Sonorex rk 52

Weighing of samples was done with the balances Sartorius Cubis^®‐series (Sartorius, Göttingen, Germany) and Mettler Toledo AB54 (Mettler‐Toledo, Gießen, Germany). The biomaterial was dried using a SP VirTris BenchTop Pro freeze dryer (SP Industries, Warminster, UK). The laboratory mill IKA Labortechnik MF10 basic (IKA^®‐Werke, Staufen, Germany) equipped with a 0.5 mm sieve was used for the comminution of fungal biomaterial. The ultrasonic baths Sonorex RK 52 (BANDELIN electronic, Berlin, Germany) and Sonorex RK 106 as well as the heating bath GFL 1042 (Gesellschaft für Labortechnik, Burgwedel, Germany) were employed for the preparation of crude extracts. Evaporation of solvents was done using a Heidolph Laborota 4000 efficient rotary evaporator (Heidolph Instruments, Schwabach, Germany) coupled to a vacuubrand PC 101 NT (VACUUBRAND, Wertheim, Germany) vacuum pump. The Vortex‐Genie 2 vortex mixer (Scientific Industries, Bohemia, New York) was used to homogenize the samples. Pipettes and pipette tips were purchased from Eppendorf (Hamburg, Germany) or STARLAB International (Hamburg, Germany).

To determine the germination rate, the temperature-adapted fungus cultures were incubated for 21 days on MPA at the corresponding temperatures in the dark. An undefined number of conidia of each fungus was suspended in a 1.5 mL tube filled with 0.5% (v/v) Tween 80^®. The tubes were placed on a vortexer for 10 s and afterwards in an ultrasonic bath (Sonorex RK 52, 35 kHz, Bandelin electronic GmbH & Co. KG) for 15 min. The conidia concentration was determined using a hemocytometer, and a conidial suspension of 1 × 10⁶ conidia/mL was made for each adapted fungus by diluting with 0.5% (v/v) Tween 80^®. The length and width were determined for 100 conidia as previously described.

To determine the pollen grain production per flower we collected five pre‐anthetic flowers from five different plant individuals each (total 25 flowers/species). We transferred the closed anthers of each flower into individual Eppendorf tubes. These were left to dry at room temperature for at least 48 h with an open lid. Afterwards, we added 200 µl glycerol to each sample and mixed it for 5 min with a laboratory mixer mill at 200 Hz (Retsch MM 200; Retsch, Haan, Germany). Samples were then placed in an ultrasonic bath (Sonorex Rk 52; Bandelin, Berlin, Germany) for 15 min each. Finally, we vortexed each sample and transferred 20 µl of the unstained mixture into a hemocytometer containing a Fuchs–Rosenthal counting chamber with 16 squares. Pollen grains were then counted in five randomly chosen large squares under a microscope (Axio, Scope.A1, Zeiss) and the total number of pollen grains per flower was calculated.
To count the number of ovules we used the same 25 flowers as for counting pollen grains. Since the ovules of Lobelia and Mimulus are minute and numerous we cut the ovary in several parts and counted the ovules in aliquots under a stereo microscope and totaled up the counts. We quantified pollen grain number and ovule number of the same flower separately to calculate ovule/pollen ratio of the individual flowers.