Effects of plant competition, seed predation, and nutrient limitation on ...

Biol Invasions DOI 10.1007/s10530-010-9769-9

ORIGINAL PAPER

Effects of plant competition, seed predation, and nutrient limitation on seedling survivorship of spotted knapweed (Centaurea stoebe) David G. Knochel • Cody Flagg • T. R. Seastedt

Received: 17 July 2009 / Accepted: 26 April 2010 Ó Springer Science+Business Media B.V. 2010

Abstract We measured seed germination and seedling survivorship of spotted knapweed, Centaurea stoebe, in a series of laboratory and field experiments to evaluate the efficacy of seed limitation as a management focus. This work was initiated 6 years after introduction of several biological control agents. The soil seed bank of the site used in this study contained a mean density of 5,848 seeds/m2 (ranging from 0 to 16,364 seeds/m2), and 92% of the seeds isolated from soils were shriveled, discolored, and/or partially decayed. Additionally, none of the intact seeds germinated, suggesting that the viable seed bank at our field study site has been exhausted. Centaurea stoebe seeds were planted into pots under a range of soil nitrogen (N) availability, with half of the pots containing a single density of previously established seedlings of a native cool-season grass, slender wheatgrass (Elymus trachycaulus). A watering regime mimicking local precipitation was applied. Spotted knapweed exhibited large biomass responses to N addition, but the presence of grasses suppressed the ability to exploit this N. Surprisingly, low soil N conditions improved knapweed survivorship in the presence of grasses. Nevertheless, recruitment and

D. G. Knochel (&) C. Flagg T. R. Seastedt Department of Ecology and Evolutionary Biology and Institute of Arctic and Alpine Research, University of Colorado, Campus Box 450, Boulder, CO 80309-0450, USA e-mail: [email protected]

biomass were still far below the levels reached in the absence of competition. To evaluate the effect of density on successful recruitment, Centaurea stoebe seed was introduced into a meadow at three densities matching reduced levels of seed production under the constraints of seed predators. These densities were sown with or without a seed mixture of native species, into an existing plant community lacking C. stoebe, and seedling recruitment was recorded over 2.5 years. Across all plots and densities sown (568–2,272 seeds m-2 year-1), seedling recruitment was less than 1%. The invasion potential of spotted knapweed was greatly diminished when realistic levels of plant competition and biological control limit seed production. We therefore conclude that a combination of seed limitation and shortage of ‘safe sites’ within undisturbed vegetation can limit densities of C. stoebe. Keywords Centaurea stoebe (spotted knapweed) Biological control Seed limitation Plant competition Nitrogen limitation Seedling recruitment

Introduction Spotted knapweed (Centaurea stoebe) is a non-native invasive plant that occupies grassland and forest

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communities over large portions of western North America, and land managers have been searching for economically feasible and sustainable methods to reduce its abundance and dominance for decades (Sheley et al. 1999). Biological control efforts have shown some success: weevil (Larinus minutus) and fly (Urophora spp.) development within flower heads of the plant are greatly reducing the reproductive output of spotted knapweed populations in North America (Story et al. 2008). However, the efficacy of seed reduction as a control mechanism on spotted knapweed populations remains uncertain. For a related Centaurea species, the annual yellow starthistle (C. solstitialis), large reductions in seed were not found to limit population growth (Garren and Strauss 2009). Thus, additional information on the effects of seed reduction for spotted knapweed is necessary. Parker (2002) demonstrated that the establishment of a species in a new community is a function of seed number and ‘safe sites’ that allow for seedling germination and survivorship. Germination and survivorship can be considered an interaction between the species and the current specific environment, including physical and chemical conditions for the seeds, as controlled by climate, the current status of the soil substrate, and the other plant species using that substrate. Thus, effects of reduced seed production (i.e., the use of seed predators on C. stoebe) on plant densities will vary as a function of climate and plant competition (Fig. 1). For example, a favorable climate for seedling recruitment could steepen the slope of the line shown in the figure, allowing higher plant densities because more seedlings survive for a given input of propagules. Alternatively, greater plant competition with establishing seedlings would decrease the slope and shift the asymptote to the right, increasing the importance of seed predators and requiring higher levels of seed production to maintain similar plant densities. The ability of plant communities to resist invasion (i.e., reduce ‘safe sites’) has been the subject of numerous studies. Experiments have focused on resistance to invasion based on species diversity and/or plant functional groups, and their interactions with resource availability (Schirman 1981; Pokorny et al. 2005; Seastedt and Suding 2007; Rinella et al. 2007; Zavaleta and Hulvey 2007; Maron and Marler 2007, 2008a, b). Constraints on the establishment of

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Seed predators ---------important------------------unimportant------

density limited by propagules

density limited by other variables

Plant density

Seed production per square meter Fig. 1 The hypothetical influence of seed production on resulting plant densities. Seed reduction by biological control insects is considered important to the left of the dashed vertical line, where densities of the plant (in this case, spotted knapweed) are limited by propagule pressure, but become less important at higher levels of seed production and where densities are limited by other variables such as the number of safe sites. The dark arrows depict how the vertical dashed line can shift left or right, and both the slope and asymptote of the line likely vary as a function of climate, soil characteristics, and plant competition

plants during or after the seedling stage, presumably due to inherent characteristics on the invaded plant community, were found to limit populations as much as limitations to population growth due to reductions in seed production (Turnbull et al. 2000). However, it is also possible for high propagule pressure to circumvent such community resistance (Holle and Simberloff 2005). We conducted a series of greenhouse and field experiments testing the ability of spotted knapweed seedlings to invade and establish under a range of competitive and resource environments. We used realistic densities of seed inputs, documented at a Colorado field site where root and canopy feeding biological control insects have reduced plant densities after several years, to elucidate whether there exists a threshold of seed production below which C. stoebe populations might be expected to collapse. The present study used seed production, as determined by the previous success of C. stoebe under seed predation, to evaluate seed germination and survivorship as a function of resource availability and plant competition. Our project had two goals. First,

