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Pollinators may not limit native seed set at puget lowland prairie restoration nurseries

Pollinators may not limit native seed set at puget lowland prairie restoration nurseries Journal of Pollination Ecology, 15(5), 2015, pp 30-37 POLLINATORS MAY NOT LIMIT NATIVE SEED SET AT PUGET LOWLAND PRAIRIE RESTORATION NURSERIES 1, 1 2 Jennie F. Husby * Carri J. LeRoy , Cheryl Fimbel The Evergreen State College, 2700 Evergreen Parkway NW, Olympia, Washington 98505, USA Center for Natural Lands Management, 120 E, Union #215, Olympia, Washington 98501 , USA Abstract—Land managers often rely on large-scale production of native seeds in nurseries for replanting into natural environments as part of restoration strategies. Nursery managers question if unmanaged insects can be sufficient to pollinate large increases in native forbs planted into young nurseries in non-native landscapes. This study investigated pollination of deltoid balsamroot (Balsamorhiza deltoidea Nutt.) and sicklekeel lupine (Lupinus albicaulis Douglas) at a native seed nursery compared to dense patches of native plants at a natural Puget lowland prairie to determine if insect visitation affected viable seed production for those two species. In 2011 and 2012, insect visitation rates were recorded for each plant species at more than 62 plots within two study areas. In 2012, seeds were collected from hand-pollinated and naturally-pollinated inflorescences and tested for viability. Overall visitation rates were significantly higher at the nursery than the prairie for both plant species. However, pollen limitation was not evident for either plant species at either site. Natural pollination by insects and supplemental hand-cross-pollination treatments did not yield different quantities of viable seed. Factors other than pollinator visitation, such as soil nutrients and seed handling practices, may be influencing seed viability, but increasing insect visitation will not likely significantly increase seed viability for these two species at this restoration nursery. Planting dense rows of native plant species may be enough to attract a sufficient amount of unmanaged insects to provide adequate pollination for seed production for some species at even young nurseries. Keywords: restoration nursery, pollen limitation, insect visitation, prairie restoration, Balsamorhiza deltoidea, Lupinus albicaulis Several aspects of pollination can influence the production of INTRODUCTION viable seeds. Insect visitation rates can positively affect pollen receipt (Engel & Irwin 2003). Differences in pollinator Pollinators play a key role in the reproduction of wild community structure can affect overall pollination plants as they are linked to viable seed production and native effectiveness (Perfectti et al. 2009) and pollinator behaviour, plant population growth. Pollinators and their activities thus such as order in which a pollinator visits flowers, can affect provide an ecosystem-wide service, especially for landscapes whether a flower is self-pollinated or cross-pollinated (Kunin with many insect-dependent forb species like prairies 1993). When plants are pollen-limited due to insufficient (Kremen et al. 2007). Even self-compatible plant species pollinator activity, they are pollinator-limited (Dieringer may rely on pollinators to provide conspecific pollen to mix 1992). genes, preventing inbreeding depression (Heschel & Page 1995; Price et al. 2008). Native seed from nurseries plays an The Puget lowland prairie ecosystem has been important role in ecosystem restoration. Ecosystems in need fragmented by coniferous forest encroachment and urban of conservation attention may be stressed by factors such as and agricultural development so that now only 2-3% of the invading species, fragmentation, and climate change; all of original habitat remains (Dunwiddie & Bakker 2011). Re- which can suppress a plant species’ population size and limit establishing native flora has been a priority of Puget lowland its reproductive ability (McCarty 2001; Vila & Weiner prairie conservation partners (Stanley et al. 2008), and these 2004; Fazzino et al. 2011; Tscheulin & Petanidou 2011). partners rely on large-scale production of native seeds in Many restoration practitioners depend on native seed grown their nurseries for replanting into the Puget lowland prairies in nurseries for repopulating plant species in natural areas. as part of their restoration strategies. Some years restoration Native plant nursery managers strive to produce large practitioners have struggled to produce large quantities of quantities of high quality seed to meet restoration demands. viable seeds for certain plant species at their largest nursery, Webster Nursery. These managers have questioned whether When plants produce fewer viable seeds than maternal or not two critical restoration species, Balsamorhiza resources allow because of insufficient quantity or quality of deltoidea Nutt. (Asteraceae) (deltoid balsamroot) and pollen, they are pollen-limited (Wagenius & Lyon 2010). Lupinus albicaulis Douglas (Fabaceae) (sicklekeel lupine), are producing the highest proportion of viable seeds possible Received 17 July 2014, accepted 09 February 2015 at this restoration nursery and if there is a way to increase the *Corresponding author; email: JennieHusby@gmail.com proportion. Results of testing by the nursery in 2013 30 March 2015 POLLINATORS AND SEED SET IN NURSERIES 31 revealed that Webster Nursery produced 1,424 g of B. pods, including those that were empty (likely due to ovule deltoidea seed with 38.8% viability, and 10,299 g of L. abortion). The L. albicaulis inflorescences tested in this albicaulis seed with 35.3% viability (S. Smith, Center for study had an average of 48.1 flowers per inflorescence. Little Natural Lands Management, pers. comm.). The cause of this is known about the pollination system of L. albicaulis. problem may be due to seed handling or storage techniques, Lupinus species in general are typically self-compatible, inadequate environmental conditions where the plants are though some require a pollinator to trigger autogamy and grown (such as soil nutrients, weather, etc.), seed predation, have increased seed set when cross-pollinated (Kaye 1999, or pollen limitation. This study addressed the latter by Kittelson & Maron 2000). investigating the status of pollination at Webster Nursery Study Areas and comparing it to dense stands of native plants at a natural Puget lowland prairie to determine if inadequate pollination Washington Department of Natural Resources (DNR) restricted viable seed production at the nursery. owns Webster Nursery, a portion of which is leased and managed by conservation partners to produce seed from More specifically, to better understand the role of native plants at a large scale for restoring Puget lowland pollination at a native seed production facility, we prairies. The plants are grown outdoors in dense rows. The investigated the following research questions: (1) Do insect native seed nursery was first established in 2008 with partial visitation rates to dense floral patches differ between a rows (~100 m) of 10 species. The rows planted with B. nursery site and a natural prairie site for two prairie forbs? deltoidea and L. albicaulis were last fertilized during their (2) Is there evidence of pollen limitation for either plant installation in 2008, are watered only by rain, and were not species at either the nursery or prairie sites? sprayed with pesticides or herbicides in 2011 or 2012 (Angela Winter, nursery manager, pers. comm. 2012). Other MATERIALS AND METHODS species grown at Webster Nursery include: hookedspur violet (Viola adunca), spring gold (Lomatium utriculatum), Study Plant Species nine-leaf biscuitroot (Lomatium triternatum), shortspur We focused this study on two native prairie plants, B. seablush (Plectritis congesta), slender cinquefoil (Potentilla deltoidea and L. albicaulis. Both plant species grow along the gracilis), harsh Indian paintbrush (Castilleja hispida), golden west coast of the United States and into Canada (USDA paintbrush (Castilleja levisecta), sea pink (Armeria Natural Resources Conservation Service 2012). These plants maritima), Nuttall’s Larkspur (Delphinium nuttallii), are both found at natural prairie sites and are being produced woolley sunflower (Eriophyllum lanatum), buttercup from seed at Webster Nursery, Tumwater, WA, USA. (Ranunculus occidentalis), Pacific lupine (Lupinus lepidus), bicolor lupine (Lupinus bicolor), farewell to spring (Clarkia Deltoid balsamroot (B. deltoidea) is a species of amoena), and camas (Camassia quamash). Farmland, open potential concern in Washington State (Washington Natural grassland, residential properties, and