Western North American Naturalist

Volume 76 Number 1 Article 2

3-31-2016

Population differentiation in early life history traits of lutea var. lutea in the Intermountain West

Lisa Hintz The Evergreen State College, [email protected]

Magdalena M. Eshleman Northwestern University, [email protected]

Alicia Foxx Northwestern University, [email protected]

Troy E. Wood United States Geological Survey, [email protected]

Andrea Kramer Chicago Botanic Garden, [email protected]

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Recommended Citation Hintz, Lisa; Eshleman, Magdalena M.; Foxx, Alicia; Wood, Troy E.; and Kramer, Andrea (2016) "Population differentiation in early life history traits of Cleome lutea var. lutea in the Intermountain West," Western North American Naturalist: Vol. 76 : No. 1 , Article 2. Available at: https://scholarsarchive.byu.edu/wnan/vol76/iss1/2

This Article is brought to you for free and open access by the Western North American Naturalist Publications at BYU ScholarsArchive. It has been accepted for inclusion in Western North American Naturalist by an authorized editor of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Western North American Naturalist 76(1), © 2016, pp. 6–17

POPULATION DIFFERENTIATION IN EARLY LIFE HISTORY TRAITS OF CLEOME LUTEA VAR. LUTEA IN THE INTERMOUNTAIN WEST

Lisa Hintz1, Magdalena M. Eshleman2, Alicia Foxx2, Troy E. Wood3, and Andrea Kramer4

ABSTRACT.—Large-scale restoration is occurring in many areas of the western United States and the use of genetically appropriate native seed is expected to increase the success of restoration efforts. Thus, determining intraspecific variation among populations and its driving forces are the first steps in successful seed sourcing. Here, we examine intraspecific variation of characters expressed in early life history stages of Cleome lutea var. lutea, an annual forb native to the western United States that has attracted increasing attention as a restoration species because it provisions diverse pollinators. We conducted a common garden experiment comprised of 9 populations sourced from across the Inter- mountain West in a climate-controlled growth chamber. We measured 10 life history and morphological traits and found significant among-population differences for 9 of them, including seed germination requirements and flowering status. With the exception of seed germination, this variation was not effectively captured by broad ecoregion delineations, nor was it significantly explained by source site climatic differences or geographic distance between sites. However, flowering status was significantly explained by latitude of the source population (P = 0.033), suggesting that among-population variation reflects divergent adaptation to photoperiod. The variation in life history traits that differentiates our study populations indicates that informed seed sourcing will be necessary when using C. lutea var. lutea for restoration. More comprehensive spatial sampling that stratifies both environmental and geographic variates is needed to determine the drivers of population differentiation and the scale of local adaptation in this species. Such sampling can be used to better inform appropriate seed sourcing decisions. Until then, a cautious approach to sourcing this species for use in restoration is indicated.

RESUMEN.—En muchas zonas del oeste de los Estados Unidos se está implementando la restauración a gran escala y se espera que el uso de semillas de plantas nativas genéticamente adecuadas aumente la probabilidad de éxito en los esfuerzos de restauración. Por lo tanto, determinar la variación intraespecífica de las poblaciones y las fuerzas que las impulsan son los primeros pasos hacia un abastecimiento exitoso de semillas. En este estudio examinamos la variación intraespecífica en los caracteres expresados en etapas tempranas de la historia vida de Cleome lutea var. lutea, una her- bácea anual nativa del oeste de los Estados Unidos. El interés en la restauración de esta especie ha aumentado debido a que provisiona a diversos polinizadores. Llevamos a cabo un experimento de jardín común con nueve poblaciones, que se originan a lo largo de la región conocida como Intermountain West, en una cámara de crecimiento con temperatura controlada. Medimos 10 rasgos de historias de vida y morfológicos y encontramos diferencias significativas entre las poblaciones en nueve de ellos, incluidos requerimientos para la germinación de las semillas y el estado de floración. Con la excepción de la germinación de las semillas, la variación no fue debidamente capturada por las delineaciones amplias de la eco-región, tampoco fue explicada significativamente por las diferencias climáticas de las áreas de origen o por las distancias geográficas entre las zonas. No obstante, el estado de floración fue explicado significativamente por la latitud de la población de origen (P = 0.033), lo que sugiere que entre poblaciones, la variación refleja una adaptación divergente al fotoperiodo. La variación en los rasgos de historias de vida que se distinguen en nuestras poblaciones de estudio indica que un abastecimiento fundamentado de semillas será necesario cuando se utilice a C. lutea var. lutea para la restauración. Es necesario un muestreo espacial más exhaustivo que estratifique ambas variables, ambientales y geográficas, para determinar las causas de la diferenciación de las poblaciones y la escala de adaptación local de esta especie. Tal muestreo podría utilizarse para proporcionar mejor información y contribuir en decisiones más adecuadas con respecto a los abastecimientos de semillas. Hasta entonces, recomendamos un enfoque cauteloso al utilizar esta especie en la restauración.

