2.1. Glasshouse experiment

Four serotinous species from the family Proteaceae, Banksia hookeriana Meisn., Banksia leptophylla A.S. George, Hakea costata Meisn. and Hakea polyanthema Diels., from the biodiverse SWA Kwongan were investigated (for details see the electronic supplementary material). For each species, canopy-stored seeds set 1–2 years prior to the investigation were collected from five sites (three for Hakea costata) of different post-fire age at eight locations near Eneabba, Western Australia (electronic supplementary material, table S1). The sites are geographically proximate (2–60 km) and with similar species composition (typical Kwongan vegetation dominated by species from the families Proteaceae and Myrtaceae) and soils (low nutrient, acidic white sands). All sites have experienced the same long-term climate while fire history may vary, as fires are patchy in size and location [27]. Distances between sites are sufficiently large such that genes are not immediately mixed through pollen and seed dispersal after fire [19,20]. All sites were last burnt at least 8 years ago, and so supported mature stands of the selected species. Sites were classified either as average-to-wet winter (HiR; high rainfall populations) or dry winter (LoR; low rainfall populations) based on total rainfall in the first winter/spring following the last fire, with ‘dry’ defined as more than 20% below the long-term average mean winter rainfall at Eneabba (electronic supplementary material, table S2).

Seeds were extracted from woody fruits and germinated at 15°C before being transferred to custom-made pots (15 cm diameter, 100 cm deep—to facilitate the natural pattern of early tap root growth). Seedlings were grown in a temperature-controlled glasshouse (air temperature ranged from 12.9°C to 36.1°C, and soil temperature ranged from 12.4°C to 31.2°C). Seedlings were watered every second day with 200 ml water for four weeks to allow seedlings to establish. Once established, seedlings from HiR and LoR sites for each species were divided into two treatment groups, with up to 60 replicates per species per group and an equal number of seedlings from each location in each group. Seedlings were subjected to either a control (mean winter rainfall at Eneabba over the past 30 years equivalent; approximately 200 ml per plant every second day) or drought regime (equivalent to a 50% decrease in mean winter rainfall at Eneabba; 100 ml per plant every second day) for two weeks and then no water over the next three months simulating severe drought. After three months of growth, half of the seedlings (up to 24, electronic supplementary material, table S4) of each species were harvested for measurement of growth and phenotypic variation in drought resistance traits. The remaining half of the seedlings were grown on without water supply in the greenhouse for a further 12 weeks with mortality recorded each week. For more details, see the electronic supplementary material.