forested areas surround Heritage Program 2012), its flowers are popular with insect the 5.3 km nursery (Tab. 1, Fig. 1). visitors, and it is a valued restoration plant. The federally endangered butterfly, Taylor’s checkerspot (Euphydryas The US Department of Defense manages Johnson editha taylori), frequently uses this plant as a nectar resource. Prairie, a natural prairie site on Joint Base Lewis-McChord. Balsamorhiza deltoidea bloomed from the last week of May Johnson is one of the few remaining Puget lowland prairies to mid-June in 2011 and from May 7 to June 1 in 2012. dominated by semi-native vegetation, and is located near This perennial has yellow, compact head inflorescences Rainier, WA. Camassia quamash is a dominant flowering containing many fertile female ray flowers and bisexual disk species and the site includes similar plant species as grown at flowers. The fruits are achenes, each with a single ovule. Webster Nursery in clumped patches throughout the fescue- Fazzino et al. (2011) documented that this plant species is dominated grassland. This prairie is subject to some self-incompatible and does not reallocate resources among recreational activity, though less military training activity flower heads. than other prairie sites located on the base (Stinson 2005). A portion of this site, including the study area, was burned in Sickle-keel lupine (L. albicaulis) provides food for August, 2011 for restoration purposes. Coniferous forest caterpillars and adults of ‘blue’ butterflies, such as the Puget and open non-native grasslands border this 7.5 km prairie blue (Plebejus icarioides blackmorei), a species of concern in site (Tab. 1, Fig. 1). Washington State, and occasionally the federally endangered Fender’s blue butterfly (Icaricia icarioides fenderi Macy) (Wilson et al. 1997). This plant is a popular floral resource TABLE 1. Percent land-use in a 1 km buffer around the for several species of native bees and provides vertical study areas in Thurston County, WA. vegetative structure on the low stature Puget prairies. Lupinus albicaulis is a perennial and bloomed from late June Land-use Webster Nursery Johnson Prairie to mid-July in 2011 and from May 29 to June 29 in 2012. The blue, papilionaceous flowers develop basally first in Open Grassland 7.0% 7.2% racemes. Each flower contains 10 monodelphous stamens Forestland 55.4% 92.8% and a simple carpel with several ovules (Hitchcock & Agriculture 14.1% ----- Cronquist 1998). An average of five ovules was presumed to Residential 23.5% ----- be in each carpel of the flowers in this study, which was calculated by counting the number of cells in the collected 32 HUSBY ET AL. J Poll Ecol 15(5) FIGURE 1. Land-use in 1km buffer around: A) Webster Nursery (46.951817 latitude, -122.962126 longitude) and B) Johnson Prairie ® ® (46.927746 latitude, -122.732272 longitude). Spatial Reference: NAD27 UTM Zone 10N. Digitized using ESRI ArcGIS 10.2 World Imagery basemap Source: ESRI, DigitalGlobe, GeoEye, i-cubed, Earthstar, Geographics, CNES/Airbus DS, USDA USGS, AEX, Getmapping, Aerogrid, IGN, IGP, swisstopo, and the GIS User Community, 2014. At Johnson prairie, both plant species have a clumped Visitation Rates distribution, spread across the site in patches. To reduce the The methods used for this study were adapted from chance of potential confounding factors differentially Arroyo et al. (1982) who recorded the number of visits to a contributing to visitation and seed set rates, only patches of known number of flowers for a set time interval. Others plants with similar densities to the planted rows at Webster (Arroyo et al. 1985; Inouye & Pyke 1988; Berry & Calvo nursery were selected at Johnson prairie for plot locations. 1989; McCall & Primack 1992) replicated this method to The selected patches contained few other flowering species allow comparisons among studies (Kearns & Inouye 1993). to reduce the chance of small scale floral competition or For this study, plots were selected to collect visitation rate facilitation happening at the Johnson prairie plots and not at data for both study plant species in 2011 and 2012. At the monoculture plots at Webster nursery. A 7,937 m Webster nursery, the target plant species were planted in macroplot was chosen at Johnson prairie in the Northeast dense rows. Plot locations were selected at Webster Nursery corner where the majority of patches of the target species by breaking each row of the target plant species into two- were. Patches of plants with similar floral densities as found meter segments. Rows were 1.5 m deep. This plot size was at the nursery were identified within the macroplot, and plot chosen, as it was the largest area that could be observed by locations were selected randomly from those patches. All one person without missing visits and entirely encompassed plots selected were spaced at least two-meters apart. most patches of the target species at Johnson prairie. Plots were then randomly selected. If a plot selected was directly At both sites, floral density was calculated for each plot by counting the number of inflorescences of the focal species adjacent to a plot previously selected, it was thrown-out and a new random number was generated to ensure at least two- in bloom and dividing that number by the area of the plot (2 meters of distance between plots. m × 1.5 m). Plot densities varied from 0.3-9.3 inflorescences per square meter for B. deltoidea, and from 4.6-82.0 inflorescences per square meter for L. albicaulis, but March 2015 POLLINATORS AND SEED SET IN NURSERIES 33 variation in plot density was similar between sites (Appendix particular morphotype visited, a value of zero was recorded 1 and 2). In 2011, six plots were selected for B. deltoidea and the trial was not discounted. and 16 plots for L. albicaulis at each location and observed Permutative two-way ANOVAs (Manly 2007) were once. In 2012, 30 plots were selected at each site for B. used to compare mean visitation rates at the nursery to the deltoidea, and each observed once (N = 30). Ten plots were prairie for each plant species in each year, and to detect if selected at each site and each sampled three times for L. there was a year by site interaction. Two tests compared albicaulis in 2012 (N = 10). After sampling B. deltoidea, we overall visitation rates (one per plant species). For B. noted that visitation rates varied more than anticipated deltoidea we ran an additional six permutative ANOVAs for during the bloom period, likely due to different insect the insect morphotypes to determine if there were effects of species being more active at certain times of the year. On location, year or their interaction on individual morphotypes. average, visits on the first day of observations were 2.68 For L. albicaulis we ran an additional three permutative visits per flower per hour higher at Webster nursery and ANOVAs for selected insect morphotypes. All analyses were 0.63 visits per flower per hour higher at Johnson prairie, conducted using Resampling Stats for Excel 2007. Alphas than on the last day of observation. Although B. deltoidea were adjusted using Bonferroni corrections to address plots were sampled relatively evenly throughout the bloom multiple comparisons. We used an alpha = 0.003 for the period, the experimental design was changed for L. albicaulis two overall visitation rate tests, an alpha = 0.008 for six in 2012 based on a recent study (Tscheulin & Petanidou separate individual insect visitation rate tests for visitors of B. 2011), to reduce the influence of variation in visitation rates. deltoidea, and an alpha = 0.017 for three separate individual Instead of recording each plot only once, we thus recorded insect visitation rate tests for visitors of L. albicaulis. visits to plots for three rounds of timed intervals and calculated a mean number of visits per flower per hour. In Pollen Limitation each of the three rounds, the order in which the ten plots Procedures for the pollen limitation experiment were were observed was randomized. adapted from methods used by Fazzino et al. (2011) who Observations took place during peak flowering times on compared seed set from naturally-pollinated B. deltoidea three days for each plant species between May 20 and July 6 inflorescences to hand-cross-pollinated inflorescences to in 2011. In 2012, observations took place between May 8 investigate pollen limitation on Puget prairies. In 2012, a and June 21 on six days for B. deltoidea and five days for L. subset of 10 plots for B. deltoidea at each site was selected albicaulis. Each observation period lasted 10 minutes per randomly from the visitation rate plots, and all plots from plot, and all observations were made between 1000 and the L. albicaulis visitation rate observations were used for the 1530 