Effects of plant competition, seed predation, and nutrient limitation

we measured the seed bank at an established spotted knapweed site that had been exposed to predation by seed-feeding weevils as well as other insects reported in the literature that are known to reduce seed production. We then evaluated the germination potential of that seed bank. Second, we tested the effect of soil resources and plant competition on the availability of ‘safe sites’. Using a greenhouse and field experiment, we applied C. stoebe seeds at densities observed under local field conditions, and measured recruitment (i.e., the germination and survivorship of a seedling through a growing season into the rosette stage). Specifically, we tested two hypotheses: (1) Recruitment increases as seed densities observed in the field increase and (2) plant competition, as modified by soil resource conditions, controls the number of ‘safe sites’, i.e., an interaction effect between plant competition and resource availability will regulate the success of recruitment.

Materials and methods Soil seed bank estimation and germination assays A soil seed bank estimate was obtained by counting seeds within the soil at a spotted knapweed infestation located approximately 15 km northwest of the city of Boulder, CO (40°070 31.1300 N 105° 190 09.6400 W). In the mid 1980s, C. stoebe was accidentally introduced to this area and has spread over 40 ha of meadows, riparian areas, and ponderosa pine [Pinus ponderosa Douglas Ex. Lawson (Pinaceae)] forest that had been burned by a wildfire in 1988. This fire apparently facilitated the invasion of other non-native forbs including dalmatian toadflax, Linaria dalmatica (L.) Mill. (Scrophulariaceae), sulfur cinquefoil, Potentilla recta L. (Rosaceae), and musk thistle, Carduus nutans L. (Asteraceae). The invaded meadow sampled in this seed bank estimate was located at 1,865 m elevation in a riparian area and contained a mix of non-native and native plants. These included the non-native crested wheatgrass, Agropyron cristatum (L.) Gaertn. (Poaceae), and the invasive cheatgrass, Bromus tectorum L. (Poaceae), in addition to the native western wheatgrass Pascopyrum smithii (Rydb.) A. Lo¨ve (Poaceae), blue grama, Bouteloua gracilis (Willd. ex Kunth) Lag. ex Griffiths (Poaceae), sideoats grama, Bouteloua

curtipendula (Michx.) Torr. (Poaceae), needle grass Hesperostipa comata (Trin. & Rupr.) Barkworth (Poaceae), wild bergamot, Monarda fistulosa L. (Lamiaceae), and spreading daisy, Erigeron flagellaris A. Gray (Asteraceae). Specialist biological control insects that likely colonized the site from nearby populations of Centaurea diffusa where they were introduced (Seastedt et al. 2007) were first observed at the site in 2001. These insects included two species of gall flies of the Urophora genus and the seed head weevil, Larinus minutus. These insects were supplemented during 2001–2005 with releases of approximately 100 Sphenoptera jugoslavica root weevils, 3,000 Larinus minutus weevils, and 2,000 Cyphocleonus achates root weevils. Seed production monitoring at the site has shown a substantial reduction between 2003 and 2009 (Knochel and Seastedt 2009). In late October 2008 after the majority of seeds had dispersed from seed heads produced that year, a single 15 m transect was positioned across the central portion of a meadow infested with spotted knapweed. The transect was placed 7 m from a permanent transect used to quantify C. stoebe abundance from 2003 to 2009 (Seastedt et al. 2007; Knochel and Seastedt 2009). At every 1-m interval, two 7 cm diameter 9 10 cm deep soil cores that included the surface litter were taken from within 0.25 m from each side of the measuring tape and combined (15 samples total). Percent cover of spotted knapweed was also visually estimated within a 1 m2 quadrat surrounding the location of each soil sample. Soil samples were returned to the lab and were allowed to air dry for 5 days, and seeds were then isolated from the soil by passing each sample through a series of soil sieves. Seeds were identified by visual inspection under a dissecting scope. All C. stoebe seeds (including any that were discolored, decayed, broken, or partially consumed) were counted from each composite soil sample and values converted to an estimate of total seeds m-2. Seeds were stored at room temperature for approximately 5 days until testing. A second visual inspection under a dissecting scope was conducted to estimate viability before the germination test. Seeds were counted as potentially viable if they appeared whole, and had an intact seed coat with a hardened pericarp that maintained rigidity under gentle pressure (crush test) (Nurse and DiTommaso 2005; Borza et al. 2007).

