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First published online October 31, 2008; doi:10.3732/ajb.0800225 American Journal of Botany 95: 1584-1595 (2008) © 2008 Botanical Society of America, Inc.

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Bromus tectorum (Poaceae) in midcontinental United States: Population genetic analysis of an ongoing invasion¹

² School of Biological Sciences, Washington State University, Pullman, Washington 99164 USA ³ Department of Biological Sciences, Boise State University, Boise, Idaho 83725 USA

Received for publication 3 July 2008. Accepted for publication 24 September 2008.

ABSTRACT

Biological invasions can be substantially influenced by the genetic sampling associated with a species’ introduction. As a result, we assessed the genetic and evolutionary consequences of the entry and spread of the invasive grass Bromus tectorum (cheatgrass) across the United States midcontinent through an analysis of 54 populations, using enzyme electrophoresis. On average, these populations display 1.04 alleles per locus (A), 4.1% percent polymorphic loci per population (%P) and an expected mean heterozygosity (H_exp) value of 0.009. Heterozygotes, which have been rarely reported for B. tectorum in North America, occur in three populations in the midcontinent and are likely novel multilocus genotypes that arose postimmigration. The midcontinent distribution of multilocus genotypes suggests that plant immigrants came directly from either the native range or the eastern United States, or both. Continued dispersal of preadapted genotypes and the assembly of populations that are genetic admixtures may enhance this invasion by increasing both the genetic diversity within populations and the selection of novel genotypes arising from occasional outcrossing. The potential for postimmigration evolution in most species points to the largely unrecognized need to block the introduction of new, potentially aggressive genotypes of an alien species already in the United States.

Key Words: allozyme • Bromus tectorum • cheatgrass • invasion • multiple introductions • Poaceae • Great Plains

A popular perception of an invasion—perhaps inadvertently fostered by chronological map projections of invasions as concentric isopleths (Hengeveld, 1989; Mack, 2005)—is a monolithic spread across a new range: unidirectional, inexorable, and composed of uniform (if not uniformed) invaders (Elton, 1958; Lonsdale, 1993; Baskin, 2002; Albright et al., 2004). This view however encapsulates the exception, not the vast majority of cases. Some invasive species do arrive as one or a few genets, even as one sex of the species (Salix fragilis in New Zealand, Roy et al., 1998; Hydrilla verticillata in Florida, Schmitz et al., 1997) or in a one-time entry (Pennisetum setaceum in Hawaii, Williams et al., 1995) and have spread locally as an expanding wave front (Salvinia molesta in Papua New Guinea, Mitchell et al., 1980; Spartina anglica in Washington, Civille et al., 2005). Entry more commonly involves repeated immigrations that occur at isolated, widely separated sites in a potential new range (Barrett and Husband, 1990; Mack and Erneberg, 2002). Furthermore, alien populations may spread independently at different rates and may be composed of diverse admixtures of genotypes drawn from widely separated locales across the species’ native range (Burdon et al., 1980; Novak and Mack, 2005). This much more common pattern holds important implications for our understanding of postimmigration evolution of invasive species (Baker and Stebbins, 1965; Lee, 2002; Allendorf and Lundquist, 2003, Novak, 2007) as well as our ability to prevent and combat invasions (Roderick and Navajas, 2003; Muller-Scharer et al., 2004).

The genetic diversity of an alien in its new range is influenced by multiple demographic and evolutionary forces; including founder events and population bottlenecks (Barrett and Kohn, 1991; Novak and Mack, 2005). These forces can diminish the genetic diversity in newly established populations and potentially increase genetic differentiation among introduced populations (Brown and Marshall, 1981; Barrett and Husband, 1990). However, other factors such as mating system, effective population size, selection in the donor range, number of introductions, novel selection pressures, gene flow, and the rate at which a species proliferates can enhance, mitigate, or even reverse the genetic consequences of founder events and population bottlenecks (Nei et al., 1975; Novak et al., 1993; Barrett and Pannell, 1999; Squirrell et al., 2001; Stepien et al., 2002; Barrett et al., 2008; Dlugosch and Parker, 2008). The character and rate in which these factors operate likely varies across the environmental mosaic in a new range. Consequently, analyses of the genetic consequences of a species’ introduction should ideally involve comprehensive, regional, rather than local, investigations (Squirrell et al., 2001).