hours. Sampling dates were chosen to be as close pollinator limitation experiment. Two similar inflorescences together as possible on days with similar temperature, cloud were chosen within each plot and marked with thread before cover, and wind conditions within an optimal range for the styles had matured. One inflorescence was left to be insect activity (temperatures ranging from 9 to 27 °C, clear naturally-pollinated, and the second inflorescence was hand- to cloudy skies with shadows present, and still air to light pollinated as well as naturally-pollinated. breeze). We assumed all flowers in bloom were receptive to Hand-pollination treatments were applied every other pollen. day to all flowers of the selected inflorescences until the The number of visits made by insects was recorded stigmas shriveled, then all inflorescences in both treatments during each 10 min period. A visit was recorded only if the were covered with a coarse mesh bag to prevent seed insect landed on the reproductive parts of a flower. If an predation. When the fruits matured, the inflorescences were insect appeared to be “nectar robbing,” where there was no collected, and the seeds were extracted and counted. potential for pollen transfer, the visit was discounted. Nectar A tetrazolium assay was used to test the seeds for robbing was rarely observed in this study. All visiting insects viability using procedures adapted from the International were identified in flight. Because identification could not Seed Testing Association (2012). Ten plump seeds were accurately be made to a species level and no local guide for randomly selected from each inflorescence for B. deltoidea, pollinator identification for this area was available, the and all seeds from each of the L. albicaulis inflorescences observed insects were grouped into morphotypes: small dark were tested. Balsamorhiza deltoidea seeds were soaked in bees (Halictidae, Colletidae: Hylaeinae, Apidae: warm water for four hours, and L. albicaulis seeds were Xylocopinae, and Andrenidae), large dark bees (Andrena sp. soaked for 24 hours. A 1% aqueous solution of 2,3,5- and Colletidae), green metallic bees (Agapostemon sp.), triphenyltetrazolium chloride was prepared and the pH cuckoo bees (Apidae: Nomadinae), honey bees (Apis adjusted to 6.8. All seed coats were pierced before soaking mellifera), bumblebees (Bombus sp.), flies (Diptera), the seeds in the tetrazolium solution. After four hours, the syrphids (Syrphidae), ants (Formicidae), wasps embryos were examined for the red staining that indicates (Hymenoptera: Apocrita), and beetles (Coleoptera) based on viability. Donovall & vanEngelsdorp (2008). It could not be determined whether visiting honeybees came from feral A permutative two-way ANOVA was also used to populations or nearby managed hives. Because cuckoo bees, compare the percentage of viable seeds produced by the flies, ants, wasps, and beetles were either absent or rare, and inflorescences of each treatment group for each plant species, potentially did not facilitate pollination, these groups were and to detect if there was a treatment by site interaction. To removed from the statistical analysis. If no insects from a determine if there was pollen limitation for either plant species at Webster Nursery or Johnson Prairie, we compared 34 HUSBY ET AL. J Poll Ecol 15(5) Pollen limitation was not evident for either plant species (A) at either site. No significant difference was found between percentage of viable seeds produced by naturally-pollinated inflorescences or hand-cross-pollinated inflorescences for B. deltoidea (SSpollen: 3232.69, P = 0.100; SSsite: 8566.90, P = 0.005; SSpollen*site: 74.13, P = 0.8021) (Appendix III) or L. albicaulis (SSpollen: 2.35, P = 0.985; SSsite: 51212.67, P < 0.001; SSpollen*site: 286.09, P = 0.724) (Fig. 3, Appendix IV). For both species, the trend in seed -------- viability was significantly higher for seeds from the Nursery -------- site (Fig. 3). -------- -------- (B) TABLE 2. Results of permutative two-way ANOVAs (SS and P-values) comparing insect visitation rates at Webster nursery and Johnson prairie for both B. deltoidea and L. albicaulis in 2011 and 2012. Significant results are in bold. All significant site effect results indicate higher visitation rates at Webster Nursery than at Johnson Prairie. Bonferroni corrections for six tests (B. deltoidea) 2 would necessitate an alpha = 0.008, and for three tests (L. -------- albicaulis) would necessitate an alpha = 0.017. 1 -------- Insect Morphotype Source SS P -------- -------- B. deltoidea Small Dark Bees Site 1.76 0.010 Nursery Prairie Nursery Prairie Year 1.36 0.206 Site × Year 0.92 0.284 Large Dark Bees Site 0.17 0.499 FIGURE 2. Overall insect visitation rates (# visits / plant / Year <0.01 1.000 hr) at Webster Nursery and Johnson Prairie for: A) Balsamorhiza Site × Year <0.01 1.000 deltoidea, and B) Lupinus albicaulis in 2011 and 2012. Open gray circles represent each visitation rate and black horizontal lines Green Metallic Bees Site 0.60 0.235 represent means +/- 1 standard error. Bonferroni correction for Year 4.83 0.165 two tests would necessitate an alpha = 0.025. Site × Year 4.83 0.141 Honey Bees Site 0.62 1.000 the percentages of viable seed produced by the hand- Year 8.12 0.170 pollinated inflorescences to the naturally pollinated Site × Year 8.12 0.170 inflorescences. All analyses were conducted using Bumblebees Site 43.00 0.002 Resampling Stats for Excel 2007. Alphas were adjusted Year 51.96 0.041 using Bonferroni corrections for two comparisons (alpha = Site × Year 25.51 0.172 0.025). Syrphids Site 0.05 0.117 RESULTS Year 0.39 0.006 Site × Year 0.39 0.002 Insect visitation rates differed between Webster Nursery L. albicaulis and Johnson Prairie, both overall and for many of the insect Small Dark Bees Site 0.01 0.013 groups. Overall visitation rates were significantly higher at Year <0.01 0.501 Webster Nursery than at Johnson Prairie for both B. Site × Year <0.01 0.698 deltoidea (Fig. 2; SSsite: 92.77, P < 0.001; SSyear: 7.46, P = 0.496; SSsite*year: 2.58, P = 0.691) (Appendix I) and L. Large Dark Bees Site <0.01 0.757 albicaulis (SSsite: 12.32, P < 0.001; SSyear; 5.49, P = Year 0.03 0.136 0.039; SSsite*year: 0.18, P = 0.715) (Appendix II). Site × Year <0.01 0.601 Bumblebees Site 13.09 0.001 Webster Nursery also had significantly higher visitation Year 6.50 0.024 rates than Johnson Prairie for several insect morphotypes visiting each of the plant species, specifically bumblebees and Site × Year 0.12 0.780 small dark bees (Tab. 2, Appendix I & II). In 2012, there No longer significant at Bonferroni-corrected alpha = 0.008 were significantly higher rates of bumblebee visits at both No longer significant at Bonferroni-corrected alpha = sites than in 2011. In addition, there were higher visitation 0.017 rates for small dark bees and bumblebees to L. albicaulis at Webster Nursery. # visits / flower / hr # visits / flower / hr March 2015 POLLINATORS AND SEED SET IN NURSERIES 35 larger bumblebees, have relatively large foraging areas (A) (Greenleaf et al. 2007). Hagen et al. (2011) found the foraging area for some Bombus sp. to be between 0.25-43.53 hectares in one to four days. It is most probable that the high densities of flowers and the many blooming species found in -------- a small area are attracting pollinators to this native plant -------- nursery. Some bees can rapidly produce more offspring in -------- response to an increase in floral resources because greater foraging efficiency means less time they are exposed to -------- predators and parasites (Goodell 2003). For some plant species, rapid accumulation of dense native plant resources at a native plant nursery may attract sufficient unmanaged insects to provide pollination services similar or higher than (B) those found in native landscapes. Restoration nurseries are -------- -------- human-altered landscapes with surpluses of native floral resources, which recent studies have found to be ideal factors in drawing diverse and abundant pollinator responses (Winfree et al. 2011). The visitation rate results of this study support the conclusion that restoration nurseries that are several years old may have sufficient unmanaged insects to pollinate many of their forbs. -------- -------- Manipulatively increasing insect visitation at Webster nursery may not be a conservation priority given a lack of evidence for pollen limitation for either plant species. In Natural Hand-crossed Natural Hand-crossed addition, no evidence was found that supplemental pollen Nursery Prairie increases viable seed production for the plants in this study. An earlier study by Fazzino et al. (2011) found that hand- FIGURE 3. Percentage of viable seeds produced by hand-cross pollinated inflorescences produced more