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All of the seeds (including the potentially nonviable ones) were then placed between filter paper soaked with tap water inside 15 individual petri dishes. These germination chambers were maintained between 20 and 30°C with 15 h daylight for 14 days. Any seeds that had not germinated after this time period were considered non-viable, as 98% of viable spotted knapweed seeds usually germinate within 6 days under similar conditions (Davis et al. 1993). Recent studies have found Larinus minutus to be the key reducer of seed production for both diffuse and spotted knapweed along the Colorado Front Range (Seastedt et al. 2007). We suspected that our past monitoring of reproductive output may underestimate seed reductions due to Larinus because we did not discriminate between partially damaged seeds and undamaged seeds in previous research. The other species occupying knapweed seed heads, Urophora affinis and U. quadrifasciata, form a gall that impedes seed production. However, viable seeds found within these capitula remain whole and are for the most part spatially separated from the Urophora galls, while seeds in closer proximity to the galls are completely destroyed and typically become fused with tissue enveloping the pupal chamber. To determine the effects of partial seed consumption by L. minutus on seed viability, we conducted a germination assay on seeds collected from capitula of mature C. stoebe at the field site. The seed used in this germination assay was obtained in August–September 2008. Three flower heads were collected on several dates from 114 randomly selected plants (N = 343) and kept at 4°C until dissection. Collections were timed with the maturation of seeds and pupation of the insects, but before seed dispersal. For each seed head, the number of seeds and presence or absence of the flower head weevil, Larinus minutus, or gall flies Urophora spp., were noted. In order to isolate effects of L. minutus on seed viability, seeds from flower heads containing live Urophora or evidence of Urophora presence (galls) were not used in the germination assay. Seeds from a subset of the collected flower heads meeting the following criteria were pooled into three categories for the germination assay: (1) Larinus present, seed damaged (n = 80 seeds), (2) Larinus present, seed not damaged (n = 100) or (3) Larinus absent, seed not damaged (n = 80). Seeds from the three categories were subdivided into groups of 20

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and subjected to the germination procedures previously described in the seed bank germination assay. Greenhouse seedling experiment In order to evaluate the effects of resource availability and plant competition on spotted knapweed recruitment, we conducted a seedling study inside the University of Colorado at Boulder greenhouse facility. The experiment began in October of 2007 and was terminated in March of 2008. Centaurea stoebe was sown individually or in combination with Elymus trachycaulus (Link) Gould ex Shinners (slender wheatgrass) into 60 3.8 l pots in a fractional factorial design (3 nutrient levels 9 2 competition conditions). Slender wheatgrass is a cool-season, short lived (3–4 years) perennial bunchgrass native to North America and found along the Colorado Front Range. The plant has rapid seedling establishment and represents a potentially useful species to establish quick plant cover and compete with invasive weeds in revegetation projects (USDA-NRCS 2009). This species was also chosen because it is often included in revegetation projects in Colorado. We filled 3.8 l pots (60 total) with soil (FAFARD Growing Mix #2; 70% peat with perlite and vermiculite, manufactured by Conrad Fafard, Agawam, MA) in October of 2007. Nine evenly spaced E. trachycaulus seeds were planted 1 cm below the soil in half (30) of the pots to establish the competition treatment. Plants were grown for 1 month with nutrient enriched water to stimulate the growth of the slender wheatgrass seedlings. These were switched to tap water prior to nutrient manipulations and introduction of spotted knapweed seed. Plants were grown under artificial lighting to expand day length to 14 h, approximating summer conditions. A density of 40 C. stoebe seeds per pot were then added evenly to the soil surface (but not buried) in all 60 pots. This corresponds to a density of 2,222 seeds m-2, above the 2003–2008 average C. stoebe seed production estimated at our field site. For the duration of the experiment, the watering schedule for all 60 pots followed spring precipitation amounts and timing from an average year in the Colorado Front Range (i.e., highly irregular rainfall followed by periods of dry weather). Daily water additions were calculated by converting actual 24-h precipitation totals (April–June 1989) to an


equivalent volume of water over the area of soil within a pot. Using this method, plants received variable water inputs and timing, and soils occasionally became dry when several days passed without addition. Soil nutrient availability was held at three levels: nitrogen (N) addition (as ammonium nitrate), no amendments, and carbon (C) addition (as sucrose) to reduce N availability (Blumenthal et al. 2003). For fertilized soils, we added 0.2 g NO3NH4 to each pot on three dates, with the first addition a week prior to the introduction of the C. stoebe seeds, then twice more at monthly intervals (a rate of approximately 11 g N m-2 year-1) The ambient level of N availability served as control pots with no added amendments. We added 4 g of sucrose to each pot on the same dates as N additions, at a rate approximately equal to 252 g C m-2 year-1, to reduce N availability. Tiller height, number of plants, and number of leaves of E. trachycaulus were quantified just prior to beginning the nutrient amendments and C. stoebe seed addition. We verified that there were no significant initial differences in the level of established native competition across pots (data not shown). Following this estimate, grass numbers and height were not quantified but total biomass was estimated at the termination of the experiment. As germination of C. stoebe occurred throughout the experiment, cumulative numbers of C. stoebe seedlings were counted at approximately 4 day intervals, with a final census during destructive sampling for plant biomass. The close proximity of seedlings and presence of grasses made it difficult to monitor mortality of individual seedlings that occurred between counts. Thus, counts reflect the sum of newly germinated plants plus those individuals surviving from the previous count. Above and below ground tissues were collected and separated by species from each of the pots upon termination of the experiment in March of 2008. Aboveground tissues were clipped at the soil surface and belowground biomass was separated from the soil and rinsed to remove all soil particles. Root biomass, particularly the fine roots of grass seedlings, was under-harvested due to the difficulty of separating roots from the soil media; however, harvest of these root tissues was consistent among treatments. All biomass was oven dried at 60°C for 48 h and weighed.

