Xten platform

Whole-genome resequencing data available for the melpomene clade from previously published work were also included [30 (link),39 (link),42 (link),44 (link),45 (link),51 (link),70 (link)–72 (link)]. For a few additional samples, 100 to 150 bp paired-end whole-genome resequencing data were generated on an Illumina X Ten platform (Novogene Co. Ltd, China; S1 Table). In addition, we downloaded, processed, and analysed a publicily available data set for H. cydno galanthus [49 (link)] with a more moderate depth of coverage (for results see S14 Fig). For the erato clade already published, whole-genome-resequencing data were used [38 (link)] (S10 Table).
The whole-genome data were mainly used for demographic reconstructions, whereas, for other analyses, the regions matching the capture regions were used.

Fresh leaves, young stems, and flowers were collected from a 500-year-old C. camphora in Wuxue (115.56 E, 29.84 N), Hubei Province, China. High-quality genomic DNA was extracted from young leaves using a DNeasy Plant Mini Kit (Qiagen, Germany). The concentration and purity of the extracted DNA were assessed using a Nanodrop 2000 spectrophotometer (Thermo, MA, USA) and Qubit 3.0 (Thermo, CA, USA). The integrity of the DNA was determined using pulsed-field electrophoresis with a 0.8% agarose gel.
For genome sequencing, approximately 20-kb SMRTbell libraries were constructed for long-read sequencing on the PacBio Sequel platform at the Biomarker Technologies Corporation, Beijing. Additionally, short-read libraries were constructed and sequenced on an Illumina X-ten platform (Illumina, CA, USA) with 150-bp paired-end reads. After quality control, 111.06 Gb of PacBio single-molecule long reads and 49.59 Gb Illumina paired-end short reads were generated and used for assembly evaluation and genome correction. RaGOO [27 (link)] was used to anchor contigs and scaffolds to the C. kanehirae reference chromosome. A Hi-C library from young leaves was constructed by the Illumina Nova platform to verify the results of chromosome allocation.

For 151 KPC-PA strains, genomic DNA was extracted using a QIAamp DNA minikit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Libraries were prepared using the TruePrep DNA library prep kit V2 for Illumina (Vazyme Biotech, Nanjing, China). Sequencing was performed on an Illumina X Ten platform (Illumina Inc., CA, USA). The 150-bp paired-end reads were generated and de novo assembled using shovill v1.1.0 (37 ) with the options “–mincov 10 –minlen 200 –trim.” SPAdes v.3.13-v.3.14 (38 (link)) was used for assembly.

Young floral buds, 1–2 mm in length (pollen mother cell stage to microspore release from the tetrad) were collected from five plants, for each line, at the same time, and immediately frozen in liquid nitrogen. Samples were stored at − 80 °C before RNA extraction. Whole genomic RNA was extracted to maximize the short RNA segments in mitochondria using TRNzol (TIANGEN Biotech., Beijing, China) method in each, with three repeats. The integrity, quality, and concentration of extracted RNA were well checked as for mitochondrial DNA.
After the DNase I (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.) digestion, three independent libraries for each sample were constructed following TruSeq Stranded Total RNA Library Prep Workflow, with Ribo-Zero procedure to remove ribosomal RNA (Illumina, San Diego, California, U.S.), and then sequenced on Illumina X Ten platform (Illumina, San Diego, California, U.S.). The library was constructed and sequenced in our lab.

Phage DNA was extracted using λ phage genomic DNA rapid extraction kit (Beijing Adlai Company, China) according to the manufacturer’s instructions. The extracted DNA was fragmented to construct libraries with 300 bp inserted length, and the 150 bp paired-end sequencing was performed on the Illumina X-ten platform (Shanghai Majorbio Technology, Shanghai, China).