sprouting seeds pollination and natural pollination at either Webster Nursery or Johnson Prairie: A) Balsamorhiza deltoidea inflorescences, and B) than naturally-pollinated inflorescences for B. deltoidea in Lupinus albicaulis inflorescences. Open gray circles represent nearby Puget lowland prairies. In contrast, the B. deltoidea percent viability and black horizontal lines represent means +/- 1 plants in this study were either not pollen-limited or the standard error. Bonferroni correction for two tests would hand-pollinated inflorescences did not receive enough necessitate an alpha = 0.025. supplemental pollen by hand to show a significant difference. The discrepancy between this earlier study and the DISCUSSION results presented here could also be due to differences in weather between study years, as poor flight weather can Characteristics of Webster Nursery appear to be dampen insect visitation (Vicens & Bosch 2000), or facilitating higher rates of insect visitation to these two plant differences in methodology. Weather data were not collected species than at the natural prairie site, Johnson Prairie. The in either study throughout the bloom periods at the study nursery is located in an area with fewer assumed floral sites, though past local weather reports indicate similar fair resources for native pollinators, though insects appear to be weather temperature ranges and precipitation levels in 2009 responding to the large influx of floral resources at the and 2012 when B. deltoidea was in bloom (U.S. Climate relatively new nursery that started in 2008. Although Data 2014). Fazzino et al. (2011) examined seed visitation was higher at the nursery site, we found no germination and we measured seed viability using lab evidence of pollen limitation and no differences in viability methods. Due to various factors that can affect germination, for seeds from either location. Restoration managers at the and the destructive properties of tetrazolium testing, these nursery site were hoping to determine methods for increasing two studies are not directly comparable. Future studies could native plant yield and this study suggests that neither involve both viability and germination testing and should increased pollinator populations nor hand pollination would increase the number of replicates to further test for pollen- increase seed viability for these two species. limitation in both plant species. Though not statistically Several studies suggest that human developed landscapes significant, our results did indicate a trend that hand- are not necessarily pollinator deprived. Matteson et al. pollinated inflorescences produced a higher percentage of (2012) found it inappropriate to generalize about landscapes viable seeds. Managers may still consider providing created by humans due to high variability in habitat supplemental pollen treatments to plants or placing managed suitability for pollinators within land-use categories. Some bee colonies on site if planning to collect seed during a year researchers found that bee abundance increases in human- with poor weather for insect visitation. constructed landscapes developed with a superabundance of In this experiment, we tested whether or not more pollen floral resources, and that a combination of natural and would increase viable seed production, though supplemental developed landscapes can provide a greater diversity of pollen does not always benefit plant reproduction. When habitat resources (Frankie et al. 2009). Some insects, such as Percent viable seeds Percent viable seeds 36 HUSBY ET AL. J Poll Ecol 15(5) Appendix IV. Number of seeds produced by Lupinus albicaulis maximum seed production is reached, there are no longer inflorescences. unfertilized ovules for additional pollen to be of benefit (Ashman et al. 2004). There can be a point of pollen REFERENCES saturation on stigmas (Cane & Schiffhauer 2003); too much pollen added too quickly could lead to an underestimation of Arroyo MTK, Armesto JJ, Primack RB (1985) Community studies pollen-limitation (Ashman et al. 2004). In fact, supplemental in pollination ecology in the high temperate Andes of central pollen negatively affected seed weight in a study on pollen Chile II. Effect of temperature on visitation rates and pollination limitation at the community level, as plants may reallocate possibilities. Plant Systematics and Evolution 149:187-203. energy and resources in response (Hegland &Totland 2008). Arroyo MT, Primack R, Armesto J (1982) Community studies in Pollen in this study was collected from separate plants on the pollination ecology in the high temperate Andes of central Chile I. opposite side of the study area as the plant that was hand- Pollination mechanisms and altitudinal variation. American pollinated to ensure cross-pollination. Visiting insects, Journal of Botany 69:82-97. however, often transfer a combination of pollen from Ashman T, Knight TM, Steets JA, Amarasekare P, Burd M, separate plants and pollen that may not be compatible from Campbell DR, Dudash MR, Johnston MO, Mazer SJ, Mitchell RJ, Morgan MT, Wilson WG (2004) Pollen limitation of plant flowers of the same plant (Wagenius & Lyon 2010), so the reproduction: Ecological and evolutionary causes and effect of increasing insect visitation may not be directly consequences. Ecology 85:2408-2421. proportional to hand-pollination treatments. Berry PE, Calvo RN (1989) Wind pollination, self-incompatibility, Finally, the percentage of viable seeds was lower at and altitudinal shifts in pollination systems in the high Andean Johnson Prairie compared to Webster nursery for both genus Espeletia (Asteraceae). American Journal of Botany species, but more dramatically so for L. albicaulis (see Fig. 76:1602-1614. 3). Plants at Johnson Prairie appeared to be smaller than at Bos MM, Veddeler D, Bogdanski AK, Klein A, Tscharntke T, Webster nursery and may have been affected by seed Steffan-Dewenter I, Tylainakis JM (2007) Caveats to quantifying predators, a pathogen, or limited by lack of irrigation. There ecosystem services: Fruit abortion blurs benefits from crop pollination. Ecological Applications 17:1841-1849. may have been differences in plant population age, soil nutrients, or microclimate that influenced these differences. Cane JH, Schiffhauer D (2003) Dose-response relationships between pollination and fruiting refine pollinator comparisons for Visitation rate is only one of many factors that may cranberry (Vaccinium macrocarpon [Ericaceae]). American influence the number of viable seeds a plant produces. Journal of Botany 90:1425-1432. Availability of resources such as soil nutrients, water, and Corbet SA (1998) Fruit and seed production in relation to light can also affect plant reproduction (Stephenson 1981; pollination and resources in Bluebell, Hyacinthoides non-scripta. Corbet 1998; Bos et al. 2007), and seed handling and Oecologia 114:349-360. storage practices can affect seed viability and germination. In Dieringer G (1992) Pollinator limitation in populations of Agalinis addition, changes in light and temperature during strictifolia (Scrophulariaceae). Bulletin of the Torrey Botanical germination can affect L. albicaulis seed viability (Morey & Club 119:131-136. Bakker 2011). We recommend that land managers turn Donovall L, vanEngelsdorp D (2008) Pennsylvania Native Bee efforts towards investigating the influence of the above Survey Citizen Scientist Pollinator Monitoring Guide. The Xerces factors on native seed production for these two critical Society for Invertebrate Conservation. [online] URL: species in future studies. Pollinator visitation and pollen- www.xerces.org/download/pdf/PA_Xerces%20Guide.pdf limitation may not be primary concerns for restoration (accessed April 2011). managers working with these two species in the Puget Dunwiddie PW, Bakker JD (2011) The future of restoration and lowland prairies of western Washington. management of prairie-oak ecosystems in the Pacific Northwest. Northwest Science 85:83-92. ACKNOWLEDGEMENTS Engel EC, Irwin RE (2003) Linking pollinator visitation rate and pollen receipt. American Journal of Botany 90:1612-1618. We thank the Center for Natural Lands Management for their Fazzino L, Kirkpatrick HE, Fimbel C (2011) Comparison of hand- support and Joint Base Lewis-McChord for permission to conduct pollinated and naturally-pollinated Puget balsamroot research on their lands. We also thank H. Elizabeth Kirkpatrick, (Balsamorhiza deltoidea Nutt.) to determine pollinator University of Puget Sound, for her suggestions on experimental limitations on South Puget Sound lowland prairies. Northwest design for pollinators. We thank Greg Dasso, The Evergreen State Science 85: 352-360. College, for helping in the lab. We also thank The Evergreen State Fontaine C, Dajoz I, Meriguet J, Loreau M (2006) Functional College Foundation and the Evergreen Sustainability Fellowship diversity of plant-pollinator interaction webs enhances the committee for their financial support. persistence of plant communities. PLoS Biology 4:0129-0135. Frankie GW, Thorp RW, Hernandez J, Rizzardi M, Ertter B, APPENDICES Pawelek JC, Witt SL, Schindler M, Coville R, Wojcik VA Additional supporting information may be found in the online (2009) Native bees are a rich natural resource in urban California version of this article: gardens. University of California. California Agriculture [online] Appendix I. 