Bromus tectorum, commonly referred to as cheatgrass or downy brome, is a diploid (2N = 14), predominately cleistogamous, annual grass. It has been introduced into temperate regions worldwide from its native range in Eurasia and North Africa (Upadhyaya et al., 1986). Distributed throughout southern Canada and the United States (USA) (Novak and Mack, 2001), the grass was first reported in eastern North America in ca. 1790 (Muhlenberg, 1793), although its presence cannot be confirmed again until 1859 (Bartlett et al., 2002). In contrast to its status in eastern North America (common but not abundant) (Upadhyaya et al., 1986; Bartlett et al., 2002), B. tectorum swiftly invaded much of western North America from ca. 1889 through 1930 (Mack, 1981).

Eastern and western USA populations of B. tectorum have not experienced the same history of introduction (Novak and Mack, 2005). At least two introduction events occurred in the eastern USA (Bartlett et al., 2002); in the western USA, a minimum of six introduction events have been documented with genetic markers (Novak and Mack, 19932001). The grass has relatively low levels of genetic diversity; however, multiple introductions and subsequent formation of admixtures within western USA populations from genetically divergent native populations (Novak and Mack, 1993) have produced higher levels of overall genetic diversity and within-population diversity than are reported for eastern USA populations (Novak et al., 1991, 1993; Bartlett et al., 2002).

Across the midcontinent of the USA, that region west of the Mississippi River and east of the Continental Divide, B. tectorum was reported regionally as "scattered" or common throughout much of the early 20th century (Over, 1932; Gates, 1940; Harrington, 1964). Spread of the grass across this region was apparently much slower than in the western USA (Clements, 1949; Klemmedson and Smith, 1964; Mack, 1981); however, since ca. 1950, the regional invasion of B. tectorum has accelerated (Heitschmidt et al., 1995; Kotanen et al., 1998; Mosley et al., 1999). The occurrence of B. tectorum in the midcontinent may stem in any combination from (1) direct introductions from the native range (Novak and Mack, 1993, 2001; Novak et al., 1993), (2) spread to the midcontinent from populations whose descendents were originally introduced in the wake of westbound settlers after 1859, or (3) spread eastward from independent Eurasian introductions along the Pacific Coast (Novak and Mack, 2001). However the route(s) of the introduction and range expansion of B. tectorum into the midcontinent of the USA, these events have directly influenced the amount and distribution of genetic diversity within and among the grass’s populations in this region.

Using enzyme electrophoresis, we have assessed the genetic and evolutionary consequences of the entry and spread of B. tectorum in the midcontinent of the USA. Specifically, we address the following questions here: (1) How much genetic diversity occurs in these midcontinental populations (2) Is there any relationship between the grass’s current genetic diversity and the region’s major native grassland communities (Kuchler, 1964; Great Plains Flora Association, 1986) and bordering forest? (3) How is genetic diversity partitioned within and among populations? (4) How does the genetic diversity and structure compare among populations in the midcontinent, other USA regions and Eurasia? (5) To what extent have specific genotypes been dispersed across the USA? (6) Can probable source (or donor) populations be identified in the native range?

MATERIALS AND METHODS

Plant material
Samples of caryopses from midcontinental populations were collected in June and July 2003. Most collection sites were along roadsides, railroad tracks, and other publicly accessible disturbed sites. Sampling was conducted throughout each population or within a 500-m² area, whichever was smaller. The mature panicle from each of 25–35 individuals was collected at each sampling location and placed in a numbered paper envelope. Harvested plants were approximately 1–3 m apart to minimize the collection of full sibs. Forty-eight populations from locations that comprehensively represented the study area were sampled for laboratory analysis. Allozyme data for six populations analyzed by Novak et al. (1991) within the midcontinent were included in this analysis. Thus, this study describes genetic diversity and structure for 54 populations of B. tectorum within this region (Fig. 1), all analyzed using the same molecular markers (described later).

