Samples collection and DNA extraction
Locations were selected to cover a large range of climatic conditions
across E. coccineum natural distribution; with three locations
(Chillán, Nahuelbuta and Curacautín) representing the northern part of
the distribution, four locations (Puerto Montt, Chiloé Norte, Chiloé Sur
and Pumalín) the center part of the distribution, and three locations
(Coyhaique, Chile Chico and Torres del Paine) the southern part of the
distribution (Fig. 1a and Supplementary table 1). These three regions
are subsequently named North, Center and South. Three to six individuals
were sampled within each location and five mature leaves were collected
per tree. A total of 42 trees were sampled. To calibrate divergence time
among E. coccineum genetic groups, two individuals ofLomatia hirsuta Diels a Proteaceae species from Embothrieae tribe
(Sauquet et al., 2009) were included as outgroup.
Whole genomic DNA extraction was carried out using the Qiagen DNeasy
Plant kit (Qiagen Inc., USA) following manufacturer’s instructions.
Foliar tissue (50 mg) was kept in Precellys24 homogenizer (Precellys,
USA) with two 1/4” ceramic spheres (MP BIOMEDICALS, USA) and AP1 buffer
until processing in the laboratory. The genomic DNA integrity was
evaluated using direct visualization in 1% agarose gels, and the DNA
was quantified using a Qubit fluorometer (Invitrogen, USA).