Isoton 2

mRuby2 fluorescence intensity of individual cells from cultures grown in the TECAN plate reader was determined using flow cytometry. Mid-exponential cultures from the plate reader were diluted in Isoton II (Beckman Coulter, Brea, CA) and the fluorescence intensity was determined for 10000 cells per sample on a BD FACSAriaII (Franklin Lakes, NJ) equipped with an 561 nm excitation laser and 582/15 nm emission filter. Data were analyzed using FlowJo v10.2 (FlowJo LLC). As expected from strains in which the mRuby2 expression system is integrated in the genome the fluorescence signal was homogeneously distributed among the yeast population (Supplementary Figure S5).

For each taxon, we averaged the number of stamens and ovules per ovary after dissecting one floral bud from 5 to 21 individual plants under a dissecting microscope totaling 59 flower buds (Table 3). For each sample, we also estimated the number of pollen grains per anther by squashing one anther randomly selected on a slide containing Isoton II (Beckman Coulter, Fullerton, CA) and counting the number of pollen grains contained in aliquots of 500 μl using a particle counter (Coulter Multisizer 3, Beckman Coulter). Pollen production per flower was estimated as the number of pollen grains per anther (mean value of three aliquots) multiplied by the number of anthers in the flower; then, we computed the pollen‐to‐ovule (P/O) ratios accordingly (Cruden, 1977). We also dissected 1–3 recently‐opened flowers from 7 to 10 individual plants per taxon to measure: (a) length of petals, (b) height of the tallest anther, (c) height of the ovary, and (d) height of the stigma; then, we computed the stigma‐anther separation (i.e., herkogamy) by subtracting the height of the tallest anther from the height of the stigma (ovary size plus style length). Stigma‐anther separation has positive values when stamens do not reach the stigma height (i.e., approach herkogamy) and negative values when the stigma is below the anthers (i.e., reverse herkogamy).

Three days after the formation of the first sporangia – checked by daily observations under a magnifying glass – or 5 days after inoculation (depending on the experiment, Table 1), newly formed sporangia were washed from each leaflet in 10 mL Isoton II (saline buffer; Beckman Coulter, Villepinte, France). Suspensions were kept in glass tubes at -20°C until the counting of sporangia number (SN) produced using a Coulter Z2 counter (Beckman Coulter) equipped with a 100-μm aperture tube. Sporangia size (SS) was determined under a microscope fitted with a camera and using the image analysis software Histolab^® v8.1.0 (Microvision Instruments, Evry, France). For this purpose, drops of the sporangial suspension were placed under the microscope and we used the camera measurement software to determine length and width of 100 randomly chosen sporangia. These measures allowed to calculate sporangia volumes, assuming a revolution ellipsoid shape for each sporangium (Philibert et al., 2011 (link)) by applying the standard equation $\frac{4\text{π}}{3}*\frac{length}{2}*{\left(\frac{width}{2}\right)}^2.$

For each MB sample, diameter distribution and concentration were quantified with a Coulter Counter Multisizer 3 (Beckmann Coulter, Krefeld, Germany). For this, 2 μL of MB solution were mixed with 20 ml of ISOTON® II (Beckman Coulter) and measured in a volumetric mode at room temperature. Wide-area optical microscopy (OM) was carried out with a Zeiss Axiovert 40 C with an LD A-Plan 40×/0.5 Ph2 objective (Carl Zeiss Microscopy GmbH, Jena, Germany) at a MB concentration of 1 × 10⁸ MB mL⁻¹. Absorbance intensity of the MB samples was recorded in the 500–900 nm wavelength range using a TECAN Infinite M200 Pro (TECAN group Ltd, Männedorf, Switzerland), where a 150 μL aliquot of 1 × 10⁹ MB mL⁻¹ solutions was collected in a triplicate.

For analysis of spore size distributions, triplicate spore preparations were made in 3 ml of 20% glycerol and suspended in Isoton II (Beckman Coulter, Brea, CA, USA). The particle volume was then measured for a minimum of 100,000 particles per sample using a Multisizer 4e Coulter counter (Beckman Coulter) equipped with a 30 µm aperture tube (size range 0.6–18 µm, aperture current 600 μA). All measurements were conducted with logarithmic spacing into 400 bins using the Multiszier 4 software (Version 4.01) but are presented on a linear x-axis for clarity. For analysis of spore efficiency, triplicate spore preparations were measured in the same way but instead counting the total number of particles in 50 µl. The dilution factor (1:500 for all samples) was then used to calculate the number of particles per µl as presented.

T. brucei blood stream form cells (Lister 427) were cultured in HMI‐9 complete medium with 10% tetracycline free heat inactivated foetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin and 2 mM l‐glutamine at 37°C with 5% CO₂ in non‐adherent culture flasks, with vented caps.78 For RNAi experiments single marker bloodstream (SMB) cells expressing T7 RNA polymerase79 were cultured under the same conditions in the continuous presence of 5 μg/mL neomycin and expression of double‐stranded RNA was induced by the addition of tetracycline at 1 μg/mL. All BSF cell lines were maintained below a culture density of 2 × 10⁶ cells/mL. To determine cell density, 100 μL aliquots were withdrawn from cultures and diluted with 10 mL isoton II (Beckman Coulter), cell number was determined with a Z2 Coulter Counter (Beckman Coulter), averaging 3 measurements. A hemocytometer was also used for counting cells. Procyclic form cells were cultured in SDM79 media supplemented with 10% FBS, penicillin, streptomycin, l‐glutamine and haemin.80 Cells were cultured in non‐vented flasks at 27°C and maintained between 1 × 10⁵ and 2 × 10⁷ cells/mL.