Field sowing experiment To evaluate the effect of propagule density on successful recruitment, Centaurea stoebe seed was introduced into an un-invaded meadow at three densities matching a gradient of levels of seed production monitored at our field site under the constraints of multiple introduced herbivores (Knochel and Seastedt 2010). The seeding experiment spanned 2.5 growing seasons (April 2007 to June 2009) in a spotted knapweed-free meadow 0.5 km downstream from the same infestation of spotted knapweed previously described in the seed bank estimate. The experimental area has a 5% slope with a slight southeast aspect, about 10 m above the stream channel. Soils at the site are derived from both igneous and sedimentary materials, and meadows along the gulch drainage-way are of mixed loamy alluvium parent material, with a stony sandy clay loam profile at 0–100 cm depth (NRCS 2009). Moderate cattle grazing within and around this study area occurred since the early 1900s, but this experiment was fenced to prevent grazing interfering with experimental treatments. During the preceding year (2006), herbivores removed the majority of standing biomass of the extant herbaceous community, and bare ground at the time of seeding comprised approximately 20% of total cover, but decreased to near 0% bare ground from 2008 to 2009. The extant plant community was composed primarily of the native grass species listed previously for this site, and these species were relatively homogenous across the area. Of note was a stand of the nonnative weed, sulfur cinquefoil (Potentilla recta), that was inconspicuous at the time of seeding but by 2009 dominated the vegetative cover in one-third of the plots. Plots with P. recta were evenly distributed among treatment groups, with 4–5 plots per treatment containing the plant. To verify that the existing seed bank contained no C. stoebe seed, a seedling emergence test was conducted in the greenhouse. Soil samples to 4 cm depth from five random locations within the experimental area were spread to a thin layer on trays and kept moist while the germination of C. stoebe seedlings was monitored over a 2 weeks period. We established 110 1 m2 plots in a fenced 35 9 15 m area with 0.5 m spacing between plots. Each plot represented a single randomized replicate

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in a fractional factorial design with C. stoebe sown at four densities in 2007 and 2008, either in monoculture or in combination with one addition of native seed added in 2007 at a single density (4,154 seeds m-2). In the C. stoebe monoculture plots and in plots with native seed addition, C. stoebe was added at a rate of 568, 1,136, or 2,272 seeds m-2 year-1, with 15 replicates per treatment combination (n = 90). An additional 20 replicate plots had native seed added but 0 C. stoebe seeds m-2 year-1 (N = 110 total). No plots were established with a treatment of 0 C. stoebe and 0 native seed. Because the majority of C. stoebe seeds require exposure to light for germination (Nolan and Upadhyaya 1988), we limited the soil seed bed preparation to light raking to promote seed–soil contact while minimizing disturbance and simulating the conditions under which spotted knapweed seeds would naturally reach the soil surface. The C. stoebe seed used in this experiment was collected in 2002 from the same population at this field site and stored at room temperature in sealed plastic. Because stored seed may have reduced germination compared to freshly harvested seed or seed remaining dormant in the soil, a soil germination assay was conducted to estimate the germination rate of this seed source prior to experimental sowing. Seeds (N = 280) were planted in groups of four just below the soil surface in 70 tree seedling containers filled with FAFARD Growing Mix #2, and soils were kept moist for 2 weeks while germination was recorded. The upslope half of each plot (0.5 m2) was lightly disturbed using a thatch rake to expose the soil bed, and on 15 April 2007, seed of C. stoebe alone or in combination with native herbaceous species were hand broadcast onto the exposed soil surface. Any surface litter was then raked back over the sown area. To facilitate seedling counts, seeds were introduced only to half (0.25 m2) of the raked subplot. To simulate a second year of spotted knapweed seed production, and because the 2002 seed source had a lower germination rate than those reported for freshly collected seed (Davis et al. 1993), on 9 April 2008 we added a second round of C. stoebe seeds to the same subplots and at the same densities. Procedures for adding seed followed those used in 2007, with the exception that in 2008 seed was broadcast onto the surface and plots were not raked, in order to limit disturbance to already established seedlings. Thus, the cumulative densities of spotted knapweed

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added after April 2008 were 0, 1,136, 2,272, or 4,544 seeds m-2. As mentioned, half of the C. stoebe addition plots were also seeded in 2007 with a mixture of native grasses and forbs at a density of 4,154 seeds 0.25 m-2. Native species and addition rates were adapted from general rangeland revegetation guidelines (Goodwin et al. 2004) to meet the specific characteristics of the field site, and to follow suggestions by Choi (2007) and Seastedt et al. (2008) to use a diversified mix of seeds to guarantee germination and survivorship across a wide range of potential rainfall conditions. The mix included common warm and cool-season grasses and forbs native to the area and found at the research site, including xeric and mesic-adapted species used for native revegetation along the Colorado Front Range (Knochel 2009). Recruitment of C. stoebe was then monitored over 2.5 growing seasons by counting the number of C. stoebe seedlings within each plot September 2007, May, June, and November of 2008, and in early June 2009. Experimental plots received full to partial sunlight and were not beneath the canopy of P. ponderosa or other woody vegetation. Precipitation during the 7 months preceding this experiment (April 2006 to March 2007) was 51.7 cm or 100.5% of the 30 year average 1971–2000 precipitation recorded in Boulder, CO, about 15 km south of field site (NOAA 2009). During the 2007 and 2008 growing seasons (April–September), precipitation was 22.7 and 30 cm (66.8 and 88.3% of average, respectively). Our seedling census ended in early June 2009, and in April–May of the 2009 growing season, precipitation was 22.8 cm (149.8% of the 15.2 cm 30 year April–May average). These precipitation data concurred with those recorded at a climate station 4 km south of the field site (CO-BO-232, 40°040 58.8000 N, 105°200 40.2000 W) at a similar elevation (CoCoRaHS 2009). In an attempt to control for reduced precipitation, plots received supplemental water on a weekly basis only during June–July 2007 when precipitation dropped below 80% of the 30 year average. Statistical analysis Linear regression analysis was used to analyze the relationship between percent C. stoebe cover and the soil seed bank estimates. Spotted knapweed seed