View larger version (52K): [in this window] [in a new window]   Fig. 1. Location of the 54 populations of Bromus tectorum analyzed within the midcontinental USA. Numbers in the figure correspond to the population designation and locations listed in Table 2. Each population was assigned to one of four geographic subregions as described in the text: I, forest; II, tallgrass prairie; III, mixed-grass prairie and IV, tallgrass prairie (according to Küchler [1964] and the Great Plains Flora Association [1986]). Although populations 20 and 21 occur inside the forest boundary, they were assigned to the tallgrass prairie subregion because of their proximity to other populations in this subregion.

Enzyme electrophoresis
Bromus tectorum caryopses were germinated at room temperature on moistened filter paper in petri dishes. Seedlings were harvested approximately 7–10 d after germination to maximize the resolution of enzyme banding patterns on the gel. Starch gel electrophoresis was conducted using the methods of Soltis et al. (1983), with modifications described by Novak et al. (1991). The 15 enzymes used here were assayed using the same buffer systems indicated in Novak et al. (1991), with the exception that isocitrate dehydrogenase (IDH), glucose-6-phosphate dehydrogenase (G6PDH) and shikimate dehydrogenase (SKDH) were visualized using system 1 of Soltis et al. (1983). Nomenclature for the resulting 25 loci and all alleles followed Novak and Mack (1993).

Multilocus genotype designations were based on identity of the allele at each locus scored. The multilocus genotype characterized by the most common combination of alleles at the 25 loci scored is termed here the most common genotype (MCG); it had the highest frequency of occurrence across both native and introduced populations (data not shown). Other multilocus genotypes were determined by specific alleles that deviated from the MCG at one to several loci and are potentially diagnostic for separate introduction events.

Data analysis
As in our previous reports on the population genetics of B. tectorum (Novak et al., 1991; Novak and Mack, 1993; Bartlett et al., 2002; Valliant et al., 2007), electrophoretic data from midcontinental populations were analyzed using the program BIOSYS-1 (Swofford and Selander, 1981). Data were entered into the program as genotype frequencies, with populations arranged hierarchically based on the four geographic subregions described. Genetic diversity within and among midcontinental populations and subregions of B. tectorum was assessed using the parameters and methods described by Novak et al. (1991), Novak and Mack (1993), Bartlett et al. (2002), and Valliant et al. (2007).

RESULTS

Genetic diversity of B. tectorum in the midcontinent
Midcontinent genetic diversity of B. tectorum was based on analysis of 1624 individuals in 54 populations (30.07 individuals/population). Across all 54 populations at the 25 loci, a total 32 alleles was observed (1.28 alleles/locus); six loci (24%) were polymorphic: Got-4, Mdh-2, Mdh-3, Pgm-1, Pgm-2, and Tpi-1 (Table 1). Only one of the six polymorphic loci (Got-4) had more than two alleles per loci. When polymorphic, Pgm-1 and Pgm-2 possessed the same allele frequencies in each population, and a similar result was observed for Mdh-2 and Mdh-3. These results suggest that alleles at each of these loci pairs are linked, most probably due to gametic disequilibrium (S. Novak and R. Mack, unpublished data). Populations from all four subregions had the Pgm-1a & Pgm-2a multilocus genotype; all other multilocus genotypes had a much more restricted geographic distribution (Fig. 2; Table 1).

View this table: [in this window] [in a new window]   Table 1. Mean of allele frequencies at polymorphic loci for 54 populations of Bromus tectorum in the midcontinent of the United States that vary relative to the most common genotype. For a description of the most common genotype, see the text. Mean values are reported by regions determined by major vegetation types that dominated this area before European settlement (I: forest, II: tallgrass prairie, III: mixed-grass prairie, and IV: shortgrass prairie) in accordance with Küchler (1964) and the Great Plains Flora Association (1986). The number of populations in each region is in parentheses after the headings. Numbers in parentheses beside the allele frequencies are the number of populations in each region that possessed that allele; a value of 1.000 indicates that all populations in the region are monomorphic for that allele. MCG: most common genotype

View larger version (27K): [in this window] [in a new window]   Fig. 2. Pie diagrams indicating the relative frequency of multilocus genotypes within each of the 54 populations of Bromus tectorum sampled in the midcontinental USA.

