factorial experiment (Ranunculaceae)

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Method synopsis

Controlled factorial experiment manipulating both water availability and pollen supplementation to test for interactive effects on plant reproduction.

Synthesized from method descriptions across 4 papers using this protocol.

Procedure as described in the canonical source

Steps below were extracted from the paper that introduces this protocol — Does pollination interact with the abiotic environment to affect plant reproduction? (2025), Annals of Botany. Implementations in other papers (listed below) may differ.

1. Apply pollen supplementation treatments
   Randomly assigned half the plots to pollen supplementation treatment. Collected supplemental outcross pollen from donor plants outside study plots using forceps, gently rubbed across stigmas of treatment plants. Removed anthers from donor plants and transferred pollen using a standard technique in pollination biology.
   Quantities: Supplemental pollen applied to all stigmas on treatment plants, pollen donors usually located further than 5m from focal plantsDuration: Three times per week throughout flowering period of each species (early July for H. fendleri, July-August for D. nuttallianum)Conditions: Applied in morning when anthers contained visible pollen, typically 08:30-10:00h
   Equipment: forceps
2. Apply experimental watering treatment
   In Study 2, established paired plots within 1.25m distance and similar slope/aspect. Randomly assigned one plot per pair to watering treatment. Applied 15L water using plastic backpack sprayer to each watering plot every Monday, Wednesday, and Friday when weather permitted.
   Quantities: 15L water per application, applied to plots averaging 8.5-9.2 plants each, 6 pairs of plots for D. nuttallianum, 10 blocks for H. fendleriDuration: Three times per week during flowering period (June 11 - July 1 for D. nuttallianum, July 15 - August 1 for H. fendleri)Conditions: Weather permitting, excluded during rain events, water sourced from nearby East River
   Equipment: 15L plastic backpack sprayer (SprayMate Storm 2.5-Gallon 18V Battery Powered Sprayer)

Typical Equipment

• SprayMate Storm 2.5-Gallon 18V Battery Powered Sprayer
• 15 L plastic backpack sprayer
• Meter SC-1 porometer

Output Measurements

• fruits per flower
• seeds per fruit
• seeds per plant
• soil moisture
• stomatal conductance

Papers Using This Protocol (3)

Species Studied With This Protocol

Places Where Used