More than a century of common garden Dudley 1996, Nagy and Rice 1997, Kawecki and reciprocal transplant studies have shown and Ebert 2004, Leimu and Fischer 2008, that most plant species that are distributed Hufford and Mazer 2012). This variation may across heterogeneous landscapes exhibit be due to phenotypic plasticity, in which case among-population variation (Langlet 1971, differences disappear when populations are

1The Evergreen State College, Olympia, WA. 2Program in Plant Biology and Conservation, Northwestern University, Evanston, IL. 3United States Geological Survey, Colorado Plateau Research Station, Flagstaff, AZ. 4Corresponding author. Chicago Botanic Garden, Glencoe, IL. E-mail: [email protected]

6 2016] POPULATION DIFFERENTIATION IN CLEOME LUTEA VAR. LUTEA 7 grown together under identical conditions resulting in unique ranges of tolerance for (Hufford and Mazer 2003, Kawecki and Ebert survival and reproduction. 2004, Johnson et al. 2012). Among-population Research on the distribution and causes of variation may also be due to genetic differ- population differentiation is important for ences that are driven by adaptation to local informing restoration practice, as it allows conditions and/or by genetic drift (Hiesey et researchers to identify appropriate seed trans- al. 1942, Nagy and Rice 1997, Hufford and fer zones that delineate the extent to which Mazer 2003). Local adaptation is determined seeds or propagules of a particular ecotype by the interaction between the strength of may be moved across the landscape with natural selection and gene flow among popu- minimal risk of maladaptation (Hufford and lations, often producing unpredictable out- Mazer 2003, Johnson et al. 2012). For a grow- comes (McKay et al. 2005). High rates of ing number of species that are commonly gene flow among populations that occupy used in restoration, seed zones have been different habitats can preclude adaptive di - empirically delineated (Knapp and Rice 1996, vergence unless selection is strong enough to Miller et al. 2011, Johnson et al. 2012). How- exclude migrants (Vergeer and Kunin 2013). ever, most species used in restoration lack However, gene flow also increases additive the genecological data needed for the devel- genetic variance segregating in a population opment of empirical seed zones (Knapp and and thus can increase the potential of popu- Rice 1994, Hufford and Mazer 2003, Johnson lations to respond to local selection (Fisher et al. 2004, Johnson et al. 2010a, 2012, 2013, 1930). Lack of gene flow may facilitate local St. Clair et al. 2013). To accommodate for adaptation particularly in large (e.g., more this lack of research, Omernik’s Level III than 1000 individuals) plant populations Ecoregions (Omernik 1987) have been sug- (Leimu and Fischer 2008), whereas genetic gested for use as minimum seed transfer drift may strongly influence the genetic zones in the Intermountain West (Johnson et structure and phenotypic divergence of al. 2010a). Recent research shows that these small populations (McKay et al. 2005). In proxy seed zones can capture at least some order to test whether intraspecific variation of the variation found among the popula- identified among populations is driven by tions of widespread perennial forbs in the natural selection (i.e., populations are adapted Great Basin (Miller et al. 2011, Kramer et al. to local conditions) or genetic drift, recipro- 2015), but that smaller Provisional Seed Zones cal transplant experiments (Hiesey et al. 1942) (Bower et al. 2014) are generally more effec- or comparisons of phenotypic to neutral tive at capturing variation. genetic divergence (Kawakami et al. 2011) are Creating a strategy that will maximize necessary. establishment and long-term survival of Regardless of whether natural selection or seeded native plant populations is of the drift is driving genetic differentiation among highest importance in any restoration project populations, the presence of intraspecific varia- (Jones and Johnson 1998, McKay et al. 2005). tion indicates that populations are heteroge- Seed germination and early establishment are neous across their range and that populations often the most significant bottlenecks to re - cannot necessarily be treated as equivalent cruitment and restoration success, and this is when sourcing plant material for restoration particularly true in the semiarid habitats of (McKay et al. 2005, Kulpa and Leger 2013). If the Great Basin and Colorado Plateau (Knapp introduced genotypes are not adapted to the and Rice 1994, James et al. 2013, Kulpa and restoration site, they may negatively impact Leger 2013). To address this, increased re - the survival and adaptive potential of the search is needed specifically on interspecific restored population, as well as any nearby and intraspecific variation of early life history populations (Jones and Johnson 1998, Hufford stages (e.g., seed stratification requirements, and Mazer 2003, Johnson et al. 2004, McKay germination, early survival, and establish- et al. 2005, Johnson et al. 2010b). However, ment) of used in restoration seed mixes. patterns of local adaptation, and the sourcing Ensuring that the source material used at decisions derived from them, are expected to restoration sites is appropriately adapted to be species specific because each species has conditions that impact fitness during early unique patterns of gene flow and adaptation life history can increase the efficiency and 8 WESTERN NORTH AMERICAN NATURALIST [Volume 76