View this table: [in this window] [in a new window]   Table 2. Measures of genetic diversity for 54 populations of Bromus tectorum in the midcontinental United States. Roman numerals refer to the regions described in Fig. 1 and Table 1. N is the number of individuals sampled in each population; A is the mean number of alleles per locus; %P is the percent polymorphic loci; Hexp is the expected mean heterozygosity; Hobs is the mean observed heterozygosity. Values of Hobs are in boldface in all populations where heterozygotes were detected. Populations 3, 4, 21, 25, 42, and 46 were analyzed by Novak et al. 1991.

View this table: [in this window] [in a new window]   Table 6. Nei’s (1978) unbiased genetic identity coefficients for populations of Bromus tectorum within and between regions in the midcontinental United States. Regions were determined by major vegetation types as described in Fig. 1 and Table 1. N is the number of populations in each region. The mean of all intraregional pairwise population comparisons appear on the diagonal, and the means of interregional population comparisons appears below the diagonal. The range of values for population pairs in each regional comparison is given in parentheses.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Unweighted pair-group method with arithmetic averaging (UPGMA) phenogram based on Nei’s (1978) unbiased genetic identity values for the 54 populations of Bromus tectorum sampled in the midcontinental USA. Postal code abbreviations are used for each population’s state location. Roman numerals indicate the geographic subregion to which each population was assigned: I, forest; II, tallgrass prairie; III, mixed-grass prairie and IV, tallgrass prairie.

View this table: [in this window] [in a new window]   Table 7. Populations of Bromus tectorum from the native range in Eurasia and populations in the midcontinental United States that share the same homozygous multilocus genotype. Multilocus genotype names used here are derived from their constituent alleles that deviate from those observed in the most common genotype (MCG). Data for native range populations are from Novak et al. (1993) and Novak and Mack (2001).

Our results reveal that the ongoing range occupation of B. tectorum in the midcontinental USA is the product of the entry of genotypes initially drawn from different parts of the native range. These introductions have not only created admixtures of genotypes in populations across this new range but have also occasionally produced novel genotypes in several populations. Perhaps most striking is the regional prominence of some multilocus genotypes (e.g., Pgm-1a & Pgm-2a) and the virtual absence of at least one other (Got-4c).

Sources, introduction, and spread of B. tectorum
Low levels of genetic variation in B. tectorum, maintained by almost complete cleistogamy, provide the opportunity to identify an original donor range and trace the introduction and spread of multilocus genotypes into the midcontinent. The most common genotype (MCG) in these midcontinent populations is also most common among European populations (Novak and Mack, 2001) and consequently provides information on a donor provenance only at a coarse geographic scale. Other multilocus genotypes detected in midcontinental populations have, however, much more restricted native ranges: Got-4c, Pgm-1a & 2a and Got-4d are known only from Bayreuth, Germany and Libochovice, Czech Republic; Vac, Hungary and Bratislava, Slovakia; and Vienna-Landstrasse, Austria, respectively (Novak and Mack, 2001). Given the origin of most of the human immigrants to North America in the 19th century, the arrival of strictly European genotypes seems predictable. Although direct introductions of B. tectorum from Europe to the USA midcontinent may have occurred, these introductions cannot be reliably identified among the multilocus genotypes detected so far because these same genotypes also occur elsewhere in North America. Exceptions appear to be the products of postimmigration outcrossing within North America (e.g., the Pgm-1a, Pgm-2a, Mdh-2b & Mdh-3b multilocus genotype) (discussed later).

The MCG is prominent in the entire midcontinent, providing no discernible molecular evidence for the direction(s) of entry of immigrants with this genotype into the region. The historical evidence, although certainly not conclusive, suggests the spread of B. tectorum from the East, where the grass became increasingly common after 1859 (Bartlett et al., 2002). If the current Eastern populations bear any genotypic similarity to populations in the 19th century, almost all immigrants carried west had a high likelihood of being the MCG because this genotype was detected in 37 of 38 eastern populations (Bartlett et al., 2002).