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PLoS USDA Natural Resources Conservation Service (2012) Plants ONE 6(5):e19997. Database. [online] URL: www.plants.usda.gov (accessed Oct 2012). Hegland SJ, Totland O (2008) Is the magnitude of pollen limitation in a plant community affected by pollinator visitation Perfectti F, Gomez JM, Bosch J (2009) The functional and plant species specialization levels? Oikos 117:883-891. consequences of diversity in plant-pollinator interactions. Oikos 118:1430-1440. Heschel MS, Page KN (1995) Inbreeding depression, environmental stress, and population size variation in scarlet gilia Price MV, Campbell DR, Waser NM, Brody AK (2008) Bridging (Ipomopsis aggregata). Conservation Biology 9:126-133. the generation gap in plants: Pollination, parental fecundity, and offspring demography. Ecology 89:1596-1604. Hitchcock CL, Cronquist A (1998) Flora of the Pacific Northwest. University of Washington Press, Seattle and London. Stanley AG, Kaye TN, Dunwiddie PW (2008) Regional strategies for restoring invaded prairies: Observations from a multisite, Inouye DW, Pyke GH (1988) Pollination in the snowy mountains collaborative research project. Native Plants Journal 9:247-254. of Australia: Comparisons with montane Colorado, USA. Australian Journal of Ecology 13:191-210. Stephenson AG (1981) Flower and fruit abortion: Proximate causes and ultimate functions. Annual Review of Ecology and International Seed Testing Association (2012) International Rules Systematics 12:253-279. for Seed Testing (2012ed.). Bassersdorf: Switzerland. Stinson DW (2005) Washington State Department of Fish and Kaye TN (1999) Obligate insect pollination of a rare plant, Wildlife. Washington State status report for the Mazama pocket Lupinus sulphureus ssp. kincaidii. Northwest Science 73:50-52. gopher, streaked horned lark, and Taylor’s checker spot. Olympia. Kearns CA, Inouye DW (1993) Techniques for Pollination Tscheulin T, Petanidou T (2011) Does spatial population Biologists. University Press of Colorado, Niwot. structure affect seed set in pollen-limited Thymus capitatus? Kittelson PM, Maron JL (2000) Outcrossing rate and inbreeding Apidologie 42:67-77. depression in the perennial yellow bush lupine, Lupinus arboreus U.S. Climate Data (2014) [online] URL: www.usclimatedata.com (fabaceae). American Journal of Botany 87:652-660. (accessed November 2014). Kremen C, Williams NM, Aizen MA, Gemmill-Herren B, LeBuhn Vicens N, Bosch J (2000) Weather-dependent pollinator activity in G, Minckley R, Packer L, Potts SG, Roulston T, Steffan- an apple orchard, with special reference to Osmia cornuta and Dewenter I, Vazquez DP, Winfree R, Adams L, Crone EE, Apis mellifera (Hymenoptera: Megachilidae and Apidae). Greenleaf SS, Keitt TH, Klein A, Regetz J, Ricketts TH (2007) Environmental Entomology 29:413-420. 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Washington Natural Heritage Program (2012) Washington Department of Natural Resources [online] URL: Matteson KC, Grace JB, Minor ES (2012) Direct and indirect http://www1.dnr.wa.gov/nhp/refdesk/lists/plantrnk.html effects of land use on floral resources and flower-visiting insects (accessed August 2012). across an urban landscape. Oikos 116:1588-1598. Wilson MV, Hammond PC, Schultz CB (1997) The Mayer C, Adler L, Armbruster S, Dafni A, Eardley C, Huang S, interdependence of native plants and Fender’s blue butterfly. In: Kevan PG, Ollerton J, Packer L, Ssymank A, Stout JC, Potts SG Kaye TN, Liston A, Love RM, Luomo D, Meinke RJ, Wilson (2011) Pollination ecology in the 21st century: Key questions for MV (eds) Conservation management of native flora and fungi. future research. Journal of Pollination Ecology 3:8-23. Native Plant Society of Oregon, Corvallis, pp 83-87. McCall C, Primack RB (1992) Influence of flower characteristics, Winfree R, Bartomeus I, Cariveau DP (2011) Native pollinators in weather, time of day, and season on insect visitation rates in three anthropogenic habitats. Annual Review of Ecology, Evolution, plant communities. American Journal of Botany 79:434-442. and Systematics 42:1-22. McCarty JP (2001) Ecological consequences of recent climate change. Conservation Biology 15:320-331. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Pollination Ecology Unpaywall

Pollinators may not limit native seed set at puget lowland prairie restoration nurseries

Journal of Pollination EcologyJan 1, 2015

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Abstract

Journal of Pollination Ecology, 15(5), 2015, pp 30-37 POLLINATORS MAY NOT LIMIT NATIVE SEED SET AT PUGET LOWLAND PRAIRIE RESTORATION NURSERIES 1, 1 2 Jennie F. Husby * Carri J. LeRoy , Cheryl Fimbel The Evergreen State College, 2700 Evergreen Parkway NW, Olympia, Washington 98505, USA Center for Natural Lands Management, 120 E, Union #215, Olympia, Washington 98501 , USA Abstract—Land managers often rely on large-scale production of native seeds in nurseries for replanting into natural environments as part of restoration strategies. Nursery managers question if unmanaged insects can be sufficient to pollinate large increases in native forbs planted into young nurseries in non-native landscapes. This study investigated pollination of deltoid balsamroot (Balsamorhiza deltoidea Nutt.) and sicklekeel lupine (Lupinus albicaulis Douglas) at a native seed nursery compared to dense patches of native plants at a natural Puget lowland prairie to determine if insect visitation affected viable seed production for those two species. In 2011 and 2012, insect visitation rates were recorded for each plant species at more than 62 plots within two study areas. In 2012, seeds were collected from hand-pollinated and naturally-pollinated inflorescences and tested for viability. Overall visitation rates were significantly higher at the nursery than the prairie for both plant species. However, pollen limitation was not evident for either plant species at either site. Natural pollination by insects and supplemental hand-cross-pollination treatments did not yield different quantities of viable seed. Factors other than pollinator visitation, such as soil nutrients and seed handling practices, may be influencing seed viability, but increasing insect visitation will not likely significantly increase seed viability for these two species at this restoration nursery. Planting dense rows of native plant species may be enough to attract a sufficient amount of unmanaged insects to provide adequate pollination for seed production for some species at even young nurseries. Keywords: restoration nursery, pollen limitation, insect visitation, prairie restoration, Balsamorhiza deltoidea, Lupinus albicaulis Several aspects of pollination can influence the production of INTRODUCTION viable seeds. Insect visitation rates can positively affect pollen receipt (Engel & Irwin 2003). Differences in pollinator Pollinators play a key role in the reproduction of wild community structure can affect overall pollination plants as they are linked to viable seed production and native effectiveness (Perfectti et al. 2009) and pollinator behaviour, plant population growth. Pollinators and their activities thus such as order in which a pollinator visits flowers, can affect provide an ecosystem-wide service, especially for landscapes whether a flower is self-pollinated or cross-pollinated (Kunin with many insect-dependent forb species like prairies 1993). When plants are pollen-limited due to insufficient (Kremen et al. 2007). Even self-compatible plant species pollinator activity, they are pollinator-limited (Dieringer may rely on pollinators to provide conspecific pollen to mix 1992). genes, preventing inbreeding depression (Heschel & Page 1995; Price et al. 2008). Native seed from nurseries plays an The Puget lowland prairie ecosystem has been important role in ecosystem restoration. Ecosystems in need fragmented by coniferous forest encroachment and urban of conservation attention may be stressed by factors such as and agricultural development so that now only 2-3% of the invading species, fragmentation, and climate change; all of original habitat remains (Dunwiddie & Bakker 2011). Re- which can suppress a