In the present study, we examined among- population variation in life history traits and morphological characters expressed in Cleome lutea Hook. var. lutea (Capparaceae; yellow beeplant or yellow spiderflower), an annual forb species targeted for increasing restoration use in the Intermountain West. We hypothe- sized that C. lutea var. lutea plants sourced from populations found in substantively differ- ent environments of this region would demon- strate significant phenotypic variation when grown in a common environment and that differences would be greater between popu- lations from different Level III Ecoregions than between those from the same ecoregion. Further, we tested whether differences among populations could be explained by geographic and/or climatic distance between populations.

METHODS Study Species: Cleome lutea var. lutea Cleome lutea var. lutea is an herbaceous Fig. 1. Map of study populations in the Great Basin and Colorado Plateau, shown with Level III Ecoregions. Popu- annual native to western North America, lation codes correspond with ecoregion abbreviations, as thriving in sandy, disturbed sites in hot, dry indicated in Table 1. climates. It grows to 1 m high, is taprooted, and produces indeterminate racemes of bright yellow flowers. Cane (2008), in a study of the success of these efforts, mitigating economic breeding biology of C. lutea, found that out- and ecological losses via short- and long-term crossed flowers did not have greater fertility plant failure (Knapp and Rice 1994, Jones and than selfed flowers; however, flowers are visited Johnson 1998, McKay et al. 2005, Gonzalo- by a wide range of insects, especially bees, Turpin and Hazard 2009, Hufford and Mazer which likely results in substantial outcross- 2012). ing in nature. Fruits develop into siliques that There is a need for large-scale restoration dehisce, dispersing seed gravitationally. Annual across much of the Intermountain West due early successional species may play impor- to habitat degradation caused by intensive tant roles following disturbance (Walker and grazing (Pellant et al. 2004), mining, oil and del Moral 2009), and C. lutea var. lutea is a gas exploration and extraction, (Schwinning et particularly important candidate for restora- al. 2008), invasive species (Floyd et al. 2006), tion use because it thrives in disturbed areas, and altered fire regimes (D’Antonio and emerges rapidly, reproduces quickly, and pro- Vitousek 1992). Restoration efforts that in - vides wildlife and pollinator forage during clude the application of native seed have the first year following seeding (Comstock been shown to significantly decrease the level and Ehleringer 1992, Cane 2008, Ogle et al. of exotic invasion and its impacts (Stohlgren 2011, Wood et al. 2015). et al. 2003, Floyd et al. 2006). Local genotypes of many native plant species (particularly Study Region, Sites, and Seed Collections forbs) are not currently available for restora- The Intermountain West is bordered by the tion in this region, but both the Colorado Rocky Mountains on the east and the Sierra Plateau Native Plant Program and the Great Nevada on the west. We focus on 4 Level III Basin Native Plant Project are working with Ecoregions within this region: the Colorado collaborators across the regions to address Plateau (CP), the Northern Basin and Range this (Peppin et al. 2010, Shaw and Jensen (NBR), the Central Basin and Range (CBR), 2014, Wood et al. 2015). and the Wasatch and Uinta Mountains (WUM; 2016] POPULATION DIFFERENTIATION IN CLEOME LUTEA VAR. LUTEA 9

see Fig. 1). These ecoregions are characterized by heterogeneous landscapes with steep gra- C) ° 0.9 dients in environmental variables known to − impact plant adaptive divergence (e.g., tem- MTWetQ perature and precipitation amount and season- ality; Bower et al. 2014) resulting, in part, from an extreme topography of basins, ranges,

and plateaus. Additionally, these ecoregions experience cold winters and a semiarid weather pattern typically featuring winter pre- cipitation (Comstock and Ehleringer 1992, mperature during the wettest quarter C) (mm) ( ° Pellant et al. 2004, Schwinning et al. 2008). Due to a summer monsoon cycle, the southern portion of the Colorado Plateau receives more annual precipitation than the northern Colo- mes. rado Plateau or the other ecoregions, and this precipitation seasonality impacts plant adap- tive strategies (Comstock and Ehleringer 1992). In combination, high rates of change in climate over short distances within and among these ecoregions have led to high floral spe- UT 2416 6.5 241 16.4 cies biodiversity and to intraspecific variation OR 965 9 282 UT 1756 8.4 247 14.8 NV 1473 10.1 159 13.4 among plant populations (Comstock and