In contrast, evidence for the paths of dispersal of the grass once it arrived in North America is provided by several of the multilocus genotypes in the midcontinent that have distinct continental distribution patterns. The Pgm-1a & Pgm-2a multilocus genotype is common across the midcontinent (24 of 54 populations) (Table 1, Fig. 2). This genotype occurs to varying degrees across North America (Novak and Mack, 2001; Bartlett et al., 2002; Valliant et al., 2007), suggesting it could have spread inland from either coast or both coasts. But the genotype is rare in the western United States (Novak et al., 1991, 1993); consequently, a West–East spread of populations with this genotype seems unlikely. Furthermore, midcontinent populations with prominent representation of the Pgm-1a & Pgm-2a multilocus genotype are concentrated in the southern Plains and adjacent Louisiana and Arkansas, much closer to populations with this genotype in Kentucky and North Carolina (Bartlett et al. 2002). Consequently, the likely route of this genotype’s spread appears to have been from the East. The Mdh-2b & Mdh-3b multilocus genotype occurs in low frequency within populations scattered across North America (Novak et al., 1993; Bartlett et al., 2002). Whether the sporadic distribution of this genotype indicates westward expansion as hypothesized for the MCG and Pgm-1a & Pgm-2a genotypes, or independent introductions from Eurasia, cannot currently be resolved.

Other multilocus genotypes have contrasting distribution patterns among the midcontinent populations compared with North American populations as a whole. Unlike the Pgm-1a & Pgm-2a multilocus genotype, the Got-4c multilocus genotype is predominant in populations throughout the West (Novak et al., 1991), yet we found this genotype in only three individuals in the midcontinent (Laramie, Wyoming [WY]). No populations in the eastern USA have been detected with the Got-4c genotype (Bartlett et al., 2002). The striking contrast in the prevalence of this genotype in Intermountain West populations compared to populations in the midcontinent holds implications for the direction of cheatgrass spread into the midcontinent. Clements (1949, p. 205) claimed that "In the past quarter of a century it [Bromus tectorum] has swept across the Palouse [Intermountain West] into the Great Plains, where it can only be checked by fostering and replacing the native perennials." This scenario seems unlikely, given that Got-4c was detected in only one population in our study area.

Genetic diversity in the midcontinent
Genetic diversity among these midcontinent cheatgrass populations has likely been influenced by the grass’s highly selfing mating system and its pattern of introduction and regional spread. The level of diversity across and within midcontinent populations (Tables 2, 3) is low, even compared to other plant species with primarily a self-pollinating mating system (Brown, 1979; Hamrick and Godt, 1990). Equally low levels of genetic diversity have, however, been reported for some introduced selfing plants (Marshall and Weiss, 1982; Warwick et al., 1984).

The hierarchical partitioning of genetic diversity in midcontinent populations is strikingly different from the grass’s known diversity in its native range (Novak and Mack, 1993, 2005). Midcontinent populations of B. tectorum have fewer alleles (32 vs. 43) at the 25 scored loci compared with native populations and less than half (six vs. 13) the polymorphic loci detected among native range populations. However, the percentage of midcontinental populations that are polymorphic is much higher than the percentage found among native populations. Populations of B. tectorum from Eurasia also have lower mean within-population genetic diversity statistics than values for populations in the midcontinent (Novak and Mack, 1993). Thus, populations in the native range have more overall genetic variation in the form of allelic richness and polymorphic loci, but more genetic diversity occurs, on average, within populations in the USA midcontinent (Novak and Mack, 1993, 2005).

Deviation from Hardy–Weinberg equilibrium
The five heterozygotes we detected among the 1624 individuals of B. tectorum sampled in our study area is higher than expected in a species that has been described as an obligate self-pollinator (McKone, 1985; Ramakrishnan et al., 2004). No heterozygotes had been observed previously in the eastern or western USA using allozymes (Novak et al., 1991; Bartlett et al., 2002), or with single sequence repeats (SSRs) (Ramakrishnan et al., 2002); heterozygotes, however, have been recently reported from Canada (Valliant et al., 2007), Nevada (Ashley and Longland, 2007), and Colorado (Kao et al., 2008). More heterozygotes were detected during subsequent progeny array analyses of midcontinent populations in which outcrossing had been observed or would be expected because of high levels of polymorphisms. Many of these heterozygotes appeared to be generated through segregation during selfing by a heterozygous maternal plant (S. J. Novak, unpublished data).

