Flowering phenology, pollination and seeding interactions in Garden Lupine (Lupinus polyphyllus) Relationer mellan blomningsfenologi, pollinering och frösättning hos blomsterlupin (Lupinus polyphyllus) Amanda Boström Faculty of Health, Science and Technology Biology Bachelor´s thesis, 15 hp Supervisor: Lutz Eckstein Examiner: Larry Greenberg 2020-11-02 Series number: 20:181 Abstract The spreading of the invasive plant Garden Lupine (Lupinus polyphyllus) has become a matter of national importance in Sweden, due to it posing a threat to native plant and pollinator diversity. The effective attraction of bumblebees (Bombus spp.) as pollinators facilitates the production of large numbers of seeds, which are key to the Garden Lupine’s success. Possible self-pollination could also provide a competitive edge for the plant. The objective of this study was to study the relationships between Garden Lupine color morphs, pollinator attraction and seeding. Inflorescences of three color morphs were studied during the flowering period, and bumblebee behavior was observed on the site. After seeding, any produced seeds were collected and analyzed, as well as experimentally germinated to provide insight into their viability. A subset of inflorescences of each color morph was prevented access to pollinators, to study potential self-pollination effects. Bumblebees preferred blue flowers over pink, but no difference in pollination between the color morphs was found. Flower color did not affect seed production or seed morphology. Self-pollinated inflorescences produced fewer seeds than those with access to pollinators, but no difference in seed morphology or germinative success between the pollination methods could be established. The results suggest that seed production and germination are less dependent on pollination than expected. The ability to germinate through self-pollination provides insight into the invasive potential of Garden Lupine, suggesting that further studies are needed to successfully counteract its spread. Sammanfattning Den invasiva växten blomsterlupin (Lupinus polyphyllus) har på senare år blivit en nationell angelägenhet i Sverige, där den hotar mångfalden av inhemska växter och pollinatörer. Blomsterlupinens framgångsrika tilldragning av framförallt humlor (Bombus spp.) som pollinatörer möjliggör det stora antalet frön som den producerar, vilket är nyckeln till dess invasiva etablering. Eventuell förmåga till självpollinering kan också utgöra en konkurrensfördel. Målet med studien var att utforska relationen mellan blomsterlupinens färgmorfer, pollinering samt fröbildning. Blomställningar av tre färgmorfer studerades under blomningsperioden. Humlornas beteende observerades också under perioden vid lupinlokalen. Efter frösättning samlades alla producerade frön upp och analyserades, varefter ett frögroningsexperiment utfördes för att belysa frönas grobarhet. I ett fältexperiment nekades en delmängd av blomställningarna tillgång till pollinatörer, för att studera eventuell självpollinering och dess effekter. Humlorna föredrog blåa blommor före rosa, men ingen skillnad i pollinering mellan färgmorferna kunde fastställas. Blommornas färg hade ingen effekt på fröproduktion eller -morfologi. Självpollinerade blomställningar producerade färre frön överlag än de med tillgång till pollinatörer, men ingen skillnad i frömorfologi eller grobarhet mellan pollineringsmetoderna kunde påvisas. Resultaten antyder att fröproduktion och frögroning hos blomsterlupin är beroende av pollinering i mindre grad än förväntat. Förmågan att gro genom självpollinering belyser blomsterlupinens invasiva potential, och antyder att fler studier behövs för att framgångsrikt motverka dess spridning. Introduction The introduction of a non-native plant species into an ecological community changes the structure and processes of that community (Bartomeus, Vilá & Santamaria, 2008). Flowering plants and pollinators are engaged in mutualistic networks of coevolution, which may be key in sustaining diversity in many communities. Invasive plants affect plant-pollinator interactions by potentially attracting native pollinators, with reportedly both positive and negative consequences for the native plants of their new community (Bartomeus, Vilá & Santamaría, 2008; Stout & Tiedeken, 2017). The ability to self-pollinate may also improve the reproductive success of invasive species, by reducing their dependency on native pollinators (Stout & Tiedeken, 2017), and the correlation of self-compatibility and a species’ propensity for colonizing has been discussed extensively (Baker, 1955). While self-fertilization is an advantage in terms of short-term survival, it is less advantageous than sexual reproduction when it comes to long- term adaptation to a new environment (Wright, Kalisz & Slotte, 2013). The flowering perennial Garden Lupine (Lupinus polyphyllus, Fabaceae) has since its introduction to Europe in the nineteenth century quickly become an invasive species, competing with native species in several countries, including Sweden (Fremstad, 2010). Originating from western North America, it was introduced to and popularized in Sweden as a garden plant, due to its colorful and ornamental inflorescences (Fremstad, 2010). Garden Lupine flowers from late May to early July in Sweden, producing primarily three different color morphs - blue, which is the most common, pink, and white (Pohtio & Teräs, 1995). The inflorescences can grow to over 50 cm, with flowers growing in whorls around a vertical stem. Each inflorescence can produce hundreds of flowers during its flowering period, and each flower has the potential of producing a seed-containing pod. Due to their height and large leaves, they tend to form a dense canopy, obstructing access to both sunlight and pollinators for other flowering plants of smaller stature (Valtonen, Jantunen & Saarinen, 2006). Inflorescences of Garden Lupine flower from the base upwards, with the inflorescence growing as the flowers mature and become pollen-offering. Members of the genus Lupinus have been shown to also produce seeds through self- pollination, albeit to a lesser degree than through open pollination (Shi, Michaels & Mitchell, 2005). The flowers of Garden Lupine attract many native pollinators, especially bumblebees (Jennersten, Berg & Lehman, 1988). This is particularly due to their lack of nectar production, hidden pollen, and complex pollen dispensing mechanism (Mossberg & Cederberg, 2012). The detrimental effects of Garden Lupine on the pollination of native species has been studied extensively, showing that during its flowering period, bumblebees seem to prefer lupines to other native, pollen-producing plants. However, the presence of Garden Lupines may also increase the number of native pollinators sustained by the plant community, with net beneficial effects for native plants outside the lupine flowering period (Jakobsson & Padrón, 2014). Garden Lupines may also adversely affect specialized pollinators of other plant species indirectly, by outcompeting their host plants (Valtonen, Jantunen & Saarinen, 2006). Due to their long tongues and heavy bodies, bumblebees are especially effective at gathering pollen from lupines, compared to other insects. Despite the lupine flowers hiding their pollen, bumblebees seem to ignore inflorescences with low amounts of available pollen and can distinguish flowers without any pollen left from those that have available pollen (Goulson, Hawson & Stout, 1998). Bumblebees show a preference towards the color of flower which they are first to encounter on a flight, providing they receive a reward from that flower (Gumbert, 2000). They are known to traverse lupine inflorescences upward along the maturation gradient, circling each open whorl. They manipulate the lupine pollen-dispensing mechanism by pressing their head against the flower banner and, using their legs and pushing the wing petals down, triggering the piston-like mechanism to release pollen out of the keel onto their stomach (Haynes & Mesler, 1984). Upon pollination, each inflorescence of Garden Lupine produces seed-containing pods, which release the seeds around the mother plant in late summer (Fremstad, 2010). Variation in seed morphology and seed number has been observed in Garden Lupine (Aniszewski, Kupari & Leinonen, 2001; Shi, Michaels & Mitchell, 2005), and the effects of seed number and size on germination success has been the subject of previous studies of several grassland plants (Jakobsson & Eriksson, 2000). The effects of seed morphology on the germination success as well as germination date of Garden Lupine has also been studied (Klinger, Eckstein, Horlemann, Otte & Ludewig, 2020). The interaction between different color morphs of the Garden Lupine, their pollination (open and self-sustained), seeding and germination has not been studied before, however. The objective of the study was to explore the following hypotheses: 1: Different color morphs of the Garden Lupine are equally visited by pollinating bumblebees. Due to previous studies on the preferences of bumblebees and the general dominance of blue lupines, I expected pollinators to show a preference towards blue inflorescences. 2: Different color morphs of the Garden Lupine have similar seed production. As stated above, as blue inflorescences are shown to be most prevalent of the Garden Lupine colors, I expected them to show an advantage in seed production. 3: Seed production of Garden Lupine after self-pollination is not significantly different from seed production after