UT 1834 8.7 185 16.5 CO 1459 10.6 243 17.6 UT 1280 12.3 236 19.1 UT 1771 10.4 215 20.9 UT 1423 8.7 297 18.3 Ehleringer 1992). State (m) ( Seeds were sourced from 5 populations in the CP Ecoregion, 2 from CBR, and 1 each from NBR and WUM (population name corresponds to the Level III Ecoregion the population is found in; Table 1). All seeds were collected between 2009 and 2013 using the protocol Elevation MAT MAP from the Bureau of Land Management’s Seeds of Success program, with many obtained through the United States Department of Agriculture’s Germplasm Resource Informa- tion Network. The Seeds of Success collection protocol stipulates that collections be made when seeds are ripe and include a minimum of 30 individuals (BLM 2012). Common Garden Experiment Plants were grown at the Chicago Botanic Garden (Glencoe, IL) between May and August 2014. For each population, 33 to 51 seeds (based on seed availability) were placed in a petri dish on 1.5% agar in cold stratifica- tion at 3 °C on 30 May 2014. After 25 d of cold stratification, the germination status of each seed was recorded for each population to assess chilling time required for germination. With the exception of NBR (no germination), at least 10% of sown seeds for each popula- 1. Study population location information, including mean annual temperature (MAT), mean annual precipitation (MAP), and te 1. Study population location information, including mean annual temperature (MAT), tion had germinated in cold stratification by

ABLE this time. On 26 June, germinated seedlings CBR1 Central Basin and Range 9/21/2009 N38.82, W111.91 CBR2 Central Basin and Range 9/21/2009 N39.32, W117.93 WUM and Uinta Mountains 9/22/2009 Wasatch N38.35, W111.52 CP1 Colorado Plateau 7/13/2011 N37.49, W112.09 CP2 Colorado Plateau 6/28/2013 N39.21, W109.05 CP3 Colorado Plateau 6/19/2013 N38.80, W109.21 CP4 Colorado Plateau 9/28/2009 N37.85, W111.37 CP5 Colorado Plateau 8/11/2013 N40.03, W109.73 NBR Northern Basin and Range 7/7/2009 N43.72, W117.35 Site code Level III Ecoregion date Latitude, longitude Seed collection T (MTWetQ), as identified by WorldClim Global Climate Data (Hijmans et al. 2005). Site codes correspond to Level III Ecoregion na as identified by WorldClim (MTWetQ), were individually weighed and randomly 10 WESTERN NORTH AMERICAN NATURALIST [Volume 76 planted into 25.4 mm × 120.65 mm cone-tainers using R version 2.15.3 software (R Core Team using a random number generator (1 seed per 2013): mean annual temperature (MAT), mean cone-tainer) with Fafard 3BC coir potting soil diurnal range (MDR), temperature seasonality mix (Conrad Fafard, Inc., Agawam, MA). As (TSeason), mean temperature wettest quarter additional seeds germinated in cold stratifica- (MTWetQ), mean annual precipitation (MAP), tion, seedlings were weighed (to the nearest precipitation seasonality (PSeason), and pre- 0.0001 g) and planted as above until 1 July, cipitation warmest quarter (PWarmQ). Mantel when 30 seeds from each population had ger- tests were run between pairwise climate dif- minated, and had been weighed and trans- ferences and geographic distance to deter- planted. These 30 transplanted seedlings from mine whether geographic distances should be each population were grown in a climate- controlled for when testing for relationships controlled growth chamber with a 12-h photo - between climatic variables and measured period at 15/10 °C day/night temperatures. traits. Mantel tests were also run between After seedlings were established under these pairwise latitude and climate differences to colder conditions they were moved to a assess whether climate should be controlled warmer 15/9-h photoperiod with 24/15 °C for when testing for relationships between day/night temperatures. Because of varying latitude and measured traits. To determine germination timing, not all seedlings received whether our study populations occupy differ- the same number of days in colder conditions ent climate space in different Level III Ecore- (range 4–7 d), but all seedlings were kept in gions, we used a t test in JMP (SAS Institute, warm conditions for 3 weeks. Soil was allowed Inc. 2014) to determine whether pairwise to dry before all plants were watered evenly differences in means among populations for as needed with tap water. each climatic variable were greater for pair- After 3 weeks in warmer conditions, 9 traits wise comparisons between populations in were measured on all 30 individuals from each different Level III Ecoregions (n = 25) rela- population (n = 29 for CP1). Measured traits tive to populations in the same ecoregions (n are as follows: aboveground biomass (to the = 11). Finally, to understand whether differ- nearest 0.0001 g after drying at 29 °C for 2 d, ences among ecoregions may be driven by divided by Growing Degree Days [GDD] to geographic distances, we also conducted a t account for potential differences in cold grow- test to determine whether pairwise geo- ing condition), height (soil to apical meristem, graphic distances were greater between popu- divided by GDD), leaf number (divided by lations in different Level III Ecoregions rela- GDD), proportion of 3-merous leaves, leaflet tive to populations in the same Level III width/length (indicating leaflet shape; taken Ecoregions. from the center leaflet of the second set of Morphological and Life History Traits Analyses true leaves), leaflet area (calculated as the area of an ellipse, which approximates the shape To test for significant differences in mea- of each leaflet), petiole length (of 1 leaf of sured traits among populations, one-way the second set of true leaves), taproot diameter ANOVA tests were run in JMP (SAS Institute, (just below soil surface), and flowering status Inc. 2014) for all normally distributed traits: (which captured any flowering event prior to aboveground biomass/GDD, height/GDD harvest, even if flowers were no longer present (square-root transformed), number of leaves/ at harvest). GDD, leaflet width/length, leaflet area, petiole length, and taproot diameter (log trans- Site Characterization formed). Analyses initially included seedling In order to identify potential geographic weight and population as fixed effects. or climatic drivers of differentiation among Seedling weight did not significantly explain study populations, pairwise geographic dis- variation for any measured trait, so it was tances were calculated using ArcGIS version removed from final analyses. Generalized 10.1 (ESRI 2012), and an analysis of climatic linear models with a Poisson or binomial differences was undertaken. For the latter, 7 distribution and log link function were used bioclimatic variables available through World- to test for the effect of seedling weight and Clim (Hijmans et al. 2005) were extracted population in explaining measured variation from BioClim rasters for all study populations in 3 traits: proportion of 3-merous leaves, 2016] POPULATION DIFFERENTIATION IN CLEOME LUTEA VAR. LUTEA 11