The extremely low level of heterozygosity we observed suggests that outcrossing is rare in B. tectorum in the USA midcontinent (Tables 2, 4). Outcrossing may, however, be occurring at slightly higher levels than we observed because outcrossing events cannot be detected in more than half the populations in the midcontinent that are monomorphic. The rarity of reported heterozygotes in the western USA, despite the overall higher levels of polymorphisms within some populations, suggests either a temporal or spatial explanation, or both, for the outcrossing detected in the midcontinent USA. Outcrossing rates in predominately selfing species can be higher for populations occurring in moister sites compared with drier sites, e.g., Hordeum spontaneum in Israel (Brown et al., 1978; Clegg, 1980). Outcrossing rates in predominately selfing species can also vary annually (Adams and Allard, 1982). As a result, the heterozygotes we detected may be due to our sampling in a rare year in which outcrossing occurred. But even extremely low levels of outcrossing may lead to the evolution of a selfing species in its new range (Allard, 1965).

Genetic differentiation in the midcontinent
The level of genetic differentiation among populations of widely distributed, predominantly selfing plant species is often high (Hamrick and Godt, 1990). Sampling error during founder events and population bottlenecks would increase genetic differentiation among populations (Brown and Marshall, 1981; Barrett and Husband, 1990; Novak and Mack, 2005). Differentiation in a new range would be compounded in a predominately selfing species with its low levels of gene flow (Novak et al., 1991). Many introduced species, including Bromus tectorum, do not appear, however, to follow this predicted pattern (Novak and Mack, 1993; DeWalt and Hamrick, 2004; Lavergne and Molofsky, 2007).

Total genetic diversity of populations in this region (H_T = 0.084; Table 5) is intermediate to values reported for populations elsewhere (see Valliant et al., 2007). Bromus tectorum in the midcontinent of the USA has a lower value of G_ST than in its populations in eastern or western North America (Novak et al., 1991; Bartlett et al. 2002). The mean G_ST value for all polymorphic loci in midcontinent North America indicates that 29% and 71% of the genetic diversity is partitioned among populations and within populations, respectively. This distribution stands in contrast to the diversity in the native range, where most (75%) genetic diversity is partitioned among populations (Novak and Mack, 1993). Among-population genetic diversity in the native range may have been transformed to within-population diversity in the midcontinent through the random assembly of admixtures of immigrants. Such transformations have been detected in other invasive species and may be a key influence on the course of invasion (Kolbe et al., 2007; Novak, 2007).

Genetic identity values among subregions in the midcontinent are as high as the values within subregions (Table 6). Thus, populations of B. tectorum from the four subregions are genetically similar, even though they are widely separated and occur in different habitats. The UPGMA phenogram demonstrates that populations are not clearly segregated by subregions (Fig. 3), results consistent with comparisons across western North America (Novak et al., 1991).

Evolutionary aspects of the invasion of B. tectorum in the midcontinent
The USA midcontinent was likely the recipient of immigrants from genotypically diverse populations in North America; direct introductions from the native range were not detected but cannot be discounted. These events apparently led to the transformation of mostly among-population diversity in the native range to within-population diversity in the introduced range. The population collected in Martin, SD, had the highest level of within-population polymorphism (%P = 20); it contains more genotypes than any other population examined in this study, including a novel, recombinant multilocus genotype not previously detected in B. tectorum (Pgm-1a, Pgm-2a, Mdh-2b & Mdh-3b). Although outcrossing in B. tectorum is apparently rare, our evidence suggests that outcrossing produced this genotype. The collection site, an old, abandoned corral, was likely the site of multiple introductions of B. tectorum in hay, feed, or adhering to livestock (ca. 1940–1980) and hogs (1981–1986) (T. L. Nelson, personal communication). This single population illustrates the degree to which transport of extraneous seed-contaminated hay and animals may spread B. tectorum genotypes, increasing within-population diversity throughout the USA midcontinent (Mack, 2003).