germination, and biomass measurements for knapweed and E. trachycaulus were examined using analysis of variance (ANOVA) procedures, and means were compared using a-posteriori RyanEinot-Gabriel-Welsch multiple-range tests (SAS 2009). To assess intraspecific competition within C. stoebe monoculture, SAS PROC GLM was used with soil N level as the categorical predictor, and number of seedlings present on the last sampling date as the continuous predictor. Across multiple sampling dates in the greenhouse seedling study, SAS PROC MIXED repeated measures ANOVA was used to assess spotted knapweed seedling counts using soil or grass neighbor treatments as between-subject fixed factors, and date as the repeated within-subject factor. Initial grass tiller measurements were not used as continuous covariates because these values did not vary among pots. A first-order autoregressive covariance structure was used in repeated measures following Akaike’s Information Criterion (AIC) and Schwarz’s Bayesian Criterion (SBC). Variables were also log transformed if necessary to fulfill assumptions of normality of variance, and then backtransformed for clarity and the relevance of values within figures. Seedling recruitment data in the sowing study contained a high number of zeros and violated assumptions of normality, so these were analyzed using a non-parametric ANOVA (Kruskal– Wallis test) and evaluated with descriptive statistics.

Results Soil seed bank and germination tests The seed bank of the study site with a spotted knapweed infestation contained a mean density of 5,848 ± 1,172 (SE) (N = 15) total C. stoebe seeds m-2 (ranging from 0 to 16,364 seeds m-2), and 92% of the seed found in soil cores (N = 386) was shriveled, discolored, and/or partially decayed. Using the seed crush test, we estimated a potential soil seed bank viability of 8% or 468 ± 94 seeds m-2 for this location in the infestation. Along the sampled transect, C. stoebe represented on average 32 ± 9% of the vegetative cover (ranged from 0 to 90%) within each 1 m2 sampling point. No significant relationship was detected between C. stoebe cover and number of C. stoebe seeds isolated from the soil samples. The

actual germination rate of all seed isolated from soil samples after 14 days was 0%. In the germination assay on seeds from the 2008 seed head collections, the germination rates were as follows: (1) Larinus present, seed damaged, 20 ± 5% (n = 80), (2) Larinus present, seed not damaged, 53 ± 7% (n = 100) and (3) Larinus absent, seed not damaged, 59 ± 8% (n = 80). Overall, the germination rate for undamaged seeds in groups 2 and 3 (56 ± 5%) was significantly higher than the rate for seeds at least partially damaged by L. minutus weevil herbivory (group 1, F1,12 = 11.26, P = 0.007). The germination rate for undamaged seeds produced in flower heads with L. minutus present that had escaped direct damage from the insect (2) was not significantly different from the germination rate for undamaged seeds in flower heads where Larinus was absent (3). Greenhouse seedling experiment Under elevated soil N conditions, C. stoebe increased its total biomass by 62 and 149% over the control and low N treatments, respectively (Fig. 2; Table 1), and decreased its root: shoot ratio by about 87% in high N soils compared with growth in the control or low soil N treatments (Fig. 2; Table 1). When planted in pots with Elymus trachycaulus grass competition, the total biomass produced by C. stoebe was reduced to near zero (Fig. 2). Elymus trachycaulus exhibited a modest total biomass response to high soil N, with an increase of 4 and 42% above control and low N soils, respectively. Similar to knapweed, the root: shoot ratio of the grasses decreased by 62 and 73% compared to control and low N soils (Fig. 2). For C. stoebe planted in monoculture, the final number of seedlings per pot, nutrient treatment, and their interaction, showed an overall significant relationship with total shoot biomass per pot (R2 = 0.90, F5,25 = 42.26, P \ 0.001), but not with root biomass. The soil nutrient by seedling density interaction term was also significant, and subsequently, the relationship of biomass to spotted knapweed seedling number in the three soil treatments was analyzed. Total shoot biomass and seedling number per pot were negatively correlated for control soils (R2 = 0.77, F1,9 = 27.42, P \ 0.001), marginally correlated within low N soils (R2 = 0.32, F1,9 = 3.84, P = 0.08), and showed no relationship in the N fertilized soil treatment.

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(A)

7 C

6

B

Total biomass (g)

A

5

C

+ +

+

4 B

+

3 A

+

2 +

1 A

0

A

presence of grasses was significantly different between all three soil treatments (F2,29 = 39.59, P \ 0.001), with numbers highest in the low N soils, lowest in high N soils, and intermediate in the control (Fig. 3). The differences between seedling recruitment in low N versus high N soils also tended to increase over time in the presence of competition (Fig. 3). During the last 20 days of sampling, % recruitment in low N and control pots continued to increase, while in high N pots the seedling recruitment stabilized. In addition, seedling germination was initially higher in the absence of competition.