TABLE 2. Results of statistical analysis for 10 traits mea- other climatic variables, linear regressions sured on plants grown in warm conditions for 3 weeks, were performed in JMP (SAS Institute, Inc. and 1 trait measurement (germination) taken after 25 d in cold stratification. Analyses of variance (ANOVA) and 2014) to test for relationships between climate generalized linear models (GLM) are shown. and population trait means for all measured traits. Linear regressions were also used to Measurement df F P identify significant relationships between lati- ANOVA tude and mean trait values for all traits. Aboveground 8 4.60 <0.0001 biomass/GDD Height/GDD 8 36.12 <0.0001 RESULTS Number of 8 9.57 <0.0001 leaves/GDD Site Characterization Leaf length 8 5.26 <0.0001 Mantel tests identified a significant correla- Leaf width 8 8.71 <0.0001 Petiole length 8 7.36 <0.0001 tion between geography and 1 Bioclim variable Taproot diameter 8 1.03 0.4104 (mean temperature wettest quarter [MTWetQ]: P = 0.0074); no other significant relationships Measurement df c2 P between geographic and climate distance, or GLM between latitude and climate distance, were Germination 8 250.13 <0.0001 Proportion of 8 58.03 <0.0001 found. Likewise, t tests revealed that climatic 3-merous leaves differences were greater between Level III Flowering status 8 212.19 <0.0001 Ecoregions than within them for only 1 of 7 Bioclim variables (MTWetQ; t27 = 4.12, P < 0.0003), with the mean distance between popu- proportion of plants in flower at harvest (pro- lations in different ecoregions being 4 times portion in flower), and proportion of seeds greater than within ecoregions (8.1 °C [SE germinated after 25 d in cold stratification 14.5] vs. 2.0 °C [SE 3.4]). A t test also con- (proportion germinated). As with ANOVA firmed that geographic distance between tests, seedling weight did not significantly populations was greater for comparisons be - explain variation for any measured trait and tween ecoregions than comparisons within was removed from final analyses. Significant ecoregions (t33 = 3.3, P < 0.0023), with the differences among populations were evaluated mean distance between populations in differ- following ANOVA with post hoc Tukey’s HSD ent ecoregions nearly twice as much as within tests in JMP, and pairwise population differ- ecoregions (478 km [SE 59] vs. 239 km [SE ences were assessed for generalized linear 42], respectively). Taken together, these results models using the Multcomp package (Hothorn indicate that our study species occupies differ- et al. 2008) in R version 2.15.3 software (R ent climate space in different ecoregions for Core Team 2013). MTWetQ, and illustrate the need to control Finally, for all traits with significant among- for distance between populations when test- population variation, we performed t tests in ing for relationships between MTWetQ and JMP (SAS Institute, Inc. 2014) to determine measured traits. whether pairwise differences in population trait means were greater for comparisons Morphological and Life History Traits Analyses between populations in the same Level III We identified significant differences among Ecoregion (n = 25) relative to comparisons populations for 9 of 10 traits (Table 2), includ- between populations in different ecoregions ing aboveground biomass/GDD (F8 = 4.6, P (n = 11). < 0.0001), height/GDD (F8 = 36.12, P < 0.0001), number of leaves/GDD (F = 9.57, Relationship between Traits, 8 P < 0.0001), leaflet width/length (F = 7.52, P Geography, Climate, and Latitude 8 < 0.0001, Fig. 2B), leaflet area (F8 = 7.85, P < For the climatic variable correlated with 0.0001), petiole length (F8 = 7.37, P < 0.0001), geography (MTWetQ), a partial Mantel test proportion of 3-merous leaves (c2 = 58.03, P was performed in R version 2.15.3 software (R < 0.0001), proportion in flower (c2 = 212.19, Core Team 2013) to test for a relationship P < 0.0001; Fig. 2C), and proportion germi- between climatic and trait distances while nated (c2 critical value = 250.13, P < 0.0001; controlling for geographic distance. For all Fig. 2A). There were no significant differences 12 WESTERN NORTH AMERICAN NATURALIST [Volume 76