plant species’ population size and limit establishing native flora has been a priority of Puget lowland its reproductive ability (McCarty 2001; Vila & Weiner prairie conservation partners (Stanley et al. 2008), and these 2004; Fazzino et al. 2011; Tscheulin & Petanidou 2011). partners rely on large-scale production of native seeds in Many restoration practitioners depend on native seed grown their nurseries for replanting into the Puget lowland prairies in nurseries for repopulating plant species in natural areas. as part of their restoration strategies. Some years restoration Native plant nursery managers strive to produce large practitioners have struggled to produce large quantities of quantities of high quality seed to meet restoration demands. viable seeds for certain plant species at their largest nursery, Webster Nursery. These managers have questioned whether When plants produce fewer viable seeds than maternal or not two critical restoration species, Balsamorhiza resources allow because of insufficient quantity or quality of deltoidea Nutt. (Asteraceae) (deltoid balsamroot) and pollen, they are pollen-limited (Wagenius & Lyon 2010). Lupinus albicaulis Douglas (Fabaceae) (sicklekeel lupine), are producing the highest proportion of viable seeds possible Received 17 July 2014, accepted 09 February 2015 at this restoration nursery and if there is a way to increase the *Corresponding author; email: JennieHusby@gmail.com proportion. Results of testing by the nursery in 2013 30 March 2015 POLLINATORS AND SEED SET IN NURSERIES 31 revealed that Webster Nursery produced 1,424 g of B. pods, including those that were empty (likely due to ovule deltoidea seed with 38.8% viability, and 10,299 g of L. abortion). The L. albicaulis inflorescences tested in this albicaulis seed with 35.3% viability (S. Smith, Center for study had an average of 48.1 flowers per inflorescence. Little Natural Lands Management, pers. comm.). The cause of this is known about the pollination system of L. albicaulis. problem may be due to seed handling or storage techniques, Lupinus species in general are typically self-compatible, inadequate environmental conditions where the plants are though some require a pollinator to trigger autogamy and grown (such as soil nutrients, weather, etc.), seed predation, have increased seed set when cross-pollinated (Kaye 1999, or pollen limitation. This study addressed the latter by Kittelson & Maron 2000). investigating the status of pollination at Webster Nursery Study Areas and comparing it to dense stands of native plants at a natural Puget lowland prairie to determine if inadequate pollination Washington Department of Natural Resources (DNR) restricted viable seed production at the nursery. owns Webster Nursery, a portion of which is leased and managed by conservation partners to produce seed from More specifically, to better understand the role of native plants at a large scale for restoring Puget lowland pollination at a native seed production facility, we prairies. The plants are grown outdoors in dense rows. The investigated the following research questions: (1) Do insect native seed nursery was first established in 2008 with partial visitation rates to dense floral patches differ between a rows (~100 m) of 10 species. The rows planted with B. nursery site and a natural prairie site for two prairie forbs? deltoidea and L. albicaulis were last fertilized during their (2) Is there evidence of pollen limitation for either plant installation in 2008, are watered only by rain, and were not species at either the nursery or prairie sites? sprayed with pesticides or herbicides in 2011 or 2012 (Angela Winter, nursery manager, pers. comm. 2012). Other MATERIALS AND METHODS species grown at Webster Nursery include: hookedspur violet (Viola adunca), spring gold (Lomatium utriculatum), Study Plant Species nine-leaf biscuitroot (Lomatium triternatum), shortspur We focused this study on two native prairie plants, B. seablush (Plectritis congesta), slender cinquefoil (Potentilla deltoidea and L. albicaulis. Both plant species grow along the gracilis), harsh Indian paintbrush (Castilleja hispida), golden west coast of the United States and into Canada (USDA paintbrush (Castilleja levisecta), sea pink (Armeria Natural Resources Conservation Service 2012). These plants maritima), Nuttall’s Larkspur (Delphinium nuttallii), are both found at natural prairie sites and are being produced woolley sunflower (Eriophyllum lanatum), buttercup from seed at Webster Nursery, Tumwater, WA, USA. (Ranunculus occidentalis), Pacific lupine (Lupinus lepidus), bicolor lupine (Lupinus bicolor), farewell to spring (Clarkia Deltoid balsamroot (B. deltoidea) is a species of amoena), and camas (Camassia quamash). Farmland, open potential concern in Washington State (Washington Natural grassland, residential properties, and forested areas surround Heritage Program 2012), its flowers are popular with insect the 5.3 km nursery (Tab. 1, Fig. 1). visitors, and it is a valued restoration plant. The federally endangered butterfly, Taylor’s checkerspot (Euphydryas The US Department of Defense manages Johnson editha taylori), frequently uses this plant as a nectar resource. Prairie, a natural prairie site on Joint Base Lewis-McChord. Balsamorhiza deltoidea bloomed from the last week of May Johnson is one of the few remaining Puget lowland prairies to mid-June in 2011 and from May 7 to June 1 in 2012. dominated by semi-native vegetation, and is located near This perennial has yellow, compact head inflorescences Rainier, WA. Camassia quamash is a dominant flowering containing many fertile female ray flowers and bisexual disk species and the site includes similar plant species as grown at flowers. The fruits are achenes, each with a single ovule. Webster Nursery in clumped patches throughout the fescue- Fazzino et al. (2011) documented that this plant species is dominated grassland. This prairie is subject to some self-incompatible and does not reallocate resources among recreational activity, though less military training activity flower heads. than other prairie sites located on the base (Stinson 2005). A portion of this site, including the study area, was burned in Sickle-keel lupine (L. albicaulis) provides food for August, 2011 for restoration purposes. Coniferous forest caterpillars and adults of ‘blue’ butterflies, such as the Puget and open non-native grasslands border this 7.5 km prairie blue (Plebejus icarioides blackmorei), a species of concern in site (Tab. 1, Fig. 1). Washington State, and occasionally the federally endangered Fender’s blue butterfly (Icaricia icarioides fenderi Macy) (Wilson et al. 1997). This plant is a popular floral resource TABLE 1. Percent land-use in a 1 km buffer around the for several species of native bees and provides vertical study areas in Thurston County, WA. vegetative structure on the low stature Puget prairies. Lupinus albicaulis is a perennial and bloomed from late June Land-use Webster Nursery Johnson Prairie to mid-July in 2011 and from May 29 to June 29 in 2012. The blue, papilionaceous flowers develop basally first in Open Grassland 7.0% 7.2% racemes. Each flower contains 10 monodelphous stamens Forestland 55.4% 92.8% and a simple carpel with several ovules (Hitchcock & Agriculture 14.1% ----- Cronquist 1998). An average of five ovules was presumed to Residential 23.5% ----- be in each carpel of the flowers in this study, which was calculated by counting the number of cells in the collected 32 HUSBY ET AL. J Poll Ecol 15(5) FIGURE 1. Land-use in 1km buffer around: A) Webster Nursery (46.951817 latitude, -122.962126 longitude) and B) Johnson Prairie ® ® (46.927746 latitude, -122.732272 longitude). Spatial Reference: NAD27 UTM Zone 10N. Digitized using ESRI ArcGIS 10.2 World Imagery basemap Source: ESRI, DigitalGlobe, GeoEye, i-cubed, Earthstar, Geographics, CNES/Airbus DS, USDA USGS, AEX, Getmapping, Aerogrid, IGN, IGP, swisstopo, and the GIS User Community, 2014. At Johnson prairie, both plant species have a clumped Visitation Rates distribution, spread across the site in patches. To reduce the The methods used for this study were adapted from chance of potential confounding factors differentially Arroyo et al. (1982) who recorded the number of visits to a contributing to visitation and seed set rates, only patches of known number of flowers for a set time interval. Others plants with similar densities to the planted rows at Webster (Arroyo et al. 1985; Inouye & Pyke 1988; Berry & Calvo nursery were selected at Johnson prairie for plot locations. 