A

Field sowing experiment (B)

2 B C

Root:shoot ratio

1.5

+ + B

B B +

1

+

+

A

0.5

A A +

+

+ A

0 Low

Control High C. stoebe alone

Low Control High C. stoebe + grass

Low Control High E. trachycaulus

Fig. 2 a Total biomass and b root to shoot ratio of C. stoebe seedlings growing alone (left panel) or with grasses (middle panel), and biomass and root:shoot of E. trachycaulus seedlings in pots also containing C. stoebe, in carbon amended (low), control, or fertilized (high) soils. Boxes represent lower and upper quartiles, (?) indicates mean, the center horizontal line is the median, and whiskers extend to the most extreme value within 1.5 times the interquartile range, with extreme points outside this boundary plotted as dark circles. Different uppercase letters indicate a statistical difference in biomass or root:shoot between soil treatments but within plant treatments, at P \ 0.05

In an analysis of seedling recruitment over time, soil N, grass presence, sampling date, and all interactions significantly affected seedling recruitment (Table 2). For C. stoebe seedlings growing without competition, on the final sampling date seedling numbers were significantly lower for plants growing in high N soils versus control or low N soils (F2,29 = 8.79, P = 0.001) (Fig. 3). Seedling recruitment in the

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The C. stoebe seed collected in 2002 and used in the field sowing experiment had a germination rate of approximately 58%. At the end of the second growing season in November 2008, C. stoebe seedling recruitment across all seeded plots was 0.066% (N = 90) (Table 3). Thirty-two seedlings occurred in 19 different plots, with 15, 8, and 9 seedlings observed in the high, medium, and low sown densities, respectively. We tested for differences in recruitment of C. stoebe only in plots where seeds were added, omitting the 20 additional plots with 0 spotted knapweed seeds m-2. Centaurea stoebe seedlings were never observed in plots that were not augmented with seeds, reflecting the fact that these plots were located in a previously uninvaded area lacking a C. stoebe seed bank. On the three previous sampling dates in 2007 and 2008, six total seedlings were observed among four plots. These six seedlings that were present in Sep-2007 were absent from the same plots a year later in Nov2008. Thus, the 32 seedlings recorded in November 2008 had all emerged during the 2008 growing season. Non-parametric ANOVA (Kruskal–Wallis test) revealed no differences in 2008 C. stoebe recruitment between the sowing densities (v2 (df = 2, N = 90) = 1.57, P = 0.46), nor between the native sowing treatment (v2 (df = 1, N = 90) = 0.71, P = 0.40). On the last sampling date in June 2009, seedling recruitment across all plots was 0.057%, slightly below the recruitment number from the previous fall. We recorded 44 total C. stoebe seedlings among 19 plots, with an average of 1.03 ± 0.35, 0.20 ± 0.12, and 0.23 ± 0.11 seedlings in the high, medium, and

Effects of plant competition, seed predation, and nutrient limitation Table 1 ANOVA results of (a) C. stoebe and (b) E. trachycaulus seedling biomass harvest for the effects of soil nutrient and competition treatments on plant response variables Variables

Soil N df

Grass presence F

P

Soil 9 grass

df

F

P

df

F

P

385.3

\0.001

1, 45

14.20

\0.001

\0.001

2, 45

4.83

0.013

(a) C. stoebe Total biomass

2, 45

6.78

0.003

1, 46

Root:shoot ratio

2, 45

66.48

\0.001

1, 46

Total biomass

2, 15

4.82

0.026

Root:shoot ratio

2, 15

28.65

(b) E. trachycaulus \0.001

38.6

N = 90) = 7.46, P = 0.024), but no difference based on the native sowing treatment (v2 (df = 1, N = 90) = 0.08, P = 0.78). Herbivore damage on the leaves of several seedlings was noted during the three growing seasons, although herbivory itself was not observed. The vast majority of seedlings recorded in plots were less than 6 cm wide, and had four total leaves (two cotyledon leaves and two true leaves). By June 2009, a few seedlings had reached a basal diameter typical of a rosette, but none appeared to be of sufficient size to initiate flowering that summer.

Table 2 Repeated measures ANOVA results of C. stoebe seedling recruitment on 17 dates over 3 months Variables

Seedling recruitment df

F

P

Soil N

2, 58

10.98

\0.001

Grass presence Grass 9 soil N

1, 59 2, 58

51.15 3.44

\0.001 0.0381

Date

16, 59

12.06

\0.001

Date 9 soil N

32, 58

4.20

\0.001

Date 9 grass presence

16, 59

9.18

\0.001

Number of C. stoebe seedlings present

low density plots, respectively (Fig. 4). Twenty-five of these seedlings germinated in the late fall of 2008 or spring of 2009, while 13 of the 32 seedlings that were present in Nov-2008 were absent from the same plots in June 2009, indicating a 40.6% mortality rate. There was a significant difference in 2009 recruitment between the sowing densities (v2 (df = 2, 30

Discussion Plant competition greatly inhibited the survival and growth of germinating seedlings in greenhouse manipulations (Fig. 2), while results from field plots

E. trachycaulus present

E. trachycaulus absent

25

B B

20

15 10 5

Control N High N Low N

C B

A Control N High N Low N

A

0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Day

Fig. 3 Cumulative Centaurea stoebe seedling recruitment (number of seedlings present) in pots without competition (left) or with E. trachycaulus competition (right), and differing in soil nitrogen availability over an 80 day period. Points and

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Day

bars are means ± 1 SE (n = 20) for each sampling date. Different uppercase letters denote a significant difference in seedling numbers between soil N treatments on the final day of sampling

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D. G. Knochel et al. Table 3 Mean percent recruitment of C. stoebe in plots seeded at three densities and with or without the addition of native grass and forb seeds, at the Boulder field site from Fall 2007–June 2009 Treatment

Observed recruitment (plants present/C. stoebe seeds sown) (mean% ± SE) Sept 2007

April 2008

June 2008a

Nov 2008

June 2009*

0.02 ± 0.008

0.001

0.003

0.066 ± 0.008

0.057 ± 0.015

2,272 m-2 year-1 (568/plot/year, n = 30)

0.02 ± 0.018

0.004

0.009

0.044 ± 0.016

0.091 ± 0.031

1,136 m-2 year-1 (284/plot/year, n = 30)