Fig. 2. Significant among-population variation (P < 0.0001): A, proportion of seeds germinated after 25 d in cold strati- fication; B, leaf width/length (cm) of the center leaflet of on leaf of the second set of true leaves; C, proportion of plants in flower after 3 weeks of growth in warm conditions. Bars shaded by Level III Ecoregion. Error bars are calculated with a 95% confidence interval of the mean. Letters denote significant differences among populations (P < 0.05).

(a = 0.05) among populations for taproot 0.028; [difference of 51.0% + 6.0% vs. 30.1% diameter. + 6.6%, respectively]). Of the 10 traits evaluated, Level III Ecore- Relationship between Traits, gion contrast type (within vs. between) was Geography, Climate, and Latitude significant for only seed germination, where population comparisons between ecoregions Results of the partial Mantel test identified were greater than within them (t26 = 2.3, P < a significant relationship for germination 2016] POPULATION DIFFERENTIATION IN CLEOME LUTEA VAR. LUTEA 13

Fig. 3. Linear regression shows that latitude explains more than 50% of the among-population variation in flowering (P = 0.033). status between climatic (mean temperature conditions of restoration (Walck et al. 2011, wettest quarter: MTWetQ) and trait distances Kulpa and Leger 2013). Because we could not while controlling for geographic distance (posi- directly test whether the differences we mea- tively correlated with MTWetQ; r = 0.363, P sured were adaptive (i.e., shaped by different = 0.002). Linear regression identified a single selection pressures at each site), we utilized significant relationship between climate and easily extractable geographic and abiotic data a measured trait, with populations from sites from source sites and identified potentially with higher mean annual temperatures (MAT) adaptive variation in 2 traits. This included having greater percent germination after 25 d chilling time required for germination, which 2 in cold stratification (F1,7 = 13.26, R = 0.65, was significantly and positively correlated P = 0.008). Finally, latitude significantly ex - with both mean temperature of the wettest plained variation in only one measured trait: quarter (MTWetQ) and mean annual tempera- plants from populations at higher latitudes ture (MAT). We also identified a significant were more likely to flower by the conclusion positive relationship between latitude and 2 of the study (F1,7 = 7.05, R = 0.5016, P = flowering status, suggesting that our study 0.033; Fig. 3). All other partial Mantel and populations may be adapted to daylength or linear regression tests were not significant some other factor that varies by latitude at each (P > 0.05). source site. Taken together, these results sug- gest that seed source may impact restoration DISCUSSION outcomes for C. lutea var. lutea. The use of proxy seed transfer zones like Populations of the annual forb C. lutea var. Level III Ecoregions has been suggested as lutea sourced from 9 sites across the Inter- potentially effective minimum transfer guide- mountain West and grown in a common envi- lines for understudied species like C. lutea var. ronment exhibited substantial and significant lutea (Kramer et al. 2015). In fact, previous variation in 9 traits expressed in early life his- common garden studies of forbs in the west- tory. Traits with significant among-population ern United States have shown that these Level differences included life history traits (e.g., III Ecoregions can capture substantial (but not proportion of seeds germinated and flowering all) phenotypic variation present in a species status) as well as morphological traits of young being targeted for restoration, and therefore plants (e.g., leaf size and plant height). These may serve as a useful minimum guide for seed traits may influence plant establishment, sur- transfer (Miller et al. 2011, Kramer et al. 2015). vival, and regeneration under the stressful Our results, however, identified differences in 14 WESTERN NORTH AMERICAN NATURALIST [Volume 76 population means that were as great within are needed to determine whether these differ- ecoregions as between them for all traits with ences are truly adaptive. significant population differentiation except Another important consideration in select- seed germination. This suggests that Level III ing source material for restoration is among- Ecoregions are too large to effectively group population variation in flowering phenology. among-population variation in this species Numerous studies have found significant varia- into transfer zones, as has been found for tion in flowering phenology among study many other forb species native to this region populations, and this variation often has a (Bower