1989; McCall & Primack 1992) replicated this method to The selected patches contained few other flowering species allow comparisons among studies (Kearns & Inouye 1993). to reduce the chance of small scale floral competition or For this study, plots were selected to collect visitation rate facilitation happening at the Johnson prairie plots and not at data for both study plant species in 2011 and 2012. At the monoculture plots at Webster nursery. A 7,937 m Webster nursery, the target plant species were planted in macroplot was chosen at Johnson prairie in the Northeast dense rows. Plot locations were selected at Webster Nursery corner where the majority of patches of the target species by breaking each row of the target plant species into two- were. Patches of plants with similar floral densities as found meter segments. Rows were 1.5 m deep. This plot size was at the nursery were identified within the macroplot, and plot chosen, as it was the largest area that could be observed by locations were selected randomly from those patches. All one person without missing visits and entirely encompassed plots selected were spaced at least two-meters apart. most patches of the target species at Johnson prairie. Plots were then randomly selected. If a plot selected was directly At both sites, floral density was calculated for each plot by counting the number of inflorescences of the focal species adjacent to a plot previously selected, it was thrown-out and a new random number was generated to ensure at least two- in bloom and dividing that number by the area of the plot (2 meters of distance between plots. m × 1.5 m). Plot densities varied from 0.3-9.3 inflorescences per square meter for B. deltoidea, and from 4.6-82.0 inflorescences per square meter for L. albicaulis, but March 2015 POLLINATORS AND SEED SET IN NURSERIES 33 variation in plot density was similar between sites (Appendix particular morphotype visited, a value of zero was recorded 1 and 2). In 2011, six plots were selected for B. deltoidea and the trial was not discounted. and 16 plots for L. albicaulis at each location and observed Permutative two-way ANOVAs (Manly 2007) were once. In 2012, 30 plots were selected at each site for B. used to compare mean visitation rates at the nursery to the deltoidea, and each observed once (N = 30). Ten plots were prairie for each plant species in each year, and to detect if selected at each site and each sampled three times for L. there was a year by site interaction. Two tests compared albicaulis in 2012 (N = 10). After sampling B. deltoidea, we overall visitation rates (one per plant species). For B. noted that visitation rates varied more than anticipated deltoidea we ran an additional six permutative ANOVAs for during the bloom period, likely due to different insect the insect morphotypes to determine if there were effects of species being more active at certain times of the year. On location, year or their interaction on individual morphotypes. average, visits on the first day of observations were 2.68 For L. albicaulis we ran an additional three permutative visits per flower per hour higher at Webster nursery and ANOVAs for selected insect morphotypes. All analyses were 0.63 visits per flower per hour higher at Johnson prairie, conducted using Resampling Stats for Excel 2007. Alphas than on the last day of observation. Although B. deltoidea were adjusted using Bonferroni corrections to address plots were sampled relatively evenly throughout the bloom multiple comparisons. We used an alpha = 0.003 for the period, the experimental design was changed for L. albicaulis two overall visitation rate tests, an alpha = 0.008 for six in 2012 based on a recent study (Tscheulin & Petanidou separate individual insect visitation rate tests for visitors of B. 2011), to reduce the influence of variation in visitation rates. deltoidea, and an alpha = 0.017 for three separate individual Instead of recording each plot only once, we thus recorded insect visitation rate tests for visitors of L. albicaulis. visits to plots for three rounds of timed intervals and calculated a mean number of visits per flower per hour. In Pollen Limitation each of the three rounds, the order in which the ten plots Procedures for the pollen limitation experiment were were observed was randomized. adapted from methods used by Fazzino et al. (2011) who Observations took place during peak flowering times on compared seed set from naturally-pollinated B. deltoidea three days for each plant species between May 20 and July 6 inflorescences to hand-cross-pollinated inflorescences to in 2011. In 2012, observations took place between May 8 investigate pollen limitation on Puget prairies. In 2012, a and June 21 on six days for B. deltoidea and five days for L. subset of 10 plots for B. deltoidea at each site was selected albicaulis. Each observation period lasted 10 minutes per randomly from the visitation rate plots, and all plots from plot, and all observations were made between 1000 and the L. albicaulis visitation rate observations were used for the 1530 hours. Sampling dates were chosen to be as close pollinator limitation experiment. Two similar inflorescences together as possible on days with similar temperature, cloud were chosen within each plot and marked with thread before cover, and wind conditions within an optimal range for the styles had matured. One inflorescence was left to be insect activity (temperatures ranging from 9 to 27 °C, clear naturally-pollinated, and the second inflorescence was hand- to cloudy skies with shadows present, and still air to light pollinated as well as naturally-pollinated. breeze). We assumed all flowers in bloom were receptive to Hand-pollination treatments were applied every other pollen. day to all flowers of the selected inflorescences until the The number of visits made by insects was recorded stigmas shriveled, then all inflorescences in both treatments during each 10 min period. A visit was recorded only if the were covered with a coarse mesh bag to prevent seed insect landed on the reproductive parts of a flower. If an predation. When the fruits matured, the inflorescences were insect appeared to be “nectar robbing,” where there was no collected, and the seeds were extracted and counted. potential for pollen transfer, the visit was discounted. Nectar A tetrazolium assay was used to test the seeds for robbing was rarely observed in this study. All visiting insects viability using procedures adapted from the International were identified in flight. Because identification could not Seed Testing Association (2012). Ten plump seeds were accurately be made to a species level and no local guide for randomly selected from each inflorescence for B. deltoidea, pollinator identification for this area was available, the and all seeds from each of the L. albicaulis inflorescences observed insects were grouped into morphotypes: small dark were tested. Balsamorhiza deltoidea seeds were soaked in bees (Halictidae, Colletidae: Hylaeinae, Apidae: warm water for four hours, and L. albicaulis seeds were Xylocopinae, and Andrenidae), large dark bees (Andrena sp. soaked for 24 hours. A 1% aqueous solution of 2,3,5- and Colletidae), green metallic bees (Agapostemon sp.), triphenyltetrazolium chloride was prepared and the pH cuckoo bees (Apidae: Nomadinae), honey bees (Apis adjusted to 6.8. All seed coats were pierced before soaking mellifera), bumblebees (Bombus sp.), flies (Diptera), the seeds in the tetrazolium solution. After four hours, the syrphids (Syrphidae), ants (Formicidae), wasps embryos were examined for the red staining that indicates (Hymenoptera: Apocrita), and beetles (Coleoptera) based on viability. Donovall & vanEngelsdorp (2008). It could not be determined whether visiting honeybees came from feral A permutative two-way ANOVA was also used to populations or nearby managed hives. Because cuckoo bees, compare the percentage of viable seeds produced by the flies, ants, wasps, and beetles were either absent or rare, and inflorescences of each treatment group for each plant species, potentially did not facilitate pollination, these groups were and to detect if there was a treatment by site interaction. To removed from the statistical analysis. If no insects from a determine if there was pollen limitation for either plant species at Webster Nursery or Johnson Prairie, we compared 34 HUSBY ET AL. J Poll Ecol 15(5) Pollen limitation was not evident for either plant species (A) at either site. No significant difference was found between percentage of viable seeds produced by naturally-pollinated inflorescences or hand-cross-pollinated inflorescences for B. deltoidea (SSpollen: 3232.69, P = 0.100; SSsite: 8566.90, P = 0.005; SSpollen*site: 74.13, P = 0.8021) (Appendix III) or L. albicaulis (SSpollen: 2.35, P = 0.985; SSsite: 51212.67, P < 0.001; SSpollen*site: 286.09, P = 0.724) (Fig. 3, Appendix IV). For both species, the trend in seed -------- viability was significantly higher for seeds from the Nursery -------- site (Fig. 3). -------- -------- (B) TABLE 2. Results of permutative two-way ANOVAs (SS and P-values) comparing insect visitation rates at Webster nursery and Johnson prairie for both B. deltoidea and L. albicaulis in 2011 and 2012. Significant results are in bold. All significant site effect results indicate higher visitation rates at Webster Nursery than at Johnson Prairie. Bonferroni corrections for six tests (B. deltoidea) 2 would necessitate an alpha = 0.008, and for three tests (L. -------- albicaulis) would necessitate an alpha = 0.017. 