2.2 Sample collection and laboratory procedures
From 2015 to 2017, we conducted a thorough sampling across the known distribution range in the south-central mountain systems of Hainan Island and we collected 217 leaf samples from 10 populations of P. heterotricha (Table 1, Figure 1). Each sampled leaf was dried in a separate plastic bag containing 20 to 30 g of silica gel. We collected two individuals with flowers and fruits as representative herbarium vouchers, and these have been deposited in HUTB, under accession numbersLing & Ren 2016091902 and Ling & Ren 2016091501 . Total genomic DNA for each sample was extracted using the CTAB method (Doyle & Doyle, 1987), and served as the template for the polymerase chain reaction. DNA quality and quantity on 0.8% agarose gels stained with 2.5 μl Goldview (Aidlab Biotechnologies Co., Ltd) was detected by AL2000 DNA maker (Aidlab Biotechnologies Co., Ltd) in DTU-48 spedtrophotometer (Hangzhou Miu Instruments Co., Ltd, China).
One nuclear ribosomal DNA (nrDNA) sequence, the ITS region comprising spacer 1, the 5.8S gene and spacer 2 (White et al. , 1990) and two chloroplast DNA (cpDNA) intron-spacer region trn L-trn F (Taberlet et al. , 1991) and ycf 1b (Dong et al. , 2015) were used in this study (Table 2). PCR reactions were set up in a volume of 25 μl composed of 20 μl ddH2O, 2.5 μl 10×Buffer, 0.5 μl 10 mM dNTPs, 0.5 μl each 5 μM primer, 0.5 μl DNA template and 0.5 μl 5 U/μl Taq polymerase (Aidlab Biotechnologies Co., Ltd). The PCR reactions were carried out on the 2720 Thermal cycler (Applied Biosystems by Life Technologies, made in Singapore) and Veriti 96-Well Thermal Cycler (Applied Biosystems by Life Technologies, made in Singapore). The PCR program for ITS1/2 and trn L-trn F was designed to an initial denaturation at 94 ºC 5 min, followed by 35 cycles of 1 min at 94 ºC, 1 min at 55 ºC, 1 min at 72 ºC, and with a final extension of 10 min at 72 ºC. Amplification of ycf 1b used the following protocol: 4 min at 94 ºC, 35 cycles of 30 s at 94 ºC, 40 s at 58 ºC, and 1 min at 72 ºC, ending with 10 min at 72 ºC. All the PCR products were verified by cataphoresis. The amplicons were sequenced by an ABI 3730 DNA Analyzer based on the BigDye Terminator Cycle Sequencing Ready Kit (Applied Biosystems, Foster City, CA) in BGI (Beijing Genomics institution).