0.02 ± 0.016

0

0

0.047 ± 0.024

0.035 ± 0.021

568 m-2 year-1 (142/plot/year, n = 30)

0

0

0

0.106 ± 0.042

0.082 ± 0.040

0

0

0

0

0

Mean of all C. stoebe addition plots (N = 90) (a) C. stoebe sowing density

0m

-2

year

-1

(0/plot, n = 20)

(b) Native species seed Added (4,154/plot, n = 65)

0.005

0

0

0.057 ± 0.021

0.057 ± 0.022

Not added (0/plot, n = 45)

0.02 ± 0.014

0.003

0.006

0.049

0.057 ± 0.019

* A significant difference in recruitment between sowing densities was detected in 2009 (Kruskal–Wallis test) a

Percent recruitment values listed from June 2008 through June 2009 were calculated based on the cumulative density of C. stoebe (1,136, 568, 284, and 0 seeds/plot) after the second C. stoebe seed addition in the spring of 2008

C. stoebe plants present after 3 years

1.6

B 1.4 1.2 1 0.8 0.6

A

0.4

A

0.2

A 0

2,000

1,000

500

0

Density of C. stoebe seeds sown · m-2 ·yr-1

Fig. 4 Total number of Centaurea stoebe seedlings present in field plots after 3 years based on densities of seed added

indicated extremely low recruitment (Fig. 4). An unexpected result from the greenhouse experiment was that the survivorship of C. stoebe seedlings in the presence of a competing grass species was enhanced by low resource availability (Fig. 3). These surviving seedlings were extremely small (Fig. 2), and their survivorship may be short-lived, but the results from this study present strong evidence that resource availability can alter competitive relationships in unexpected ways (Rand 2004). Interactions with mycorrhizae fungi could have influenced these outcomes (Marler et al. 1999), but we have no data to

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support or refute this. The resource conditions that potentially weaken resistance of plant communities to C. stoebe invasion deserves further study, as many temperate ecosystems are N limited. These results support other work showing that in some cases, low N conditions could increase the success of spotted knapweed invasion (Mangold and Sheley 2008). Overall, however, the presence of competing grasses greatly reduced C. stoebe seedling biomass (Fig. 2), and decreased recruitment by 43% across all sampling dates regardless of soil N availability. The ability of a cool season bunchgrass to out-compete spotted knapweed in conditions of surplus N is reflected in both the seedling biomass responses (Fig. 2) and seedling recruitment over time (Fig. 3). We realize grass seedlings were well established before the addition of C. stoebe seed, and priority effects could significantly change the competitive outcomes had seeds of the two species been added concurrently. However, we used this greenhouse experiment as a complement to the field portion of this study in an attempt to assess the importance of resource availability in moderating competitive relationships and invasibility of spotted knapweed into existing plant communities. By establishing the grasses first, we were investigating spotted knapweed’s ability to invade rangelands inhabited by perennial plant communities and with lower levels of disturbance. The densities and abundance of spotted knapweed in some regions of the west may be largely


a symptom of excessive or unsustainable land use over multiple decades, combined with the absence of natural enemies, rather than being primarily the result of characteristics that make this plant as an aggressive invader (e.g., Hill and Germino 2005). These introduced herbivores greatly reduce seed production, and if seed reductions significantly contribute to reductions in C. stoebe densities as we have documented at our field site (Knochel and Seastedt 2009), then it would be worthwhile to understand how native plant competition potentially governs the manner in which reduced seed densities may lead to population collapse. Centaurea stoebe also experienced intraspecific competition and self-thinning (sensu Yoda et al. 1963; Weller 1987), based on the significant negative correlation between C. stoebe biomass per pot seeded at a density of 2,000 seeds m-2 versus seedling density per pot. The responses of seedlings to resource availability that were demonstrated here, as well as the ability of C. stoebe to benefit (in numbers, but not in biomass) under low N conditions when in the presence of competition, are findings not previously reported in the literature. We realize that soil N availability is probably secondary in importance to light (Nolan and Upadhyaya 1988; Hill and Germino 2005) and water (Hill et al. 2006; Maron and Marler 2008a, b) in controlling seedling recruitment. Nevertheless, N was found to modify seedling performance by directly increasing spotted knapweed growth, and indirectly through its positive effects on neighboring grasses. Thus, similar to the factors that control diffuse knapweed recruitment, increased water availability (Blumenthal 2009), or disturbances that reduce competing vegetation (Seastedt and Suding 2007), appear to exacerbate any advantages conferred by growth responses of spotted knapweed to higher soil N levels. These results add to the literature suggesting that ‘safe sites’ are a function of plant competition and soil resource interactions. Our study also supports the model that Centaurea species exhibit a surprising range of growth and can respond opportunistically to favorable environmental conditions, but are nonetheless still limited by absolute resource availability as moderated by the presence of plant competition. Generally, it might be expected that higher resources would improve the success of non-native plant invasions and perhaps lessen the importance of