1 -------- Insect Morphotype Source SS P -------- -------- B. deltoidea Small Dark Bees Site 1.76 0.010 Nursery Prairie Nursery Prairie Year 1.36 0.206 Site × Year 0.92 0.284 Large Dark Bees Site 0.17 0.499 FIGURE 2. Overall insect visitation rates (# visits / plant / Year <0.01 1.000 hr) at Webster Nursery and Johnson Prairie for: A) Balsamorhiza Site × Year <0.01 1.000 deltoidea, and B) Lupinus albicaulis in 2011 and 2012. Open gray circles represent each visitation rate and black horizontal lines Green Metallic Bees Site 0.60 0.235 represent means +/- 1 standard error. Bonferroni correction for Year 4.83 0.165 two tests would necessitate an alpha = 0.025. Site × Year 4.83 0.141 Honey Bees Site 0.62 1.000 the percentages of viable seed produced by the hand- Year 8.12 0.170 pollinated inflorescences to the naturally pollinated Site × Year 8.12 0.170 inflorescences. All analyses were conducted using Bumblebees Site 43.00 0.002 Resampling Stats for Excel 2007. Alphas were adjusted Year 51.96 0.041 using Bonferroni corrections for two comparisons (alpha = Site × Year 25.51 0.172 0.025). Syrphids Site 0.05 0.117 RESULTS Year 0.39 0.006 Site × Year 0.39 0.002 Insect visitation rates differed between Webster Nursery L. albicaulis and Johnson Prairie, both overall and for many of the insect Small Dark Bees Site 0.01 0.013 groups. Overall visitation rates were significantly higher at Year <0.01 0.501 Webster Nursery than at Johnson Prairie for both B. Site × Year <0.01 0.698 deltoidea (Fig. 2; SSsite: 92.77, P < 0.001; SSyear: 7.46, P = 0.496; SSsite*year: 2.58, P = 0.691) (Appendix I) and L. Large Dark Bees Site <0.01 0.757 albicaulis (SSsite: 12.32, P < 0.001; SSyear; 5.49, P = Year 0.03 0.136 0.039; SSsite*year: 0.18, P = 0.715) (Appendix II). Site × Year <0.01 0.601 Bumblebees Site 13.09 0.001 Webster Nursery also had significantly higher visitation Year 6.50 0.024 rates than Johnson Prairie for several insect morphotypes visiting each of the plant species, specifically bumblebees and Site × Year 0.12 0.780 small dark bees (Tab. 2, Appendix I & II). In 2012, there No longer significant at Bonferroni-corrected alpha = 0.008 were significantly higher rates of bumblebee visits at both No longer significant at Bonferroni-corrected alpha = sites than in 2011. In addition, there were higher visitation 0.017 rates for small dark bees and bumblebees to L. albicaulis at Webster Nursery. # visits / flower / hr # visits / flower / hr March 2015 POLLINATORS AND SEED SET IN NURSERIES 35 larger bumblebees, have relatively large foraging areas (A) (Greenleaf et al. 2007). Hagen et al. (2011) found the foraging area for some Bombus sp. to be between 0.25-43.53 hectares in one to four days. It is most probable that the high densities of flowers and the many blooming species found in -------- a small area are attracting pollinators to this native plant -------- nursery. Some bees can rapidly produce more offspring in -------- response to an increase in floral resources because greater foraging efficiency means less time they are exposed to -------- predators and parasites (Goodell 2003). For some plant species, rapid accumulation of dense native plant resources at a native plant nursery may attract sufficient unmanaged insects to provide pollination services similar or higher than (B) those found in native landscapes. Restoration nurseries are -------- -------- human-altered landscapes with surpluses of native floral resources, which recent studies have found to be ideal factors in drawing diverse and abundant pollinator responses (Winfree et al. 2011). The visitation rate results of this study support the conclusion that restoration nurseries that are several years old may have sufficient unmanaged insects to pollinate many of their forbs. -------- -------- Manipulatively increasing insect visitation at Webster nursery may not be a conservation priority given a lack of evidence for pollen limitation for either plant species. In Natural Hand-crossed Natural Hand-crossed addition, no evidence was found that supplemental pollen Nursery Prairie increases viable seed production for the plants in this study. An earlier study by Fazzino et al. (2011) found that hand- FIGURE 3. Percentage of viable seeds produced by hand-cross pollinated inflorescences produced more sprouting seeds pollination and natural pollination at either Webster Nursery or Johnson Prairie: A) Balsamorhiza deltoidea inflorescences, and B) than naturally-pollinated inflorescences for B. deltoidea in Lupinus albicaulis inflorescences. Open gray circles represent nearby Puget lowland prairies. In contrast, the B. deltoidea percent viability and black horizontal lines represent means +/- 1 plants in this study were either not pollen-limited or the standard error. Bonferroni correction for two tests would hand-pollinated inflorescences did not receive enough necessitate an alpha = 0.025. supplemental pollen by hand to show a significant difference. The discrepancy between this earlier study and the DISCUSSION results presented here could also be due to differences in weather between study years, as poor flight weather can Characteristics of Webster Nursery appear to be dampen insect visitation (Vicens & Bosch 2000), or facilitating higher rates of insect visitation to these two plant differences in methodology. Weather data were not collected species than at the natural prairie site, Johnson Prairie. The in either study throughout the bloom periods at the study nursery is located in an area with fewer assumed floral sites, though past local weather reports indicate similar fair resources for native pollinators, though insects appear to be weather temperature ranges and precipitation levels in 2009 responding to the large influx of floral resources at the and 2012 when B. deltoidea was in bloom (U.S. Climate relatively new nursery that started in 2008. Although Data 2014). Fazzino et al. (2011) examined seed visitation was higher at the nursery site, we found no germination and we measured seed viability using lab evidence of pollen limitation and no differences in viability methods. Due to various factors that can affect germination, for seeds from either location. Restoration managers at the and the destructive properties of tetrazolium testing, these nursery site were hoping to determine methods for increasing two studies are not directly comparable. Future studies could native plant yield and this study suggests that neither involve both viability and germination testing and should increased pollinator populations nor hand pollination would increase the number of replicates to further test for pollen- increase seed viability for these two species. limitation in both plant species. Though not statistically Several studies suggest that human developed landscapes significant, our results did indicate a trend that hand- are not necessarily pollinator deprived. Matteson et al. pollinated inflorescences produced a higher percentage of (2012) found it inappropriate to generalize about landscapes viable seeds. Managers may still consider providing created by humans due to high variability in habitat supplemental pollen treatments to plants or placing managed suitability for pollinators within land-use categories. Some bee colonies on site if planning to collect seed during a year researchers found that bee abundance increases in human- with poor weather for insect visitation. constructed landscapes developed with a superabundance of In this experiment, we tested whether or not more pollen floral resources, and that a combination of natural and would increase viable seed production, though supplemental developed landscapes can provide a greater diversity of pollen does not always benefit plant reproduction. When habitat resources (Frankie et al. 2009). Some insects, such as Percent viable seeds Percent viable seeds 36 HUSBY ET AL. J Poll Ecol 15(5) Appendix IV. Number of seeds produced by Lupinus albicaulis maximum seed production is reached, there are no longer inflorescences. unfertilized ovules for additional pollen to be of benefit (Ashman et al. 2004). 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Published: Jan 1, 2015

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