Recombination of multilocus genotypes could signal postimmigration evolution within B. tectorum in its new USA range (cf. Avena barbata in California, Clegg and Allard [1972], Garcia et al. [1989]; Phalaris arundinacea in eastern USA, Lavergne and Molofsky [2007]). Populations of cheatgrass in the midcontinent appear instead to be composed primarily of preadapted multilocus genotype combinations from the native range. Genotype distributions of the introduced Capsella bursa-pastoris in North America are also likely the product of both chance sampling in the native range and the canalization of genotypes by natural selection (Neuffer and Hurka (1999). This scenario may explain the genotype distribution of B. tectorum within subregions in the USA midcontinent and across North America.

Low levels of genetic variation and the almost total cleistogamy in B. tectorum could lead to an evolutionary dead end for the species’ introduced populations (Barrett and Shore, 1987; Schoen and Brown, 1991). Multiple processes appear however to have shaped the population genetics of B. tectorum in the USA midcontinent. Multiple introductions have increased the grass’s overall genetic diversity throughout this region, and subsequent spread and mixing of genotypes have increased the within-population genetic diversity. Selection may be occurring within midcontinent populations for preadapted and potentially more invasive genotypes, such as Pgm-1a & Pgm-2a. Rare outcrossing events among genotypes, that were once isolated in the native range, could further increase the potential for local adaptation (Clements et al., 2004).

Future spread of B. tectorum in the midcontinent?
The future of B. tectorum in the midcontinent is still unfolding, including at elevations where it had not previously been reported (Brown and Rowe, 2004). Introductions of additional immigrant genotypes in this new range could accelerate or extend this invasion. For example, whether more plants with the Got-4c genotype arrive in this region and become invasive is an intriguing issue. Although plants with Got-4c are major contributors to the invasion in the Intermountain West, the genotype is rare east of the Rockies. Perhaps it has arrived only recently from the West, and this sole eastern Wyoming population with the Got-4c genotype is but a vanguard that could spark a new round of proliferation. Careful investigation of the occurrence of this genotype in the midcontinent and its role in the ongoing invasion of cheatgrass in this region is warranted.

As we described, invasions are often viewed as initiated by a single entry of immigrants. Most invasions are instead the product of recurring arrivals of genetically different immigrants; each wave of immigration brings the potential to enhance the invasion through the assembly of unique admixtures as well as the creation of novel recombinations of genotypes. Bromus tectorum in the midcontinental USA along with other immigrant species (Garcia et al., 1989; Kolbe et al., 2007; Lavergne and Molofsky, 2007) illustrates both these genetic opportunities for an invader and the resulting need for regulations that consider the evolution of introduced species (Cox, 2004). Quite aside from the well-recognized need to prevent the entry of unwanted new species into a new range (Westbrooks and Eplee, 1999), there is apparent need to revise quarantine practices to address the largely unrecognized threat from the introduction of new, potentially aggressive genotypes of an alien species already in the USA.

FOOTNOTES

¹ The authors thank especially J. Schachner and N. Mack for their invaluable assistance in collecting these populations along with C. Brown, S. Gray, M. Haferkamp, P. Sorensen, and D. Williams. Laboratory assistance was provided by E. Murnigham, K. Burdon, A. Hodzic, and A. Ulappa. J. Nelson, M. M. Brooke, M. T. Valliant, S. Foster, C. L. Kinter, S. Louda, M. Minton, S. G. Mortenson, R. R. Pattison, A. V. Novak, S. N. Novak, M. I. Novak, C. Stern, and M. Webster provided much appreciated additional help. The authors especially thank R. Scott who prepared the final version of all figures. The manuscript was improved through the comments of several anonymous reviewers. This research was funded substantially through support from the Betty W. Higinbotham Trust at Washington State University, with additional funding provided by the Faculty Research Grant Program at Boise State University. An earlier version of this manuscript was completed while S.J.N. was on sabbatical leave at the CSIRO European Laboratory in Montferrier-sur-Lez, France, and he is grateful for the generous use of this facility.

⁴ Author for correspondence (e-mail: snovak{at}boisestate.edu)

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