propagule inputs. In Fig. 1, this would shift the dashed line toward the left and lessen the breadth of conditions under which ongoing seed predation is important (i.e., high plant densities are maintained even with low seed production). This is because over time, high growth and fecundity of the few seedlings that survive may balance reduced propagule inputs (Garren and Strauss 2009). However, in our greenhouse experiment, when high soil resources occurred in the presence of competing vegetation, increased growth of the native competitor greatly reduced the ability of C. stoebe seedlings to benefit from those resources. In essence, the effects of seed predators might become important under a wider range of seeds produced. In this case, plant densities are reduced through seed predation because seedling survivorship is strongly regulated by the interactions of plant competition and soil resources. In Fig. 1, high soil resource availability would instead shift the dashed line toward the right. In the field plots, extremely low seedling recruitment observed over 2.5 growing seasons was consistent with the findings in Montana by Pokorny et al. (2005). The amount of seed potentially lost from plots due to climatic factors or predation (Jensen and Six 2006) is unknown, but even with considerable loss, recruitment would be expected to be much greater given the high numbers of seed added. Because so few C. stoebe seedlings survived over the experimental period, it was difficult to rigorously assess the influence of propagule density on recruitment. Recruitment was equally low across all densities of sown seed, except in 2009 when high density plots had almost triple the number of seedlings as medium and low density plots. We also found no difference between recruitment of C. stoebe sown in monoculture versus recruitment in the presence of seeded natives. Although the presence of native seedlings was not monitored, it is likely that these species experienced similarly low recruitment in the presence of established vegetation. These results show that, under the environmental conditions found at our study site, spotted knapweed is greatly inhibited from establishing in the presence of significant plant competition. While not part of this study, disturbance from fire and cattle grazing activities prior to our research may have provided additional safe sites for the establishment and spread of C. stoebe throughout this drainage.

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The flower weevil Larinus minutus, in addition to gall flies (Urophora spp.) and the root feeder, Cyphocleonus achates, have greatly reduced plant reproductive output over a 7-year period at this field site, as reflected in estimates of seed rain (Knochel and Seastedt 2009) and the diminished viable seed bank reported here. The majority of seeds within a flower head are either partially or completely consumed by developing L. minutus larvae. Seastedt et al. (2007) demonstrated that seed production per flower head is reduced by over 50% by the presence of L. minutus, and the number of flower heads produced per plant attacked by C. achates is reduced as well (Knochel and Seastedt 2010). Seeds damaged but not consumed by L. minutus exhibited a germination rate of approximately 20%, while germination of undamaged seed was 60%, a germination rate close to that found in another seedling survivorship study on C. stoebe (Pokorny et al. 2005). Our seed bank survey found substantial numbers of spotted knapweed seed in the soil under a moderately dense stand of spotted knapweed. Surprisingly, the vast majority of seed in the soil was damaged or decayed, and none of the seeds that appeared viable upon visual or physical inspection actually germinated. Thus, we suggest that future viability estimates of spotted knapweed seed banks should go beyond visual inspection and use germination tests. In another seed bank estimate conducted at two sites in Montana, Jacobs and Sheley (1998) found an average of 5% viability of the seed bank in areas where knapweed density was near 109 stems m-2 and total seeds in the soil reached 55,000 m-2. Similarly, Story et al. (2008) determined that seed banks contained from 19 to 281 seeds m-2 depending on knapweed density, and estimated the critical threshold for population maintenance in their sites in the Northwestern US to be 160 seeds m-2. Using the overall seedling survivorship estimated over 2.5 years (0.057%) in the field sowing experiment, we calculated that it will take an input of roughly 2,710 seeds m-2 year-1 to produce 1 plant m-2 in the presence of established vegetation. This survivorship estimate is based on the cumulative experimental seed input of 4,454 seeds m-2 over 2 years. In contrast, a study that added a total of 7,500 C. stoebe seeds/m2 over 2 years to plots varying in species diversity and water addition, recruitment was much higher: approximately 200 seedlings m-2, or 2.6%, survived by the second year (Maron and Marler

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2008a, b). This number extrapolates to an input of just 38 seeds m-2 to produce one plant after 2 years. In that experiment, however, the area was previously used for an organic farm and known to be high in soil nutrients, and C. stoebe appears to have been seeded into plots with larger gaps in the vegetation than those used here. Lastly, Pokorny et al. (2005) added 2,000 seeds m-2 to plots either intact or with functional group removals. In the intact plots, seedling recruitment was near zero. Clearly, site-specific conditions exhibit large effects on germination and seedling survival rates, and findings from areas with undisturbed soils (i.e., free from intense animal or human activities, and with extant vegetation occupying a majority of open ground) suggest that a very strong limitation on spotted knapweed recruitment can occur. Centaurea stoebe, unlike other species in the Centaurea genus (Sheley et al. 1983), may have an innate or induced seed dormancy that is at least partially due to soil burial and light inhibition (Nolan and Upadhyaya 1988). Depending on the strength and interaction of a variety of environmental, biotic, and soil microsite conditions, a small portion of C. stoebe seeds can germinate after eight or more years in the soil seedbank (Davis et al. 1993; Nolan and Upadhyaya 1988). However, results from the population studied here indicate that despite dormancy mechanisms, the viable seed bank has been largely exhausted approximately 15 years after the area was invaded, and 7 years following the arrival of biological control herbivores. We therefore conclude that a combination of seed limitation and shortage of ‘safe sites’ within intact vegetation can limit densities of C. stoebe, a finding consistent with Story et al. (2008). Herbivore-caused reductions in seed output, combined with a paucity of surviving C. stoebe seedlings in the presence of plant competition, is believed to effectively constrain invasions and population growth of spotted knapweed in western rangelands. Acknowledgments We thank Christine Fairbanks, Kali Blevins, Justin Feis, and Dr. Nataly Ascarrunz for field and lab help. Drs. Deane Bowers, William Bowman, Carol Wessman, and Susan Beatty provided valuable input on drafts of the manuscript. We also thank Linda and Sergio Sanabria for use of their land to conduct research, and Tom Lemieux for assistance in the greenhouse. This work has been funded by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 06-03618.


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