et al. 2014, Kramer et al. 2015). For C. genetic basis (Wilczek et al. 2010). A recent lutea var. lutea, the use of smaller Provisional meta-analysis indicated that variation in flow- Seed Transfer Zones or Level IV Ecoregions ering phenology is frequently under selection, may be warranted, and should be tested in particularly at higher latitudes (Munguía- future studies. In order to determine specific Rosas et al. 2011). In many cases, this means seed sourcing recommendations for C. lutea populations are often adapted to site-specific var. lutea in the Intermountain West, further environmental cues like photoperiod and tem- research incorporating additional source popu- perature, with populations in short-season lations and a more extensive common garden habitats flowering earlier than those in long- study will be necessary (e.g., Johnson et al. season habitats (Ray and Alexander 1966, 2013). Stinchcombe et al. 2004, Franke et al. 2006, Seed germination is a critical limiting step Ågren and Schemske 2012). It is therefore in restoration (Larson et al. 2015). If among- important to ensure that environmental cues population variation in seed germination timing of a source site match those at a restoration is present and adaptive, restoration sourcing site. This is particularly true for annual plant decisions should incorporate knowledge of species, as they have only 1 growing season this variation in order to maximize the chances interval to produce seed. Additionally, seasonal that seeds used in restoration are able to ger- mismatches between animal-pollinated plants minate at the right time. We found extensive and pollinators in a habitat can lead to among-population variation in seed germina- reduced seed set (Kudo and Ida 2013). Al - tion requirements in C. lutea var. lutea. This though C. lutea var. lutea is capable of pro- variation may be adaptive, as it was signifi- ducing viable seeds without pollinator visits cantly explained by climate at each source site (autogamy), flowers visited by pollinators pro- (specifically MAT and MTWetQ). In general, duce significantly more seeds (Cane 2008). We populations from colder climates displayed were not able to determine whether the greater dormancy and required more chilling among-population variation we measured in time to germinate. These results are consistent flowering status of C. lutea var. lutea is driven with seed germination studies of other species by selection, but we did find that plants from across the Intermountain West (e.g., Penste- higher latitudes were significantly more likely mon and Eriogonum species [Kramer et al. to be flowering at the conclusion of our 2015] and Allium species [Phillips et al. 2010]). study than those from lower latitudes, re- Similar variation in seed dormancy has been gardless of the climate or ecoregion from shown to be adaptive (Huang et al. 2010) but which they were sourced. Additional research may also be due, at least in part, to plastic that manipulates temperatures and photo - responses experienced during seed matura- periods, and assesses days to flower rather tion at each study site (Munir et al. 2001). For than flowering status at a single point in time, example, extensive research on seed dor- is needed to better understand how different mancy in another annual plant species (Ara- populations respond to different environmental bidopsis thaliana) has shown that dormancy is cues. Finally, to determine whether these dif- both adaptive and driven by a combination of ferences are adaptive, reciprocal transplant genetic and environmental factors (Debieu et studies, or other methods that account for al. 2013). Additional common garden research neutral differentiation, are needed. is needed in order to determine the genetic Additional work is needed to understand and environmental basis of seed germination whether our limited ability to explain signifi- variation in C. lutea var. lutea, and reciprocal cant among-population variation in morpho- transplant studies or molecular genetic studies logical traits is an artifact of sampling or 2016] POPULATION DIFFERENTIATION IN CLEOME LUTEA VAR. LUTEA 15 whether this variation can be explained by research on C. lutea var. lutea to guide restora- other (untested) biotic or abiotic factors at tion sourcing would benefit from more thor- source sites. Native plant populations in the oughly incorporating the possibility of maternal Great Basin, where precipitation largely falls effects into the study design. during the coldest months of the year, are gen- Application to Restoration erally adapted to minimum temperature and aridity (Comstock and Ehleringer 1992). This The significant among-population differen- pattern is less clear in the Colorado