. 2012) compared to the relative genetic homogeneity common in commercial crops. In this case genetic variability not only increases the likelihood of seed production, but also has the potential to enhance establishment success and increase the habitat range of exotic species (Lockwood et al. 2005).

Surprisingly, none of the accessions were affected by the level of competition, which not only contradicts much of the literature suggesting the need for weed management at establishment (Lewandowski et al. 2003), but supports the conclusion that these species are fast growing and highly competitive (Raghu et al. 2006). The high seedling establishment of ‘PowerCane’ in the Lc and lower seedling establishment numbers in the Hc, appears to contradict our recent findings in which only 0.1% of emerged ‘PowerCane’ seedlings survived after six months (Smith and Barney 2014b). However, our earlier study indicated the majority of ‘PowerCane’ seedlings emerged in areas of available bare ground and low resident plant competition such as agricultural fields and forest understories. However, annual weed species dominated our Hc receiving area, which created open spaces and the availability of bare ground during the winter months and early spring, coinciding with annual seed dispersal for many of these late flowering species. The small, light seed and ciliate lemma (Gleason 1952) of Miscanthus spp. is valuable for dispersal. However, the ciliate lemmas appear to interfere with the ability of Miscanthus seed to make important soil contact required for germination, which is evident in areas of high resident plant competition and litter (Smith and Barney 2014b). According to Quinn et al. (2011), the anemochorous M. sinensis spikelets have been shown to disperse an average of 50 m. Therefore, open areas of low resident plant competition near production sites will likely be the most susceptible, suggesting that our Lc plots represent a worst case scenario. This information will be critical for identifying susceptible habitats near cultivated fields and will be important considerations for management.

Despite the utility of trait-based research for helping to make associations and guide management, traits do not confer absolute predictability. Invasions will always be contingent on a number of interacting factors (Barney et al. 2008; Dredovsky et al. 2012). Hence, our experimental design was critical in the interpretation of our results. Clearly this geographic location, habitat and treatment factors were ideal for a species such as S. halepense; a species intentionally introduced for agronomic purposes, which now flourishes in agricultural and anthropogenic systems of the southeastern U.S. (Warwick and Black 1983). However, despite the reputation of A. donax as an aggressive riparian invader, this species appeared to be constrained by the non-riparian landscape.

The selection of our invasive and noninvasive taxa was a novel methodology used to make important relative comparisons. The ten species selected in this study are of similar life form, and all of them, including S. halepense (Nackley et al. 2013), have the potential to be used as a feedstock in bioenergy production. Comparisons of ‘PowerCane’ to specific taxa, S. halepense for example, across an invasive spectrum, provided meaningful information. However, making all pairwise comparisons would not be ecologically and statistically meaningful; therefore, our goal was to also find and examine broader trends within invasive groups. Interestingly, our positive control species were selected on the basis of a past history of invasiveness, a robust predictor of future invasiveness (Davis 2009). However, multivariate analysis failed to indicate a relationship between our measured functional traits and our invasive groups (Figure 4). After three growing seasons, our ranking of fitness metrics also fails to show the anticipated consistent gradient of positive (high fitness) to negative (low fitness). This suggests that functional traits vary in their relationship to invasiveness (Dredovsky et al. 2012). However, meta-analysis reveals that traits such as fitness, size, and growth rate were significantly higher for exotic invasive species when compared with the traits values of non-invasive species (van Kleunen et al. 2010). It is imperative to remember that invasiveness is not a “one size fits all” scenario, which is made evident in our study. Traits vary with life stage and environmental conditions and the importance of any given trait will therefore also vary (Davis 2009; Pyšek and Richardson 2007). While our positive and negative invasive groups may not have clustered as we hypothesized; the use of numerous species and accessions allows us to evaluate the usefulness of traits across an invasive spectrum.

Unfortunately the loss of our Kentucky and Alabama sites limits our ability to generalize across a broader geographic range. The ability to observe relative comparisons across a gradient of species reinforces the fact that it is the important interaction of species and habitat that result in invasive populations (Barney and Whitlow 2008). Our results indicate that several candidate feedstocks have the ability to move from the cultivated field, but it should be noted that we only recorded spread to < 5 m from the field edge in three years. On average, more than 100 culms m^-2 were observed in every measured quadrat in the Lc receiving area of S. halepense, naturalized M. sinensis, P. virgatum and ‘PowerCane’. Conversely, only S. halepense and naturalized M. sinensis maintained high numbers in the Hc receiving area when P. virgatum and M. × giganteus ‘PowerCane’ showed a dramatic decrease in culm numbers.

Bioenergy crop movement beyond the cultivated field would not be novel to agronomic crops because feral escapes are known for most row crops (DiTomaso and Healey 2007). Yet due to their economic and social importance, crops are not frequently discussed in the invasion literature. Our experiment covers the first three years of the establishment phase, and unexpected and nonlinear changes may manifest in subsequent stages. Our results suggest that at least short-range movement away from the cultivated field is probable for fertile bioenergy feedstocks. It should be noted that no species in this study were detected outside our trial boundary, which predominately consisted of a mowed perennial border. Further study, across broader geographic locations and continued research will be necessary to further determine acceptable risk and management planning.

The use of ‘PowerCane’ or other fertile M. × giganteus germplasm could improve grower adoption but the invasive potential and ecosystem impacts of widespread cultivation still require further evaluation including the determination of climatic limitations of M. × giganteus and other bioenergy crop seedlings. The ability to contextualize our results suggests that M. × giganteus ‘PowerCane’ did not have the highly competitive seedling establishment potential of S. halepense. Alternatively, in this growing region sterile cultivars provide a lower risk option, but require additional economic investment. The scrutiny that has been applied to bioenergy crops indicates that we have moved beyond the once cavalier approach toward species introduction. These efforts should continue in order to reduce unwanted and unintentional invasive spread. Nascent populations or seedlings may be easily overlooked. However, management at the seedling or early growth stage will likely increase the chances of successful control (Stephens and Sutherland 1999). Our relative methodology and results from this study can help us prepare for industry development while helping to minimize risk and mitigate invasive spread.

Thanks to Ryan Dougherty, Eugene Dollete, John Halcomb, Matt Ho, Daniel Tekiela, Elise Benhase, Phillip Cox, and Carissa Ervine for help during installation, data collection, and harvest of this experiment. Thanks to Mendel Biotechnology, Inc for donation of M. × giganteus ‘PowerCane’ and ‘Nagara’ plugs and Dr. Erik Sacks for naturalized M. sinensis seed. The USDA graciously provided the two Saccharum spp.