Plateau. tiation we identified in life history and mor- For example, many source populations in Utah phological traits of C. lutea var. lutea from the and Colorado experience a seasonal monsoon Intermountain West indicates that seed source gradient, with southern populations experi- may be an important predictor of restoration encing an increase in precipitation during the success for this species. While seeds of this hot and otherwise dry summer months (Com- species are currently available for purchase stock and Ehleringer 1992, Schwinning et al. from commercial native seed vendors in the 2008). This means that plant populations may Intermountain West, very little is known be adapting to differences in seasonal aridity about where these seeds were sourced from, across their distribution. In all cases, climate, making it challenging to determine the most soil type, and numerous other untested abiotic appropriate location for their use. For exam- and biotic factors may be driving measured ple, we identified a significant relationship differences. between cold stratification requirements for The variation measured among study popu- seed germination and mean annual tempera- lations may also be due, at least in part, to ture (MAT). This suggests that restoration genetic drift. In self-compatible species like efforts would benefit from minimizing differ- C. lutea var. lutea, it is not clear whether local ences in MAT between source and restoration adaptation or genetic drift is more likely to sites to ensure that seed dormancy is broken play a greater role in population differentia- at the right time. While our results do not tion. For example, selfing may indirectly pro- allow us to prescribe specific seed sourcing mote local adaptation by limiting gene flow guidelines for this species, they do indicate that would otherwise counter adaptive diver- that Level III Ecoregions are not an effective gence (Linhart and Grant 1996). Yet selfing is guide for seed sourcing. Provisional Seed also associated with smaller population sizes, Zones, which are smaller and incorporate indicating a role for genetic drift in population high-resolution climate data on aridity and differentiation, and these opposing forces may mean monthly winter temperature, may pro- cancel each other out (Hereford 2010). How- vide better sourcing guides for the use of ever, many of our study populations contained this species in restorations. Additional re - hundreds of individuals, and, while controlled search with more study populations from crosses do not suggest that there is selection throughout the range of the species is neces- against selfing, cross-pollination within and sary to determine the most ecologically and between populations is likely facilitated by the economically appropriate restoration sourcing >140 bee species documented to visit this approach for this species. species in southern Utah alone (Cane 2008). A final cause of the differentiation we iden- ACKNOWLEDGMENTS tified may be environmental maternal effects rather than, or in addition to, adaptive or We are grateful to Shannon Still for valu- random genetic causes. While time and fund- able statistics help and to Grace Guarraia ing limitations did not allow us to directly and Jada Washington for project assistance and assess or control for effects of maternal provi- data acquisition. Thanks also to seed pro - sioning due to environmental variables on viders, including the USDA Germplasm Re - our study populations (e.g., producing F1 seed source Information Network, Patty West and under common conditions to use in common other collectors for the BLM Seeds of Success garden studies), research has shown that Program, and Steve Parr at the Upper Colorado maternal effects can influence variation ex- Environmental Plant Center. The Kramer– hibited by plants at early life history stages Havens lab group and Dr. Dylan Fischer pro- (Bischoff and Müller-Schärer 2010). Future vided valuable feedback on previous versions 16 WESTERN NORTH AMERICAN NATURALIST [Volume 76 of this manuscript. Funding was provided by HIESEY, W.M., J. CLAUSEN, AND D.D. KECK. 1942. Rela- the Bureau of Land Management’s Plant tions between climate and intraspecific variation in plants. American Naturalist 76:5–22. Conservation Program and National Science HIJMANS, R.J., S.E. CAMERON, J.L. PARRA, P.G. JONES, AND Foundation REU Grant 0648972. A. JARVIS. 2005. Very high resolution interpolated climate surfaces for global land areas. International LITERATURE CITED Journal of Climatology 25:1965–1978. HOTHORN, T., F. BRETZ, AND P. W ESTFALL. 2008. Simulta- neous inference in general parametric models. Bio- ÅGREN, J., AND D.W. SCHEMSKE. 2012. 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